Genetically engineered cells expressing c-x-c chemokine receptor type 4, and uses thereof

Abstract

Provided are genetically engineered induced pluripotent stem cells (iPSCs) and derivative cells thereof that express C-X-C chemokine receptor type 4 (CXCR4). Also provided are uses of the CXCR4 positive iPSCs or derivative cells thereof for allogenic cell therapy.

Description

[0001] GENETICALLY ENGINEERED CELLS EXPRESSING C-X-C CHEMOKTNE RECEPTOR TYPE 4, AND USES THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 599,520 filed November 15, 2023, and U.S. Provisional Patent Application No. 63 / 569,849 filed March 26, 2024, each of which is incorporated by reference herein in its entirety.

[0004] TECHNICAL FIELD

[0005] This application provides genetically engineered induced pluripotent stem cells (iPSCs) and derivative cells thereof that express C-X-C chemokine receptor type 4 (CXCR4). Also provided are uses of the CXCR4 positive iPSCs or derivative cells thereof for allogenic cell therapy. Also provided are related vectors, polynucleotides, and pharmaceutical compositions.

[0006] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0007] This application contains a sequence listing, which is submitted electronically via Patent Center as an XML formatted sequence listing with a file name “066461-26WO1 SequenceListing_ST26.xml” having a file size of 310 kilobytes, and a creation date of November 11, 2024. The sequence listing submitted via Patent Center is part of the specification and is herein incorporated by reference in its entirety.

[0008] INCORPORATION BY REFERENCE

[0009] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BACKGROUND

[0010] Induced pluripotent stem cell (iPSC)-derived effector cells are emerging as a promising new approach for cancer immunotherapy. iPSCs can be generated by reprogramming adult somatic cells into a pluripotent state similar to embryonic stem cells. These iPSCs can then be differentiated into various effector immune cells like natural killer (NK) cells, T cells, and macrophages. The potential advantages of using iPSC-derived effector cells include their unlimited expansion capability and the ability to genetically engineer them to recognize and kill cancer cells.

[0011] However, a major challenge with iPSC-derived effector cell therapies is ensuring the cells can efficiently migrate to and infiltrate the tumor site. Without proper migration, the antitumor cytotoxic effect of the transferred cells are diminished. Effector cells rely on chemokines and chemokine receptors to migrate towards and into tumors. But in vitro generated effector cells can lack appropriate chemokine receptor expression, limiting their ability to respond to chemokine gradients produced by tumors.

[0012] To address these challenges, embodiments of the present disclosure are designed to increase depth and durability of response by engineering various immune cells (like T cells and NK cells) cells to express wild-type or mutated forms of CXCR4 to improve their trafficking and infiltration into tumors. CXCR4 is chemokine receptor known to be specific to the chemokine stromal-derived-factor- 1 (SDF-1). Interaction of CXCR4 with SDF-1 is generally involved in chemotaxis of lymphocytes specifically important for hematopoietic stem cell migration to bone marrow. Embodiments of the present disclosure demonstrate that the expression of CXCR4 in differentiated iNKs and iT cells not only improves migration of the cells to the bone marrow in mice, but also significantly improves tumor killing in a human leukemia xenograft mouse models.

[0013] BRIEF SUMMARY

[0014] In some aspect, the disclosure provides a primary cell, an induced pluripotent stem cell (iPSC) or a derivative cell thereof comprising at least one exogenous polynucleotide encoding: (i) one or more chimeric antigen receptors (CARs) comprising an antigen binding domain targeting at least one tumor antigen, and at least one intracellular domain; and (ii) C-X-C chemokine receptor type 4 (CXCR4) or a fragment or variant thereof. In some embodiments, the primary cell, the induced pluripotent stem cell (iPSC) or the derivative cell thereof optionally comprises at least one of: (i) a deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5, RFXAP genes, (ii) an exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G), (iii) an exogenous polynucleotide encoding a natural killer (NK) cell receptor immunoglobulin gamma Fc region receptor III (FcγRIII, cluster of differentiation 16 (CD 16)) and / or an NKG2D protein, (iv) a deletion or reduced expression of one or more of NKG2A CD70, CD38 or CD33 genes, (v) an exogeneous polynucleotide encoding a cytokine, (vi) an exogenous polynucleotide encoding a safety switch; (vii) an exogeneous polynucleotide encoding a PSMA cell tracer; and (viii) an exogeneous polynucleotide encoding a membrane bound IL- 12 polypeptide. In some embodiments, the at least one tumor antigen comprises a first tumor antigen and a second tumor antigen, and wherein: (i) the one or more CARs consists of a dual-targeting CAR comprising a first antigen- binding domain targeting the first tumor antigen and a second antigen binding domain targeting the second tumor antigen; or (ii) the one or more CARs comprises a plurality of CARs including a first CAR and a second CAR, wherein the first CAR comprises the first antigen binding domain targeting the first tumor antigen, and wherein the second CAR comprises a second antigen binding domain targeting the second tumor antigen. In some embodiments, the first tumor antigen is selected from Table 2. In some embodiments, the second tumor antigen is selected from Table 2. In some embodiments, the first antigen binding domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 2, 4, and 158-193. In some embodiments, the first antigen binding domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 194-229. In some embodiments, the second antigen binding domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 2, 4, and 158-193. In some embodiments, the second antigen binding domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 194-229. In some embodiments, in the CAR, at least one of: (i) a signal peptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 or 143; (ii) the at least one extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 2, 4, and 158-193; (iii) a hinge comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 21 and 22; (iv) a transmembrane domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 23 and 24; (v) the intracellular domain comprises a signaling and / or a co-stimulatory domain (i) comprising amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 6, 8-20, and 156, or (ii) encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 152-155. In some embodiments, in the CAR, at least one of: (i) the signal peptide comprises amino acids having the sequence of SEQ ID NO: 1 or 143; (ii) the at least one extracellular domain comprises amino acids having the sequence of one of SEQ ID NOs: 2, 4, and 158-193; (iii) the hinge comprises amino acids having the sequence of SEQ ID NO: 21 or 22; (iv) the transmembrane domain comprises amino acids having the sequence of SEQ ID NO: 23 or 24; (v) the intracellular domain comprises a signaling domain and / or a co-stimulatory domain (i) comprising amino acids having the sequence of one or more of SEQ ID NOs: 6, 8-20, and 156, or (ii) encoded by a polynucleotide having the sequence of one or more of SEQ ID NOs: 152-155. In some embodiments, the CXCR4 or a fragment or variant thereof is selected from the group consisting of wild-type CXCR4 and a mutated variant of CXCR4. In some embodiments, the wild-type CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 134. In some embodiments, the wild-type CXCR4 is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 142. In some embodiments, the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 134 with a deletion of the C-terminal domain between 10 and 20 amino acid residues. In some embodiments, the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 134 with one or more mutations selected from the group consisting of R334X, G336X, E343X, S341fs, S339fs342X, S338X, E343K, T328X, R144A, E179A, and E262A. In some embodiments, the mutated variant of CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 134-141. In some embodiments, the cytokine comprises an interleukin 15 (IL- 15) or a membrane bound IL-15, where all or a functional portion of the IL-15 protein is fused to all or a portion of a transmembrane protein that anchors the expressed IL- 15 as a cell membrane- bound polypeptide (mbIL15). In some embodiments, the IL-15 comprises an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope and an IL- 15, and wherein the inactivated cell surface receptor and the IL-15 are operably linked by an autoprotease peptide. In some embodiments, the IL- 15 comprises (i) a fusion polypeptide comprising an IL-15 and an IL-15 receptor alpha (IL-15Ra), or (ii) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 145. In some embodiments, the IL-15 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 72. In some embodiments, the primary cell, the iPSC, or the derivative cell comprises the deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes. In some embodiments, the primary cell, the iPSC, or the derivative cell comprises an exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G). In some embodiments, the CD 16 is a CD 16 variant protein. In some embodiments, the CD 16 variant protein is a high affinity CD 16 variant. In some embodiments, the CD 16 variant protein is a non-cleavable CD 16 variant. In some embodiments, the CD16 variant protein comprises wild-type CD16 comprising amino acids having the sequence of SEQ ID NO: 86 and one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, T220A. In some embodiments, the CD 16 variant protein comprises amino acids having at least 90% sequence identity to any one of SEQ ID NOs: 85 and 86. In some embodiments, the CD 16 protein and the NKG2D protein are operably linked by an autoprotease peptide. In some embodiments, the NKG2D protein is a wildtype NKG2D protein. Tn some embodiments, the NKG2D protein comprises amino acids having at least 90% sequence identity to SEQ ID NO: 89. In some embodiments, the autoprotease peptide is selected from the group consisting of a porcine tesehovirus-1 2A (P2A) peptide, a foot-and-mouth disease virus 2A (F2A) peptide, an Equine Rhinitis A Virus (ERAV) 2A (E2A) peptide, a Thosea asigna virus 2A (T2A) peptide, a cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and a Flacherie Virus 2A (BmIFV2A) peptide. In some embodiments, the autoprotease peptide is a P2A peptide comprising amino acids having at least 90% sequence identity to SEQ ID NO: 91. In some embodiments, the exogenous polynucleotide encoding the CD 16 protein and the NKG2D protein comprises a nucleic acid having at least 90% sequence identity to SEQ ID NO: 91. In some embodiments, the primary cell, the iPSC, or the derivative cell comprises (i) an exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL- 12) comprising a first polypeptide comprising an IL-12 alpha subunit p35 or a polypeptide at least 90% similar thereto, a second polypeptide comprising an IL-12 beta subunit p40 or a polypeptide at least 90% similar thereto, and a transmembrane domain fused to the terminus of the first and / or second IL- 12 subunit polypeptide. In some embodiments, the exogenous polynucleotide(s) are integrated into the chromosome of the cell at one or more loci selected from the group consisting of AAVS1, B2M, CBL-B, CCR5, CD33, CD38, CD70, CIITA, CIS, CISH, CLYBL, Collagen, CTLA4 , GAPDH, HTRP, LAG3, NKG2A, NKG2D, NLRC5, PD1, RFX5, RFXANK, RFXAP, ROSA26, RUNX1, SOCS2, TAP2, Tapasin, TAPBP, TAPI, TCR a or b constant region, TIGIT, TIM3, and TRAC genes, provided at least one of the exogenous polynucleotides is integrated at a locus of a gene selected from the group consisting of AAVS1, B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes to thereby result in a deletion or reduced expression of the gene. In some embodiments, the exogenous polynucleotide(s) are integrated into the chromosome of the cell at one or more loci selected from the group consisting of AAVS1, B2M, CIITA, CD33, and CLYBL. In some embodiments, the primary cell, the iPSC, or the derivative cell comprises a deletion or reduced expression of one or both of the B2M and the CIITA genes. In some embodiments, the primary cell, the iPSC, or the derivative cell comprises a deletion or reduced expression of both of the B2M and the CIITA genes. In some embodiments, (i) the primary cell is a T-cell or an NK cell, or (ii) the derivative cell is a T-cell or an NK cell. In some embodiments, (i) the primary cell is a T-cell, or (ii) the derivative cell is an iPSC-derived T-cell. In some embodiments, (i) the primary cell is an NK cell, or (ii) the derivative cell is an iPSC- derived NK cell. In some embodiments, the iPSC is reprogrammed from whole peripheral blood mononuclear cells (PBMCs). In some embodiments, the iPSC is derived from a re- programmed T-cell. In some embodiments, the primary cell, the iPSC, or the derivative cell further comprises an exogenous polynucleotide encoding a safety switch. In some embodiments, the safety switch comprises an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope. In some embodiments, the monoclonal antibody specific epitope is selected from a group of epitopes specifically recognized by ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, tremelimumab, ofatumumab, panitumumab, and ustekinumab. In some embodiments, the inactivated cell surface receptor is a truncated epithelial growth factor (tEGFR) variant. In some embodiments, the tEGFR variant consists of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 71. In some embodiments, the safety switch comprises (i) an intracellular domain comprising a herpes simplex virus thymidine kinase (HSV-TK) or (ii) an inducible Caspase 9 (iCasp9). In some embodiments, the PSMA cell tracer comprises an extracellular domain comprising a PSMA extracellular domain or fragment thereof. In some embodiments, the primary cell, the iPSC, or the derivative cell comprises a combined artificial cell death / reporter system polypeptide comprising an intracellular domain comprising a herpes simplex virus thymidine kinase (HSV-TK) and a linker, a transmembrane region, and an extracellular domain comprising the PSMA extracellular domain or fragment thereof. In some embodiments, (i) the HSV-TK comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 146 or 147, or (ii) the iCasp9 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 148 or 149. In some embodiments, the combined artificial cell death / reporter system polypeptide comprises the HSV-TK fused to a truncated variant PSMA polypeptide via the linker. In some embodiments, the truncated variant PSMA polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 150. In some embodiments, the linker comprises an autoprotease peptide selected from the group consisting of a P2A peptide, a T2A peptide, an E2A peptide, and a F2A peptide. In some embodiments, the artificial cell death / reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 151. 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: 234-236. In some embodiments, the artificial cell death / reporter system polypeptide is encoded by nucleic acids 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: 237-239. In some embodiments, the HLA-E comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 66, or the HLA-G comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 69. In some embodiments, (i) the exogenous polynucleotide encodes the one or more CARs comprising the antigen binding domain targeting the at least one tumor antigen selected from Table 2; (ii) the exogenous polynucleotide encoding CXCR4 (a) comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 142, or (b) encodes a CXCR4 polypeptide comprising amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 134-141; (iii) the exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G) comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 67 and 70; (iv) the exogenous polynucleotide encoding an NK cell receptor immunoglobulin gamma Fc region receptor III (FcγRIII, cluster of differentiation 16 (CD16)) and / or an NKG2D protein comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 84, 88, and 90; (v) the exogeneous polynucleotide encoding a cytokine comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 240; (vi) the exogenous polynucleotide encoding a safety switch comprises a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NO: 237-239; and / or (vii) the exogeneous polynucleotide encoding a PSMA cell tracer comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 150. In some embodiments, (i) the exogenous polynucleotide encodes the one or more CARs comprising the antigen binding domain targeting the at least one tumor antigen selected from Table 2; (ii) the exogenous polynucleotide encoding the CXCR4 (a) comprises polynucleotides having the sequence of SEQ ID NO: 142, or (b) encodes a CXCR4 polypeptide comprising amino acids having the sequence of one of SEQ ID NOs: 134- 141; (iii) the exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G) comprises polynucleotides having the sequence SEQ ID NO: 67 and 70; (iv) the exogenous polynucleotide encoding an FcyRIII receptor and / or an NKG2D protein comprises polynucleotides having the sequence of SEQ ID NO: 84, 88, and 90; (v) the exogeneous polynucleotide encoding the cytokine comprises polynucleotides having the sequence of SEQ ID NO: 240; and / or (vi) the exogenous polynucleotide encoding the safety switch comprises polynucleotides having the sequence of one of SEQ ID NOs: 237-239; and / or (vii) the exogenous polynucleotide encoding the PSMA cell tracer comprises polynucleotides having the sequence of one of SEQ ID NOs: 150. In some embodiments, each of the exogenous polynucleotides are integrated into the chromosome of the cell at a locus independently selected from the group consisting of AAVS1, B2M, CBL-B, CCR5, CD33, CD38, CD70, CIITA, CIS, CISH, CLYBL, Collagen, CTLA4 , GAPDH, HTRP, LAG3, NKG2A, NKG2D, NLRC5, PD1, RFX5, RFXANK, RFXAP, ROSA26, RUNX1, SOCS2, TAP2, Tapasin, TAPBP, TAPI, TCR a or b constant region, TIGTT, TIM3, and TRAC. In some embodiments, (i) the exogenous polynucleotide encoding the one or more CARs is integrated at a locus of the AAVS1 gene, the CD33 gene, or the CLYBL gene; (ii) the exogenous polynucleotide encoding the CXCR4 is integrated at a locus of the CLYBL gene; (iii) the exogenous polynucleotide encoding a HLA-E)and / or HLA-G is integrated at a locus of the B2M gene; (iv) the exogenous polynucleotide encoding an FcyRIII receptor and / or an NKG2D protein is integrated at a locus of the CD70 gene; (v) the exogeneous polynucleotide encoding the cytokine is integrated at the locus of the NKG2A gene; (vi) the primary cell, the iPSC or the derivative cell thereof comprises a deletion or reduced expression of the CIITA gene; and, optionally (vii) the exogenous polynucleotide encoding the safety switch or the PSMA is integrated at the locus of the CIITA gene. In some embodiments, the derivative cell is a natural killer (NK) cell or a T cell. In some embodiments, the derivative cell is a natural killer (NK) cell. In some embodiments, the derivative cell is a T cell. In some embodiments, the T cell is (i) a gamma delta T cell, or (ii) an alpha beta T cell. In some embodiments, the T cell is a gamma delta Vγ9 / Vδ1 T cell.

[0015] In some aspects, the disclosure provides a composition comprising a primary cell, an iPSC, or a derivative cell of the present disclosure. In some embodiments, the composition further comprises or is used in combination with, one or more therapeutic agents selected from the group consisting of a small-molecule therapeutic agent, a biologic, a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), a siRNA, an oligonucleotide, mononuclear blood cells, a vector comprising one or more polynucleotides of interest, an antibody, a chemotherapeutic agent, a radioactive moiety, and an immunomodulatory drug (IMiD).

[0016] In some aspects, the disclosure provides a method of treating a subject comprising administering the derivative cell, the primary cell, or the composition of the present disclosure to a subject in need thereof. In some embodiments, the subject has cancer, and the cancer is selected from the group consisting of leukemias, such as AML, CML, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), and chronic lymphocytic leukemia (CLL), lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, and follicular lymphoma, and solid cancers such as sarcomas, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer, ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer, cancer of the gall bladder and biliary tracts, thyroid cancer, thymus cancer, cancer of bone, and cerebral cancer, as well as cancer of unknown primary (CUP). In some embodiments, the cancer is a B-cell malignancy, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B- ALL), chronic lymphocytic leukemia (CLL), or non-Hodgkin lymphoma, follicular lymphoma. In some embodiments, the subject has minimal residual disease (MRD) after an initial cancer treatment. In some embodiments, the subject has no minimal residual disease (MRD) after one or more cancer treatments or repeated dosing.

[0017] In some aspects, the present disclosure comprises a method of manufacturing the derivative cell of the present disclosure comprising differentiating an iPSC of the present disclosure under conditions for cell differentiation to thereby obtain the derivative cell. In some embodiments, the iPSC is obtained by genomic engineering an unmodified iPSC, wherein the genomic engineering comprises targeted editing. In some embodiments, the targeted editing comprises deletion, insertion, or in / del carried out by CRISPR, ZFN, TALEN, homing nuclease, homology recombination, or any other functional variation of these methods. In some aspects, the present disclosure provides a method of differentiating an induced pluripotent stem cell (iPSC) into an NK cell, comprising subjecting the iPSCs to a differentiation protocol including culturing the cells in a medium containing a recombinant human IL- 12 for the final 24 hours of culturing under the differentiation protocol. In some embodiments, the recombinant IL-12 comprises IL12p70.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings.

[0020] FIGs. 1A-B show CXCR4 levels in (A) untransduced iPSC-derived NK (iNK) cell lines, and (B) iNK cell lines transduced with lentivirus designed to express wild-type CXCR4. Briefly, an iPSC line expressing a chimeric antigen receptor (CAR) against a tumor cell antigen was engineered to express the human wild type CXCR4 protein. The wild type human CXCR4 sequence was specifically engineered into the CLYBL locus of the iPSC line through homology -directed-repair. The iPSC lines were differentiated to NK cells and the resulting NK cells analyzed for CXCR4 levels by flow Cytometry. CXCR4 was found to be expressed in 42.4% of the engineered iNK cells IPSC 1613) versus 2.4% of the iNK cells not engineered to express CXCR4.

[0021] FIG. 2 shows expressing CXCR4 in iNK cells (iPSC 1613) significantly improves tumor killing in a Human leukemia xenograft mouse model, as compared to an iNK control cell line (iPSC611). iPSC611 is an iPSC line expressing chimeric antigen receptor targeting tumor cells, and iPSC1613 is the iPSC611 cell that was engineered to express human CXCR4. Both lines had exogenous EGFR / IL15 integrated at the CIITA locus, HLAE integrated at the β2M locus, an anti-CD19 CAR integrated at the AAVS1 locus, and CXCR4 integrated at the CLYBL locus. Both lines were differentiated to NK cells and tested in a human leukemia xenograft model. Tumor cells engineered to express luciferase were injected into mice intravenously (Day 0). At day 1, 10 million iNK cells were injected per mouse. Tumor burden was tracked through whole body bioluminescent imagine (BLI). At day 14, iNK cells expressing CXCR4 (iPSC 1613) had significantly improved tumor growth inhibition (TGI) versus NK cells without CXCR4 engineering (IPSC611) as measured by BLI.

[0022] FIGs. 3A-B show flow cytometry results of iNK levels as measured through enumeration of CD56+ cells (an NK specific marker) collected from (A) the blood and (B) the bone marrow in mice injected with CXCR4 negative iNK cells (iPSC611) and CXCR4 positive iNK cells (iPSC 1613). Results show that CXCR4 drives significant numbers of iNK cells into the bone marrow in mice (with and without the tumor being present). In FIG. 3A, no measurable increase of NK cells were observed in the blood of mice injected with CXCR4 positive iNK cells. The increase in CXCR4 negative cells in mice that also have tumor cells injected was not statistically significant. In FIG. 3B, a statistically significant increase in NK cells were observed in mouse bone marrow samples (each sample = 1 femur) that received CXCR4+ NK cells. The differences in the CXCR4+ mice with and without tumors was not found to be statistically significant. FIG. 4 shows surface CXCR4 expression data in cells of the present disclosure that are engineered to express wild-type CXCR4, as assessed by FACS. CXCR4-positive (+) iNK (iPSC1613) underwent engineering for expression of the human wild type CXCR4 protein via insertion into the CLYBL locus of the iPSC line through homology- directed repair. Following differentiation to iNK, this resulted in this particular bulk population of CXCR4+iNK with heterogenous expression of CXCR4 at 42.2%. Endogenous expression of CXCR4 in the CXCR4-negative (-) iNK cells (iPSC611) was 2.4%.

[0023] FIG. 5 shows surface CXCR4 expression data in cells of the present disclosure that are engineered to express wild-type CXCR4, as assessed by FACS. iPSC611 cells were genetically modified to express a transgene for wild-type CXCR4 (iPSC1613). Subsequent differentiation yielded a bulk population of iPSC1613 with heterogenous expression of CXCR4 at 47.7%. The endogenous expression of CXCR4 of iPSC611 was 4.74%.

[0024] FIG. 6 shows cytotoxicity of CD 19 CAR iNK cells iPSC611 (CXCR4-negative, circle) and iPSC1613 (CXCR4-positive, square), and CAR-negative CXCR4-negative effector cells (triangle) towards NucLight Red expressing NALM6 tumor line. Effector cells were co-cultured with target tumor cells at a 1: 1 effector-to-target cell ratio. Target cell growth was measured by Total Integrated Intensity readings from Incucyte Live-Cell Imaging system every 3 hours for 72 hours total. Figure curves represent target cell growth curves when co-cultured with effector cells, normalized to target cell growth curves when target cells are cultured alone. iPSC611 and iPSC1613 cells were equally potent in killing CD19-expressing target cells, indicating the CXCR4 transgene does not impact cytotoxicity in this assay.

[0025] FIG. 7 shows the CXCR4 expression in CAR iNKs differentiated to express a dual -targeting CAR (iNKs differentiated from iPSC2984), following lentiviral transduction of CXCR4 at the hematopoietic progenitor stage of differentiation, and subsequent differentiation to iNK. iPS cells expressing a dual -targeting anti-CD33 / anti- CD123 CAR (iPSC2984) were transduced with a lentivirus-containing sequence encoding for WT CXCR4 upon integration into the cell line genome. Hematopoietic progenitor cells (HPCs) derived from iPSC2984 were transduced with the WT CXCR4 lentivirus and then differentiated 14 days to iNK cells. The lentivirus-transduced cells (CAR-iNKCXCR4+) were determined to be 54.5% CXCR4-positive versus 3.29% for non- transduced cells (CAR-iNKCXCR4-).

[0026] FIGs. 8A-B show migration of CXCR4-positive cells (iNKCXCR4+ and Jurkat) and CXCR4-negative cells (iNKCXCR4- and M0LM13) expressing a dual-targeting CAR (iNKs differentiated from iPSC2984) in a transwell assay format. Transwell plates were coated with rat tail collagen, and test cells added to the upper chamber of the plates. Assay medium containing C-X-C motif chemokine 12 (CXCL12) was added to the lower chamber of the plates. Plates were incubated for 4 hours before being assessed by FACS for cell quantification. Chemotactic Index (Chi) was calculated as the number of cells in experimental wells (400 ng CXCL12) divided by the average of cells in the baseline wells (0 ng CXCL12). Shown are (A) raw cell counts at migration assay completion, and (B) calculated Chemotactic Index (Chi), which indicate pronounced migration of CXCR4-positive cells in the presence of CXCL12.

[0027] FIGs. 9A-B show cytotoxicity of CAR iNK cells that are CXCR4-negative (open shape) and CXCR4-positive (filled shape), and also express a dual-targeting CAR (iNKs differentiated from iPSC2984), towards NucLight Red expressing antigen-positive (diamond) and antigen-negative (triangle) tumor lines. Effector cells were co-cultured with target tumor cells at (A) 1 : 1 and (B) 2:1 effector-to-target cell ratios. Target cell growth was measured by Total Integrated Intensity readings from Incucyte Live-Cell Imaging system every 3 hours for 72 hours total. Figure curves represent target cell growth curves when co-cultured with effector cells, normalized to target cell growth curves when target cells are cultured alone. CXCR4-positive and CXCR4-negative CAR iNK cells were equally potent in killing antigen-expressing target cells, indicating the CXCR4 transgene does not impact cytotoxicity in this assay. No cytotoxicity against antigen-negative target cells was observed.

[0028] FIG. 10 depicts the study design for in vivo testing of CXCR4-positive and CXCR4-negative CAR-iNK cells that express a dual-targeting CAR (iNKs differentiated from iPSC2984). Luciferase-labeled tumor cells expressing CAR-specific antigen were intravenously (IV) injected into NOD SCID Gamma (NSG) mice on Day -4 (D-4). On Day 0 (DO), mice were intravenously injected with CXCR4-positive or -negative CAR- iNK. Mice underwent bioluminescent imaging to monitor tumor burden on Day -1 and twice weekly thereafter. On Day 9 (D9), tumor growth inhibition (TGI) was calculated, and mice were humanely euthanized for blood FACS analysis and histology sampling.

[0029] FIG. 11 shows in vivo tumor burden by bioluminescent imaging (BLI). Tumor- bearing mice were either untreated controls (open circle), or intravenously injected with CXCR4-positive (filled diamond) and CXCR4-negative (open diamond) CAR-iNK cells engineered to express a dual-targeting CAR (iNKs differentiated from iPSC2984) on Day 0, and tumor burden monitored twice weekly. On the final study day, percent tumor growth inhibition was calculated for each treatment as (l-[treated BLI / control BLI]) x 100. CXCR4-positive CAR-iNK elicited significant efficacy compared to CXCR4 negative CAR-iNK (p=0.0363) and untreated controls (p=0.0061).

[0030] FIG. 12 shows bone marrow presence of CAR-iNK cells engineered to express a dual-targeting CAR (iNKs differentiated from iPSC2984) in the presence and absence of tumor, assessed by FACS. CXCR4-positive and CXCR4-negative CAR-iNK cells were intravenously injected into tumor-bearing and non-tumor bearing mice. Nine days later, mice were sampled for bone marrow aspirates. Both tumor-bearing and non-tumor- bearing mice that received CXCR4-negative CAR-iNK did not have iNK present in the bone marrow. Significant numbers of iNK were present in the bone marrow of both tumor-bearing (p<0.0001) and non-tumor-bearing mice (p=0.0018) that received CXCR4-positive CAR-iNK.

[0031] FIG. 13 shows positive immunohistochemical (IHC) staining for C-X-C motif chemokine 12 (CXCL12) in mouse bone marrow.

[0032] FIG. 14 shows immunohistochemical (IHC) staining for iNK cells in mouse tissues from mice dosed with CXCR4-positive and CXCR4-negative CAR-iNK cells engineered to express a dual-targeting CAR (iNKs differentiated from iPSC2984). Mice that received CXCR4-positive CAR-iNK had greater tissue iNK infiltration (bone, spleen, liver, and lung), indicating enhanced migration as a result of CXCR4 engineering of iNK.

[0033] FIGs. 15A-B show CXCR4 levels in (A) iPSC-derived T (iT) cells engineered to express CXCR4, and (B) iT cells with endogenous CXCR4. Briefly, an iPSC line expressing a chimeric antigen receptor (CAR) against a tumor cell antigen was engineered to express the human wild type CXCR4 protein. The wild type human CXCR4 sequence was specifically engineered into the CLYBL locus of the iPSC line through homology-directed-repair. The iPSC lines were differentiated to T cells and the resulting T cells analyzed for CXCR4 levels by flow Cytometry. CXCR4 was found to be

[0034] 5 expressed in 91.4% of the engineered iT cells) versus 62.5% of the iT cells not engineered to express CXCR4 (i.e., expressing only endogenous CXCR4).

[0035] FIG. 16 shows chemotaxis of iT cells in vitro. iT Cells with either endogenous levels of CXCR4 Expression (left) or enhanced CXCR4 expression driven by transgene expression (right) were tested for chemotaxis to the chemokine SDF-1 in a transwell fO assay. iT cells were tested for migration through a membrane barrier either in the presence or absence of the chemokine. Chemotaxis was measured as the amount of cells in the bottom of the well that migrated through the membrane barrier.

[0036] FIG. 17 shows the presence of iT cells in the bone marrow of mice, as measured using FACS. NSG mice with established disseminated M0LM13 xenografts were intravenously injected with 7.5x10^6 CXCR4 transgene-positive or -negative iPSC- derived gamma delta iT cells per mouse. Nine days following injection, femurs were sampled for flow cytometry detection of iT cells. iT cells with CXCR4 transgene were found in greater numbers in the bone marrow than iT cells without the CXCR4 transgene.

[0037] FIGs. 18A-C show (A) the presence of iT cells in the bone marrow of mice, as

[0038] 20 measured using IHC. NSG mice with established disseminated M0LM13 xenografts were intravenously injected with 7.5x10^6 CXCR4 transgene-positive or -negative iPSC- derived gamma delta iT cells per mouse. Nine days following injection, femurs were sampled for immunohistochemical detection of T cells using a pan CD3 antibody (LN10). iT cells with CXCR4 transgene were found in greater numbers in the bone marrow than iT cells without the CXCR4 transgene; (B) anti-CD3 stained IHC image of bone marrow tissue from mice that were administered CXCR4 transgene-positive iPSC- derived gamma delta iT cells; and (C) anti-CD3 stained IHC image of bone marrow tissue from mice that were administered CXCR4 transgene-negative iPSC-derived gamma delta iT cells.

[0039] 30

[0040] DETAILED DESCRIPTION Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this application pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification.

[0042] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0043] Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Likewise, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

[0044] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are intended to be encompassed by the application.

[0045] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present), and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0046] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0047] As used herein, the term “consists of,” or variations such as “consist of’ or “consisting of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, but that no additional integer or group of integers can be added to the specified method, structure, or composition.

[0048] As used herein, the term “consists essentially of,” or variations such as “consist essentially of’ or “consisting essentially of,” as used throughout the specification and claims, indicate 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 materially change the basic or novel properties of the specified method, structure or composition. See M.P.E.P. § 2111.03.

[0049] As used herein, “subject” means any animal, preferably a mammal, most preferably a human. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably a human. It should also be understood that the terms “about,” “approximately,” “generally,” “substantially,” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.

[0050] The term "chimeric antigen receptor” or “CAR” refers to engineered receptors, which are grafted onto cells. In general, a CAR of the present disclosure comprises one or more extracellular domains comprising the antigen binding domain(s), one or more intracellular domains comprising one or more costimulatory and / or signaling domains, a hinge, a signal peptide, and / or a transmembrane domain. The antigen binding domain of the CAR targets specific antigens. The targeting regions may comprise full length heavy chain, Fab fragments, scFvs, divalent single chain antibodies or diabodies, each of which are specific to the target antigen (e.g., a tumor antigen of Table 2). The antigen binding domain can be derived from the same species or a different species for or in which the CAR will be used in.

[0051] The terms “C-X-C CHEMOKINE RECEPTOR TYPE 4” and “CXCR4” refer to a G protein-coupled receptor that binds the chemokine CXCL12. It plays important roles in embryogenesis, hematopoiesis, organogenesis, vascularization, and tumorigenesis. The wild type CXCR4 receptor contains 352 amino acids and has seven transmembrane domains, with an extracellular N-terminus and intracellular C-terminus. Upon binding CXCL12, CXCR4 activates downstream signaling pathways such as Ras / MAPK and PI3K / Akt that regulate cell migration, proliferation, and survival.

[0052] The terms “binder” and “specifically binds” or “specific for” with respect to an antigen-binding domain of a ligand like an antibody, of a fragment thereof or of a CAR refer to an antigen-binding domain which recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in a sample. An antigen-binding domain that binds specifically to an antigen from one species may bind also to that antigen from another species. This cross-species reactivity is not contrary to the definition of that antigen-binding domain as specific. An antigen-binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.). This cross reactivity is not contrary to the definition of that antigen-binding domain as specific.

[0053] The terms “engineered cell” and “genetically modified cell” as used herein can be used interchangeably. The terms mean containing and / or expressing a foreign gene or nucleic acid sequence which in turn modifies the genotype or phenotype of the cell or its progeny. Especially, the terms refer to cells, preferentially T cells which are manipulated by recombinant methods well known in the art to express stably or transiently peptides or proteins which are not expressed in these cells in the natural state. For example, T cells are engineered to express an artificial construct such as a chimeric antigen receptor on their cell surface. For example, the sequences encoding the CAR may be delivered into cells using a retroviral or lentiviral vector.

[0054] The term “primary cell” refers to a cell isolated directly from a multicellular organism. Primary cells typically have undergone very few population doublings and are therefore more representative of the main functional component of the tissue from which they are derived in comparison to continuous (tumor or artificially immortalized) cell lines. In some cases, primary cells are cells that have been isolated and then used immediately. In other cases, primary cells cannot divide indefinitely and thus cannot be cultured for long periods of time in vitro. In certain embodiments of the present disclosure, a primary cell can comprise a primary immune cell (e.g., a primary T-cell)

[0055] The term “target” as used herein refers to an antigen or epitope associated with a cell that should be recognized specifically by an antigen binding domain, e g., an antigen binding domain of an antibody or of a CAR. The antigen or epitope for antibody recognition can be bound to the cell surface but also be secreted, part of the extracellular membrane, or shed from the cell.

[0056] As used herein, the term “dual-targeting” refers to a protein (e.g , a chimeric protein) capable of binding to two different antigens. Specifically, a dual -targeting protein of the present di sclosure (e.g , a C AR having two or more tumor or cancer antigen binding domains) does not naturally occur and is produced by a genetic engineering method or other method. In one embodiment, a primary ceil, an engineered iPSC or derivative cell of the present disclosure can comprise one or more exogenous polynucleotides encoding a CAR having a first antigen binding domain that specifically binds CD19 and a second antigen binding domain that specifically binds CD22. This is in contrast with other examples of the present disclosure wherein a primary cell, an engineered iPSC or derivative cell comprises one or more polynucleotides encoding a first CAR having a first antigen binding domain that specifically binds CD33 and a second CAR having a second antigen binding domain that specifically binds CD123.

[0057] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences (e.g., CAR polypeptides and the CAR polynucleotides that encode them), refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0058] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, 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 for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0059] Optimal alignment of sequences for comparison can be conducted, e.g., 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 search for similarity method of Pearson & Lipman, Proc. Nat ’I. Acad. Set. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that are 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, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul etal, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0060] Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring 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 defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N= -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0061] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which 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 smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0062] 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, for example, where the two peptides 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.

[0063] As used herein, the term “isolated” means a biological component (such as a nucleic acid, peptide, protein, or cell) has been substantially separated, 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. Nucleic acids, peptides, proteins, and cells that have been “isolated” thus include nucleic acids, peptides, proteins, and cells purified by standard purification methods and purification methods described herein. “Isolated” nucleic acids, peptides, proteins, and cells can be part of a composition and still be isolated if the composition is not part of the native environment of the nucleic acid, peptide, protein, or cell. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[0064] As used herein, the term “polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double- stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0065] A “construct” refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo. A “vector,” as used herein refers to any nucleic acid construct capable of directing the delivery or transfer of a foreign genetic material to target cells, where it can be replicated and / or expressed. The term “vector” as used herein comprises the construct to be delivered. A vector can be a linear or a circular molecule. A vector can be integrating or non-integrating. The major types of vectors include, but are not limited to, plasmids, episomal vector, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, Sendai virus vector, and the like.

[0066] By “integration” it is meant that one or more nucleotides of a construct is stably inserted into the cellular genome, i.e., covalently linked to the nucleic acid sequence within the cell's chromosomal DNA. By “targeted integration” it is meant that the nucleotide(s) of a construct is inserted into the cell's chromosomal or mitochondrial DNA at a pre-selected site or “integration site”. The term “integration” as used herein further refers to a process involving insertion of one or more exogenous sequences or nucleotides of the construct, with or without deletion of an endogenous sequence or nucleotide at the integration site. In the case, where there is a deletion at the insertion site, “integration” can further comprise replacement of the endogenous sequence or a nucleotide that is deleted with the one or more inserted nucleotides.

[0067] As used herein, the term “exogenous” is intended to mean that the referenced molecule or the referenced activity is introduced into, or non-native to, the host cell. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non- chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the cell. The term “endogenous” refers to a referenced molecule or activity that is present in the host cell in its native form. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid natively contained within the cell and not exogenously introduced.

[0068] As used herein, a “gene of interest” or “a polynucleotide sequence of interest” is a DNA sequence that is transcribed into RNA and in some instances translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. A gene or polynucleotide of interest can include, but is 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 may encode an miRNA, an shRNA, a native polypeptide (i.e. a polypeptide found in nature) or fragment thereof; a variant polypeptide (i.e. a mutant of the native polypeptide having less than 100% sequence identity with the native polypeptide) or fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, and the like.

[0069] “Operably-linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably-linked with a coding sequence or functional RNA when it is capable of affecting the expression of that coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.

[0070] The term “expression” as used herein, refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term also encompasses translation of 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 a host cell, into the extracellular milieu such as the growth medium of a cell culture or anchored to the cell membrane.

[0071] As used herein, the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule comprised of amino acids and can be recognized as a protein by those of skill in the art. The conventional one-letter or three-letter code for amino acid residues is used herein. The terms “peptide,” “polypeptide,” and “protein” can be used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0072] The peptide sequences described herein are written according to the usual convention whereby the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of the amino acids are known, it is the L- form of the amino acid that is represented unless otherwise expressly indicated.

[0073] As used herein, the term “engineered immune cell” refers to an immune cell, also referred to as an immune effector cell, that has been genetically modified by the addition of exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.

[0074] Overview

[0075] The present disclosure provides engineered cells (and compositions comprising such cells) expressing wild-type or mutant forms of CXCR4. In certain aspects, the present disclosure provides a primary cell, an induced pluripotent stem cell (iPSC) or a derivative cell thereof comprising at least one exogenous polynucleotide. In certain embodiments, the at least one exogenous polynucleotide can encode one or more chimeric antigen receptors (CARs) comprising an antigen binding domain targeting at least one tumor antigen, and at least one intracellular domain. In certain embodiments, the at least one exogenous polynucleotide can encode a C-X-C chemokine receptor type 4 (CXCR4) or a fragment or variant thereof. In certain embodiments, the at least one exogenous polynucleotide can encode (i) one or more chimeric antigen receptors (CARs) comprising an antigen binding domain targeting at least one tumor antigen, and at least one intracellular domain, and (ii) a C-X-C chemokine receptor type 4 (CXCR4) or a fragment or variant thereof. In certain embodiments, the at least one exogenous polynucleotide can encode (i) one or more chimeric antigen receptors (CARs) comprising an antigen binding domain targeting at least one tumor antigen, and at least one intracellular domain, (ii) a C-X-C chemokine receptor type 4 (CXCR4) or a fragment or variant thereof, and (iii) at least one of: a deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5, RFXAP genes; an exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G); an exogenous polynucleotide encoding a natural killer (NK) cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD 16)) and / or an NKG2D protein; a deletion or reduced expression of one or more of NKG2A CD70, CD38 or CD33 genes; an exogeneous polynucleotide encoding a cytokine; an exogenous polynucleotide encoding a safety switch; an exogeneous polynucleotide encoding a PSMA cell tracer; and an exogeneous polynucleotide encoding a membrane bound IL-12 polypeptide.

[0076] I. Induced Pluripotent Stem Cells (iPSCs) And Immune Effector Cells IPSCs have unlimited self-renewing capacity. Use of iPSCs enables cellular engineering to produce a controlled cell bank of modified cells that can be expanded and differentiated into desired immune effector cells, supplying large amounts of homogeneous allogeneic therapeutic products.

[0077] Provided herein are genetically engineered iPSCs and derivative cells thereof. The selected genomic modifications provided herein enhance the therapeutic properties of the derivative cells. The derivative cells are functionally improved and suitable for allogenic off-the-shelf cell therapies following a combination of selective modalities being introduced to the cells at the level of iPSC through genomic engineering. This approach can help to reduce the side effects mediated by CRS / GVHD and prevent long-term autoimmunity while providing excellent efficacy.

[0078] As used herein, the term "differentiation" is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the features of a specialized cell. Specialized cells include, for example, a blood cell or a muscle cell. A differentiated or differentiation- induced cell is one that has taken on a more specialized ("committed") position within the lineage of a cell. The term "committed", when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. As used herein, the term "pluripotent" refers to the ability of a cell to form all lineages of the body or soma or the embryo proper. For example, embryonic stem cells are a type of pluripotent stem cells that are able to form cells from each of the three germs layers, the ectoderm, the mesoderm, and the endoderm. Pluripotency is a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell or EpiSC), which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e g., an embryonic stem cell).

[0079] As used herein, the terms "reprogramming" or "dedifferentiation" refers to a method of increasing the potency of a cell or dedifferentiating the cell to a less differentiated state. For example, a cell that has an increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in the non-reprogrammed state. In other words, a reprogrammed cell is one that is in a less differentiated state than the same cell in a non-reprogrammed state.

[0080] As used herein, the term "induced pluripotent stem cells" or, iPSCs, means that the stem cells are produced from differentiated adult, neonatal or fetal cells that have been induced or changed or reprogrammed into cells capable of differentiating into tissues of all three germ or dermal layers: mesoderm, endoderm, and ectoderm. The iPSCs produced do not refer to cells as they are found in nature. The term “hematopoietic stem and progenitor cells,” “hematopoietic stem cells,” “hematopoietic progenitor cells,” or “hematopoietic precursor cells” or “HPCs” refers to cells which are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation. Hematopoietic stem cells include, for example, multipotent hematopoietic stem cells (hematoblasts), myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (T cells, B cells, NK cells). As used herein, “CD34+ hematopoietic progenitor cell” refers to an HPC that expresses CD34 on its surface.

[0081] As used herein, the term “immune cell” or “immune effector cell” refers to a cell that is involved in an immune response. Immune response includes, for example, the promotion of an immune effector response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytes.

[0082] As used herein, the terms “T lymphocyte” and “T cell” are used interchangeably and refer to a type of white blood cell that completes maturation in the thymus and that has various roles in the immune system. A T cell can have the roles including, e.g., the identification of specific foreign antigens in the body and the activation and deactivation of other immune cells. A T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. The T cell can be CD3+ cells. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells (e.g., Thl and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulator T cells, gamma delta T cells (gd T cells), and the like. Additional types of helper T cells include cells such as Th3 (Treg), Thl7, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tern cells and TEMRA cells). The T cell can also refer to a genetically engineered T cell, such as a T cell modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). The T cell can also be differentiated from a stem cell or progenitor cell.

[0083] “CD4+ T cells” refers to a subset of T cells that express CD4 on their surface and are associated with cell-mediated immune response. They are characterized by the secretion profdes following stimulation, which may include secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4 and IL10. “CD4” are 55-kD glycoproteins originally defined as differentiation antigens on T-lymphocytes, but also found on other cells including monocytes / macrophages. CD4 antigens are members of the immunoglobulin supergene family and are implicated as associative recognition elements in MHC (major histocompatibility complex) class Il-restricted immune responses. On T- lymphocytes they define the helper / inducer subset.

[0084] “CD8+ T cells” refers to a subset of T cells which express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells. “CD8” molecules are differentiation antigens found on thymocytes and on cytotoxic and suppressor T- lymphocytes. CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I- restricted interactions.

[0085] As used herein, the term “NK cell” or “natural killer cell” 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 chains). The NK cell can also refer to a genetically engineered NK cell, such as a NK cell modified to express a chimeric antigen receptor (CAR). The NK cell can also be differentiated from a stem cell or progenitor cell.

[0086] The induced pluripotent stem cell (iPSC) parental cell lines may be generated from peripheral blood mononuclear cells (PBMCs) or T-cells using any known method for introducing re-programming factors into non-pluripotent cells such as the episomal plasmid-based process as previously described in U.S. Pat. Nos. 8,546,140; 9,644,184; 9,328,332; and 8,765,470, the complete disclosures of which are incorporated herein by reference. The reprogramming factors may be in a form of polynucleotides, and thus are introduced to the non-pluripotent cells by vectors such as a retrovirus, a Sendai virus, an adenovirus, an episome, and a mini-circle. In particular embodiments, the one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, the one or more polynucleotides introduced by an episomal vector. In various other embodiments, the one or more polynucleotides are introduced by a Sendai viral vector. In some embodiments, the iPSC’s are clonal iPSC’s or are obtained from a pool of iPSCs and the genome edits are introduced by making one or more targeted integration and / or in / del at one or more selected sites. In another embodiment, the iPSC’s are obtained from human T cells having antigen specificity and a reconstituted TCR gene (hereinafter, also refer to as "T-iPS” cells) as described in US Pat. Nos. 9206394 and 10787642 hereby incorporated by reference into the present application.

[0087] According to a particular aspect, the application relates to an induced pluripotent stem cell (iPSC) cell or a derivative cell thereof comprising: (i) one or more exogenous polynucleotides encoding a chimeric antigen receptor (CAR) and a CXCR4 polypeptide; (ii) an exogenous polynucleotide encoding a truncated epithelial growth factor receptor (tEGFR) variant and an interleukin- 15 (IL- 15), wherein the tEGFR variant and IL- 15 are operably linked by an autoprotease peptide sequence, such as the porcine tesehovirus-1 2A (P2A); and (iii) a deletion or reduced expression of B2M and CIITA genes.

[0088] II. CXCR4 Expression

[0089] CXCR4 is a G protein-coupled receptor that binds the chemokine CXCL12. It plays important roles in embryogenesis, hematopoiesis, organogenesis, vascularization, and tumorigenesis. The wild type CXCR4 receptor contains 352 amino acids and has seven transmembrane domains, with an extracellular N-terminus and intracellular C- terminus. Upon binding CXCL12, CXCR4 activates downstream signaling pathways such as Ras / MAPK and PI3K / Akt that regulate cell migration, proliferation, and survival.

[0090] There are several known naturally occurring mutations in the CXCR4 gene that result in altered receptor function. For example, the WHIM syndrome mutation CXCR4- E343K leads to impaired receptor downregulation and enhanced cellular responses to CXCL12. Patients with WHIM syndrome exhibit warts, hypogammaglobulinemia, recurrent bacterial infections, and myelokathexis (retention of mature neutrophils in bone marrow). The enhanced CXCL12 / CXCR4 signaling causes abnormal neutrophil retention and B cell lymphopenia. Another noteworthy CXCR4 mutation is CXCR4-R334X, which causes truncation of the C-terminal tail of the receptor. This mutation impairs CXCL12- mediated chemotaxis and calcium flux, highlighting the importance of the C-tail in G protein coupling and downstream signaling. Truncation of the CXCR4 C-tail (CXCR4- T328) can reduce CXCL12 binding affinity but maintains signaling capacity. Mutating two extracellular loop glutamic acid residues (CXCR4-E179A / E262A) impairs CXCL12 binding and signaling. Substitution of the conserved DRY motif in the second intracellular loop (CXCR4-R144A) also abrogates CXCL12-induced signaling.

[0091] In addition to natural and engineered mutations, CXCR4 signaling capacity and expression levels are affected in various disease states. For example, in warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, mutations lead to increased surface expression and impaired downregulation of CXCR4. In cancer, CXCR4 is upregulated in at least 23 different types of malignancies. The increased CXCR4 expression in cancer cells promotes metastasis and angiogenesis. Thus, CXCR4 is an important therapeutic target in both WHIM syndrome and various cancers.

[0092] CXCR4 is also known to be specific to the chemokine stromal-derived-factor- 1 (SDF-1). Interaction of CXCR4 with SDF-1 can be involved in chemotaxis of lymphocytes specifically important for hematopoietic stem cell migration to bone marrow. Enforced human CXCR4 expression in primary derived NK or T cells has been shown to improve migration of the cells to the bone marrow. The present disclosure provides embodiments in which wild type human CXCR4 has been introduced into iPS- derived cells expressing a CAR, demonstrating that the expression of CXCR4 in differentiated iPSC-derived effector cells (i) improves both migration of the effector cells to the bone marrow (e.g., in mice), and (ii) significantly improves tumor killing (e.g., in a Human leukemia xenograft mouse model).

[0093] In certain embodiments, a primary cell, an iPSC or a derivative cell thereof according to the present disclosure can comprise one or more exogenous polynucleotides encoding CXCR4, and the CXCR4 or a fragment or variant thereof is selected from the group consisting of wild-type CXCR4 and a mutated variant of CXCR4. In certain embodiments, the CXCR4 is wild-type CXCR4. In certain embodiments, the CXCR4 is wild-type CXCR4 and comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 134. In certain embodiments, the wild-type CXCR4 is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 142. In other embodiments, the CXCR4 is a mutated variant of CXCR4. In certain embodiments, the CXCR4 is a mutated variant of CXCR4 and the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 134 with a deletion of the C-terminal domain between 10 and 20 amino acid residues. In a particular embodiment, a CXCR4 with the deletion in the C-terminal domain corresponds to the deletion of the 10, 11, 12, 13, 14, 15, 16, 17, 18, 16, 17, 18, 19, or 20 amino acid residues in the C-terminal domain of the CXCR4 polypeptide. In certain embodiments, the CXCR4 is a mutated variant of CXCR4 and the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 134 with one or more mutations selected from the group consisting of R334X, G336X, E343X, S341fs, S339fs342X, S338X, E343K, T328X, R144A, E179A, and E262A. In certain embodiments, the CXCR4 is a mutated variant of CXCR4 and the mutated variant of CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 134-141.

[0094] In some embodiments, the CXCR4 mutation is a CXCR4wlummutation. The term "CXCR4whimmutation" refers to the autosomal dominant mutation associated with the rare combined primary immunodeficiency Warts, Hypogammaglobulinemia, Infections and Myelokathexis (WHIM) Syndrome (WS) which have been linked to inherited autosomal dominant gain-of-function mutations in CXCR4 (see, e.g., Kawai, T. & Malech, H. L. WHIM syndrome: congenital immune deficiency disease. Current Opinion in Hematology 16, 20-26 (2009); and Dotta, L., Tassone, L. & Badolato, R. Clinical and genetic features of Warts, Hypogammaglobulinemia, Infections and Myelokathexis (WHIM) syndrome. Curr. Mol. Med. 11, 317-325 (2011), each of which is hereby incorporated by reference herein in its entirety). These mutations result in the distal truncation of the C-term of CXCR4 and a desensitization- and internalization-resistant receptor in response to CXCL12 (see, e.g., Hernandez, P. A. et al. Mutations in the chemokine receptor gene CXCR4 are associated with WHIM syndrome, a combined immunodeficiency disease. Nat Genet 34, 70-74 (2003); and Balabanian, K. et al. Proper desensitization of CXCR4 is required for lymphocyte development and peripheral compartmentalization in mice. Blood 119, 5722-5730 (2012), each of which is hereby incorporated by reference herein in its entirety). Patients also exhibit a severe, chronic pan-leukopenia with neutrophils, naive T cells and mature recirculating B cells being most affected (see, e.g., Gulino, A. V. Altered leukocyte response to CXCL12 in patients with warts hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome. Blood 104, 444-452 (2004), which is hereby incorporated by reference herein in its entirety). The different autosomal dominant gain-of-function mutations in CXCR4 associated with Whim syndrome include: (i) R334X (gene mutation 1000C>T) the most frequent mutation resulting in carboxy- terminal truncation of 19 residues (Hernandez, et al.); (ii) G336X, (gene mutation 1000C>T) resulting in carboxy-terminus truncation of 17 residues (Gulino et al.); (iii) E343X (gene mutation 1027G>T) resulting in carboxy- terminus truncation of the receptor of 10 residues (Hernandez, et al.); (iv) S339fs342X, (gene mutation 1016-1017delCT) resulting in carboxy-terminus truncation of the receptor of 13 residues and addition of 3 additional amino acid residues (Hernandez, et al.; and Liu, Q. et al. WHIM syndrome caused by a single amino acid substitution in the carboxy- tail of chemokine receptor CXCR4. Blood 120, 181-189 (2012), which is hereby incorporated by reference herein in its entirety); (v) S338X (gene mutation 1013OG), resulting in carboxy -terminus truncation of the receptor of 12 residues (Balabanian, K. et al. WHIM syndromes with different genetic anomalies are accounted for by impaired CXCR4 desensitization to CXCL12. Blood 105, 2449-2457 (2005), which is hereby incorporated by reference herein in its entirety); and (vi) E343K (nucleic acid mutation 1027G>A) (Liu, et al.).

[0095] The different autosomal dominant gain-of-function mutations in CXCR4 associated with Whim syndrome are located in the C-terminal domain of the CXCR4 receptor (see Table 1), a domain responsible for regulation of the receptor (internalization / inactivation). Accordingly the C-terminal domain of the CXCR4 receptor, which is deleted in several Whim mutation could also be directly deleted to obtain the same biological effect (gain-of- function of CXCR4 associated with the long term CD8 memory response). Biological activity of CD8 cells according to the invention (gain-of- function of CXCR4) can be measured for example with chemokine receptor internalization assay or cell migration after adding a CXCR4 agonist. Table 1.

[0096]

[0097]

[0098]

[0099] III. Chimeric Antigen Receptor (CAR) Expression

[0100] In some embodiments, an iPSC or derivative cell thereof contains an exogenous polynucleotide encoding a chimeric antigen receptor (CAR), such as a CAR targeting a tumor antigen. In some instances, the recombinant CAR polypeptide includes at least an extracellular domain that binds specifically to an antigen (or more than one antigen), a transmembrane domain and an intracellular signaling domain. In some instances, the recombinant CAR polypeptide includes a signal peptide, an extracellular domain that binds specifically to an antigen (or more than one antigen), a transmembrane domain, an intracellular signaling domain and one or more co-stimulatory domains. In other instances, the recombinant CAR polypeptide includes a signal peptide, an extracellular domain that binds specifically to an antigen (or more than one antigen), a hinge region, a transmembrane domain, an intracellular signaling domain and one or more co-stimulatory domains.

[0101] A. Extracellular domains

[0102] In certain embodiments, an extracellular domain of a CAR includes an antibody, an antibody fragment, an antigen-binding domain and / or an antigen-binding fragment. The antigen binding fragment can, for example, be an antibody or antigen-binding fragment thereof that specifically binds a tumor antigen. In some embodiments, the antigen-binding domains or fragments possess one or more desirable functional properties including, but not limited to, high-affinity binding to a tumor antigen, high specificity to a tumor antigen, the ability to stimulate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and / or antibody-dependent cellular- mediated cytotoxicity (ADCC) against cells expressing a tumor antigen, and the ability to inhibit tumor growth in subjects in need thereof and in animal models when administered alone or in combination with other anti-cancer therapies.

[0103] In some embodiments, antibodies or antibody fragments suitable for use in the CAR include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, polypeptide-Fc fusions, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuticals ("SMIPsTM"), intrabodies, minibodies, single domain antibody variable domains, nanobodies, VHHs, diabodies, tandem diabodies (TandAb®), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antigen-specific TCR), and epitope- binding fragments of any of the above. Antibodies and / or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0104] In some embodiments, the antigen-binding fragment is an Fab fragment, an 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, a Fd' fragment, a Fd fragment, a heavy chain variable region, an isolated complementarity determining region (CDR), a diabody, a triabody, or a decabody. In some embodiments, the antigen-binding fragment is an scFv fragment. In some embodiments, the antigen- binding fragment is a VHH.

[0105] In some embodiments, the extracellular domain of the CAR is a single-domain antibody or nanobody. In some embodiments, the extracellular domain is a VHH. In some embodiments, the extracellular domain is an scFv. i. Antigen-binding domains

[0106] In some embodiments, an antigen-binding domain of a CAR binds to a target antigen. The antigen-binding domain may bind to more than one antigen or more than one epitope in an antigen. For example, the antigen-binding domain may bind to 2, 3, 4, 5, 6, 7, 8 or more antigens. As another example, the antigen-binding domain may bind 2, 3, 4, 5, 6, 7, 8 or more epitopes in the same antigen.

[0107] The choice of antigen-binding domain may depend upon the type and number of antigens that define the surface of a target cell. For example, the antigen-binding domain may be chosen to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state. In some embodiments, CAR can be genetically modified to target a tumor antigen of interest by way of engineering a desired antigen- binding domain that specifically binds to an antigen (e.g., on a tumor cell). Non-limiting examples of cell surface markers that may act as targets for the antigen-binding domain in the CAR include those associated with tumor cells or autoimmune diseases.

[0108] In some embodiments, the antigen-binding domain binds to at least one tumor antigen or autoimmune antigen. In some embodiments, the antigen-binding domain binds to at least one tumor antigen or autoimmune antigen selected from one or more of Tables 2-4.

[0109] 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.

[0110] In some embodiments, the antigen-binding domain binds to at least one autoimmune antigen. In some embodiments, the antigen-binding domain binds to two or more autoimmune antigens. In some embodiments, the two or more autoimmune antigens are associated with the same autoimmune disease. In some embodiments, the two or more autoimmune antigens are associated with different autoimmune diseases.

[0111] 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 hematologic malignancy. Non-limiting examples of tumor antigens associated with glioblastoma include HER2, EGFRvIII, EGFR, CD133, PDGFRA, FGFR1, FGFR3, MET, CD70, ROBO1 and IL13Ra2. Non- limiting examples of tumor antigens associated with ovarian cancer include FOLR1, FSHR, MUC16, MUC1, Mesothelin, CA125, EpCAM, EGFR, PDGFRa, Nectin-4 and B7H4. Non-limiting examples of the tumor antigens associated with cervical cancer or head and neck cancer include GD2, MUC1, Mesothelin, HER2, and EGFR. Non-limiting examples of tumor antigen 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 CD 19, 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 cancer include CD70 and FOLR1.

[0112] Additional examples of antigens that may be targeted by the antigen-binding domain include, but are not limited to, alpha-fetoprotein, A3, antigen specific for A33 antibody, Ba 733, BrE3-antigen, carbonic anhydrase EX, CD1, CDla, 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, EphAl, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphAlO, EphBl, EphB2, EphB3, EphB4, EphB6, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, hypoxia inducible factor (HIF-I), la, IL-2, IL-6, IL-8, insulin growth factor- 1 (IGF-I), KC4-antigen, KS-1 -antigen, KS1-4, Le-Y, macrophage inhibition factor (MIF), MAGE, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody, placental growth factor, p53, prostatic acid phosphatase, PSA, PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin, 17-lA-antigen, an angiogenesis marker, an oncogene marker or an oncogene product.

[0113] In some embodiments, the antigen is associated with an autoimmune disease or disorder. Such antigens may be derived from cell receptors and cells which produce “self ’-directed antibodies. 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-Barre syndrome, Crohn's disease or ulcerative colitis.

[0114] In some embodiments, autoimmune antigens that may be targeted by the CAR include, but are not limited to, platelet antigens, myelin protein antigen, Sm antigens in snRNPs, islet cell antigen, rheumatoid factor, and anticitrullinated protein, citrullinated proteins and peptides such as CCP-1, CCP-2 (cyclical citrullinated peptides), fibrinogen, fibrin, vimentin, filaggrin, collagen I and II peptides, alpha-enolase, translation initiation factor 4G1, perinuclear factor, keratin, Sa (cytoskeletal protein vimentin), components of articular cartilage such as collagen II, IX, and XI, circulating serum proteins such as RFs (IgG, IgM), fibrinogen, plasminogen, ferritin, nuclear components such as RA33 / hnRNP A2, Sm, eukaryotic translation elongation factor 1 alpha 1, stress proteins such as HSP- 65, -70, -90, BiP, inflammatory / immune factors such as B7-H1, IL-1 alpha, and IL-8, enzymes such as calpastatin, alpha-enolase, aldolase-A, dipeptidyl peptidase, osteopontin, glucose-6-phosphate isomerase, receptors such as lipocortin 1, neutrophil nuclear proteins such as lactoferrin and 25-35 kD nuclear protein, granular proteins such as bactericidal permeability increasing protein (BPI), elastase, cathepsin G, myeloperoxidase, proteinase 3, platelet antigens, myelin protein antigen, islet cell antigen, rheumatoid factor, histones, ribosomal P proteins, cardiolipin, vimentin, nucleic acids such as dsDNA, ssDNA, and RNA, ribonuclear particles and proteins such as Sm antigens (including but not limited to SmD's and SmB' / B), U1RNP, A2 / B1 hnRNP, Ro (SSA), and La (SSB) antigens.

[0115] Non-limiting exemplary antigen targets are provided in Tables 2-4. Table 2 provides antigen binding domains that bind to exemplary antigen targets. The antigen- binding domain may comprise a VH sequence, a VL sequence, and / or CDRs thereof, such as those described in the cited publications, the contents of each publication are incorporated herein by reference in their entirety for all purposes.

[0116] Table 2.

[0117]

[0118]

[0119]

[0120]

[0121]

[0122] Table 3 provides exemplary antigen targets. The antigen-binding domain may comprise 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 publication are incorporated herein by reference in their entirety for all purposes.

[0123] Table 3.

[0124] Table 4 provides exemplary antigen targets. The antigen-binding domain may comprise 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 publication are incorporated herein by reference in their entirety for all purposes.

[0125] Table 4. ii. Linkers

[0126] In some embodiments, an scFv fragment of an extracellular domain of a CAR includes a linker between the VH and VL domains. The linker can be a peptide linker and may include any naturally occurring amino acid. Exemplary amino acids that may be included into the linker are Gly, Ser Pro, Thr, Glu, Lys, Arg, Ile, Leu, His and Phe. The linker should have a length that is adequate to connect the VH and the VL in such a way that they form the correct conformation relative to one another so that they retain the desired activity, such as binding to an antigen. The linker may be about 5-50 amino acids long. In some embodiments, the linker is about 10-40 amino acids long. In some embodiments, the linker is about 10-35 amino acids long. In some embodiments, the linker is about 10-30 amino acids long. In some embodiments, the linker is about 10-25 amino acids long. In some embodiments, the linker is about 10-20 amino acids long. In some embodiments, the linker is about 15-20 amino acids long. Exemplary linkers that may 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.

[0127] In some embodiments, the linker is a Whitlow linker. In one embodiment, the Whitlow linker includes 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 PCT / US2021 / 072646.

[0128] In another embodiment, the linker is a (G4S)3linker. In one embodiment, the (G4S)3linker includes the amino acid sequence set forth in SEQ ID NO: 25, 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: 25 of PCT / US2021 / 072646.

[0129] Other linker sequences may include portions of immunoglobulin hinge area, CL or CHI derived from any immunoglobulin heavy or light chain isotype. Exemplary linkers that may be used include any of SEQ ID NOs: 26-56 in Table 1 of PCT / US2021 / 072646, the disclosure including the sequence listing is incorporated herein by reference. Additional linkers are described for example in W02019 / 060695, incorporated by reference herein in its entirety.

[0130] The linkers described herein including SEQ ID NOs: 40-73 of Table 4 can be used in any of the polypeptides provided including those containing CD 16, NKG2D, IL- 15, IL-15Ra, HLA-E, HLA-G, HSV-TK, PSMA, and the like.

[0131] Table 5 provides exemplary linkers (SEQ ID NOs: 40-73), which correspond to SEQ ID NOs: 3 and 25-56 of US Application No. 17 / 657,803 filed April 4, 2022, the contents of which are incorporated herein by reference in its entirety. Table 5.

[0132] B. Signal peptides

[0133] In some embodiments, a CAR polypeptide includes a signal peptide (e.g., a leader peptide or localization peptide). The signal peptide may be positioned at the N-terminus of the extracellular domain. The signal peptide may be optionally cleaved from the extracellular domain during cellular processing and localization of the CAR to the cellular membrane. Any of various signal peptide sequences known to one of skill in the art may be used. Non-limiting examples of signal peptides from which the sequence may be derived include granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), FcεR, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8α, or any of various other proteins secreted by T cells. In some embodiments, the signal sequence is compatible with the secretory pathway of a T cell. In certain embodiments, the signal sequence is derived from a human immunoglobulin heavy chain.

[0134] In some embodiments, the signal sequence is derived from GMCSFR. In one embodiment, the GMCSFR signal sequence includes 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 set forth in PCT / US2021 / 072646, the disclosure of which is incorporated herein by reference.

[0135] Table 6.

[0136] C. Hinge regions

[0137] In some embodiments, a CAR polypeptide includes a hinge region (e g., a spacer region) between an extracellular domain and a transmembrane domain, such that the extracellular domain, hinge region, and transmembrane domain are in frame with each other.

[0138] A hinge region may contain up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. A hinge region may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally occurring spacer region sequence, or may be an entirely synthetic spacer region sequence. Non-limiting examples of hinge regions include a part of human CD8a chain, partial extracellular domain of CD28, FcγRIIIa receptor, IgG, IgM, IgA, IgD, IgE, an Ig hinge, or functional fragment thereof. In some embodiments, additional linking amino acids are added to the hinge region to ensure that the antigen-binding domain is an optimal distance from the transmembrane domain. In some embodiments, when the hinge region is derived from an immunoglobulin, the region may be mutated to prevent Fc receptor binding.

[0139] In some embodiments, the hinge region includes a hinge domain of a recognized protein. The hinge domain may be derived from CD8a, CD28, or an immunoglobulin (IgG). For example, the IgG hinge may be from IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera thereof.

[0140] In some embodiments, the hinge domain comprises an immunoglobulin IgG hinge or functional fragment thereof. In certain embodiments, the IgG hinge is from IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera thereof. In various embodiments, the hinge domain comprises the CHI, CH2, CH3 and / or hinge region of the immunoglobulin. In many embodiments, the hinge domain comprises the core hinge region of the 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 include EPKSCDKTHTCPPCP (SEQ ID NO: 57 of PCT / US2021 / 072646) from IgGl, ERKCCVECPPCP (SEQ ID NO: 58 of PCT / US2021 / 072646) from IgG2, ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)s (SEQ ID NO: 59 of PCT / US2021 / 072646) from IgG3, and ESKYGPPCPSCP (SEQ ID NO: 60 of PCT / US2021 / 072646) from IgG4 (see also Wypych et al., JBC 2008 283(23): 16194-16205, which is incorporated herein by reference in its entirety for all purposes). In many embodiments, the hinge domain is a fragment of the immunoglobulin hinge.

[0141] 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 PCT / US2021 / 072646, the disclosure of which is incorporated herein by reference.

[0142] 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 PCT / US2021 / 072646, the disclosure of which is incorporated herein by reference.

[0143] In some embodiments, the transmembrane domain and / or hinge domain is derived from CD8 or CD28. In some embodiments, both the transmembrane domain and hinge domain are derived from CD8. In some embodiments, both the transmembrane domain and hinge domain are derived from CD28.

[0144] D. Cytoplasmic domains including co-stimulatory domains

[0145] In some aspects, a CAR polypeptide includes a cytoplasmic or intracellular domain, which contains at least one intracellular signaling domain. In some embodiments, a cytoplasmic domain also comprises one or more co-stimulatory signaling domains.

[0146] The cytoplasmic domain is responsible for activation of at least one of the normal effector functions (e.g., specialized function) of the host cell (e.g., T cell) in which the CAR has been placed in. The term “effector function” refers to a specialized function of a cell. Effector function of a T-cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. A signaling domain can include a portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire signaling domain is present, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. An intracellular signaling domain includes any truncated portion of the signaling domain sufficient to transduce the effector function signal. Non-limiting examples of signaling domains which can be used include, e.g., signaling domains derived from DAP 10, DAP12, Fc epsilon receptor I y chain (FCER1G), FcR , CD3δ, CD3ε, CD3γ, CD3ζ, CD5, CD22, CD226, CD66d, CD79A, CD79B, IL18R1, and IL18RAP.

[0147] In some embodiments, the cytoplasmic domain comprises 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 PCT / US2021 / 072646, the disclosure of which is incorporated herein by reference in its entirety.

[0148] 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 from CD28, 41BB, IL2Rb, CD40, 0X40 (CD 134), CD80, CD86, CD27, ICOS, NKG2D, DAP10, DAP12, 2B4 (CD244), BTLA, CD30, GITR, CD226, CD79A, and HVEM in its entirety.

[0149] In some embodiments, the co-stimulatory signaling domain is derived from 4- 1BB. In one embodiment, the 4- IBB 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 PCT / US2021 / 072646, the disclosure of which is incorporated herein by reference in its entirety.

[0150] In some embodiments, the co-stimulatory signaling domain is derived from IL2Rb . In one embodiment, the IL2Rb 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 PCT / US2021 / 072646, the disclosure of which is incorporated herein by reference in its entirety.

[0151] In some embodiments, the co-stimulatory signaling domain is derived from CD40. In one embodiment, the CD40 co-stimulatory signaling domain includes 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 PCT / US2021 / 072646, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the co-stimulatory signaling domain is derived from 0X40. In one embodiment, the 0X40 co-stimulatory signaling domain includes 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 PCT / US2021 / 072646, the disclosure of which is incorporated herein by reference in its entirety.

[0152] In some embodiments, the co-stimulatory signaling domain is derived from CD80. In one embodiment, the CD80 co-stimulatory signaling domain includes 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 PCT / US2021 / 072646, the disclosure of which is incorporated herein by reference in its entirety.

[0153] In some embodiments, the co-stimulatory signaling domain is derived from CD86. In one embodiment, the CD86 co-stimulatory signaling domain includes 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 PCT / US2021 / 072646, the disclosure of which is incorporated herein by reference in its entirety.

[0154] In some embodiments, the co-stimulatory signaling domain is derived from CD27. In one embodiment, the CD27 co-stimulatory signaling domain includes 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 PCT / US2021 / 072646, the disclosure of which is incorporated herein by reference in its entirety.

[0155] In some embodiments, the co-stimulatory signaling domain is derived from ICOS. In one embodiment, the ICOS co-stimulatory signaling domain includes 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 PCT7US2021 / 072646, the disclosure of which is incorporated herein by reference in its entirety.

[0156] In some embodiments, the co-stimulatory signaling domain is derived from NKG2D. In one embodiment, the NKG2D co-stimulatory signaling domain includes 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 PCT / US2021 / 072646, the disclosure of which is incorporated herein by reference in its entirety.

[0157] In some embodiments, the co-stimulatory signaling domain is derived from DAP 10. In one embodiment, the DAP 10 co-stimulatory signaling domain includes 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 PCT / US2021 / 072646, the disclosure of which is incorporated herein by reference in its entirety.

[0158] In some embodiments, the co-stimulatory signaling domain is derived from DAP 12. In one embodiment, the DAP 12 co-stimulatory signaling domain includes 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 PCT7US2021 / 072646, the disclosure of which is incorporated herein by reference in its entirety.

[0159] In some embodiments, the co-stimulatory signaling domain is derived from 2B4 (CD244). In one embodiment, the 2B4 (CD244) co-stimulatory signaling domain includes 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 PCT / US2021 / 072646, the disclosure of which is incorporated herein by reference in its entirety.

[0160] In some embodiments, the CAR polypeptide includes one costimulatory signaling domains. In many embodiments, the CAR includes 2 or more costimulatory signaling domains. In various embodiments, the CAR includes 2, 3, 4, 5, 6, or more costimulatory signaling domains.

[0161] In some embodiments, the signaling domain(s) and co-stimulatory signaling domain(s) can be placed in any order. In some embodiments, the signaling domain is upstream of the co-stimulatory signaling domains. In some embodiments, the signaling domain is downstream from the co-stimulatory signaling domains. In the cases where two or more co-stimulatory domains are included, the order of the co-stimulatory signaling domains could be switched.

[0162] Non-limiting exemplary CAR polypeptide sequences and polynucleotide sequences, and portions thereof, are provided in Table 7.

[0163] Table 7.

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185] IV. Artificial Cell Death Polypeptide

[0186] According to embodiments of the application, an iPSC or a derivative cell thereof may comprise a second exogenous polynucleotide encoding an artificial cell death polypeptide.

[0187] As used herein, the term "artificial cell death polypeptide” refers to an engineered protein designed to prevent potential toxicity or otherwise adverse effects of a cell therapy. The artificial cell death polypeptide could 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 instance, the artificial cell death polypeptide is activated by an exogenous molecule, e.g., an antibody, that when activated, triggers apoptosis and / or cell death of a therapeutic cell.

[0188] In certain embodiments, an artificial cell death polypeptide comprises an inactivated cell surface receptor that comprises an epitope specifically recognized by an antibody, particularly a monoclonal antibody, which is also referred to herein as a monoclonal antibody-specific epitope. When expressed by iPSCs or derivative cells thereof, the inactivated cell surface receptor is signaling inactive or significantly impaired, but can still be specifically recognized by an antibody. The specific binding of the antibody to the inactivated cell surface receptor enables the elimination of the iPSCs or derivative cells thereof by ADCC and / or ADCP mechanisms, as well as direct killing with antibody drug conjugates with toxins or radionuclides.

[0189] In certain embodiments, the inactivated cell surface receptor comprises an epitope that is selected from epitopes specifically recognized by an antibody, including but not limited to, ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab, ofatumumab, panitumumab, or ustekinumab. In certain embodiments, the inactivated cell surface receptor comprises an epitope that is specifically recognized by cetuximab. In certain embodiments, the inactivated cell surface receptor comprises an epitope that is specifically recognized by trastuzumab. In certain embodiments, the inactivated cell surface receptor comprises an epitope that is specifically recognized by bevacizumab. In certain embodiments, the inactivated cell surface receptor comprises an epitope that is specifically recognized by avelumab. In certain embodiments, the inactivated cell surface receptor comprises an epitope that is specifically recognized by ipilimumab.

[0190] Epidermal growth factor receptor, also known as EGFR, ErbBl and HER1, is a cell-surface receptor for members of the epidermal growth factor family of extracellular ligands. As used herein, “truncated EGFR,” “tEGFR,” “short EGFR” or “sEGFR” refers to an inactive EGFR variant that lacks the EGF-binding domains and the intracellular signaling domains of the EGFR. An exemplary tEGFR variant contains residues 322-333 of domain 2, all of domains 3 and 4 and the transmembrane domain of the native EGFR sequence containing the cetuximab binding epitope. Expression of the tEGFR variant on the cell surface enables cell elimination by an antibody that specifically binds to the tEGFR, such as cetuximab (Erbitux®), as needed. Due to the absence of the EGF-binding domains and intracellular signaling domains, tEGFR is inactive when expressed by iPSCs or derivative cell thereof.

[0191] An exemplary inactivated cell surface receptor of the application comprises a tEGFR variant. In certain embodiments, expression of the inactivated cell surface receptor in an engineered immune cell expressing a chimeric antigen receptor (CAR) induces cell suicide of the engineered immune cell when the cell is contacted with an anti-EGFR antibody. Methods of using inactivated cell surface receptors are described in WO20 19 / 070856, WO2019 / 023396, WO2018 / 058002, the disclosure of which is incorporated herein by reference. For example, a subject who has previously received an engineered immune cell of the present disclosure that comprises a heterologous polynucleotide encoding an inactivated cell surface receptor comprising a tEGFR variant can be administered an anti-EGFR antibody in an amount effective to ablate in the subject the previously administered engineered immune cell.

[0192] In certain embodiments, the anti-EGFR antibody is cetuximab, matuzumab, necitumumab or panitumumab, preferably the anti-EGFR antibody is cetuximab.

[0193] In certain embodiments, the tEGFR variant comprises or consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 71, preferably the amino acid sequence of SEQ ID NO: 71.

[0194] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of CD79b, such as an epitope specifically recognized by polatuzumab vedotin. In certain embodiments, the CD79b epitope comprises or consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 78, preferably the amino acid sequence of SEQ ID NO: 78.

[0195] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of CD20, such as an epitope specifically recognized by rituximab. In certain embodiments, the CD20 epitope comprises or consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 80, preferably the amino acid sequence of SEQ ID NO: 80.

[0196] In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of Her 2 receptor or ErbB, such as an epitope specifically recognized by trastuzumab. In certain embodiments, the monoclonal antibody-specific epitope comprises or consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 82, preferably the amino acid sequence of SEQ ID NO: 82.

[0197] V. Cytokine Expression

[0198] In some embodiments the iPSC or a derivative cell thereof optionally comprises an exogenous polynucleotide encoding a cytokine, such as interleukin- 15 or interleukin- 2.

[0199] As used herein “Interleukin- 15” or “IL- 15” refers to a cytokine that regulates T and NK cell activation and proliferation, or a functional portion thereof. A “functional portion” (“biologically active portion”) of a cytokine refers to a portion of the cytokine that retains one or more functions of full length or mature cytokine. Such functions for IL- 15 include the promotion of NK cell survival, regulation of NK cell and T cell activation and proliferation as well as the support of NK cell development from hematopoietic stem cells. As will be appreciated by those of skill in the art, the sequence of a variety of IL-15 molecules are known in the art. In certain embodiments, the IL-15 is a wild-type IL-15. In certain embodiments, the IL-15 is a human IL-15. In certain embodiments, the IL- 15 comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 72, preferably the amino acid sequence of SEQ ID NO: 72.

[0200] In some embodiments, the IL-15 is a membrane bound form, where all or a functional portion of the IL- 15 protein is fused to all or a portion of a transmembrane protein that anchors the expressed IL- 15 as a cell membrane-bound polypeptide (mbIL15)”, for example the construct described in US Patent US9629877B2, hereby incorporated by reference into the present application.

[0201] As used herein “Interleukin-2” refers to a cytokine that regulates T and NK cell activation and proliferation, or a functional portion thereof. In certain embodiments, the IL-2 is a wild-type IL-2. In certain embodiments, the IL-2 is a human IL-2. In certain embodiments, the IL-2 comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 76, preferably the amino acid sequence of SEQ ID NO: 76.

[0202] In certain embodiments, an inactivated cell surface receptor comprises a monoclonal antibody-specific epitope operably linked to a cytokine, preferably by an autoprotease peptide sequence. Examples of the autoprotease peptide include, but are not limited to, a peptide sequence selected from the group consisting of porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), and a combination thereof. In one embodiment, the autoprotease peptide is an autoprotease peptide of porcine tesehovirus-1 2A (P2A). In certain embodiments, the autoprotease peptide comprises an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 73, preferably the amino acid sequence of SEQ ID NO: 73.

[0203] In certain embodiments, an inactivated cell surface receptor comprises a truncated epithelial growth factor receptor (tEGFR) variant operably linked to an interleukin- 15 (IL-15) or IL-2 by an autoprotease peptide sequence. In a particular embodiment, the inactivated cell surface receptor comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 74, preferably the amino acid sequence of SEQ ID NO: 74.

[0204] In some embodiments, an inactivated cell surface receptor further comprises a signal sequence. In certain embodiments, the signal sequence comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 77, preferably the amino acid sequence of SEQ ID NO: 77.

[0205] In some embodiments, an inactivated cell surface receptor further comprises a hinge domain. In some embodiments, the hinge domain is derived from CD8. 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 with SEQ ID NO: 21.

[0206] In certain embodiments, an inactivated cell surface receptor further comprises a transmembrane domain. In some embodiments, the transmembrane domain is derived from CD8. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 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 95, at least 96, at least 97, at least 98 or at least 99%, sequence identity with SEQ ID NO: 23.

[0207] In certain embodiment, an inactivated cell surface receptor comprises one or more epitopes specifically recognized by an antibody in its extracellular domain, a transmembrane region and a cytoplasmic domain. In some embodiments, the inactivated cell surface receptor further comprises a hinge region between the epitope(s) and the transmembrane region. In some embodiments, the inactivated cell surface receptor comprises more than one epitope specifically recognized by an antibody, the epitopes can have the same or different amino acid sequences, and the epitopes can be linked together via a peptide linker, such as a flexible peptide linker have the sequence of (GGGGS)n, wherein n is an integer of 1-8 (SEQ ID NO: 25). In some embodiments, the inactivated cell surface receptor further comprises a cytokine, such as an IL-15 or IL-2. In certain embodiments, the cytokine is in the cytoplasmic domain of the inactivated cell surface receptor. Preferably, the cytokine is operably linked to the epitope(s) specifically recognized by an antibody, directly or indirectly, via an autoprotease peptide sequence, such as those described herein. In some embodiments, the cytokine is indirectly linked to the epitope(s) by connecting to the transmembrane region via the autoprotease peptide sequence.

[0208] Non-limiting exemplary inactivated cell surface receptor regions and sequences are provided in Table 8.

[0209] Table 8.

[0210]

[0211]

[0212] In a particular embodiment, the inactivated cell surface receptor comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 79, preferably the amino acid sequence of SEQ ID NO: 79.

[0213] In a particular embodiment, the inactivated cell surface receptor comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 81, preferably the amino acid sequence of SEQ ID NO: 81.

[0214] In a particular embodiment, the inactivated cell surface receptor comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 83, preferably the amino acid sequence of SEQ ID NO: 83.

[0215] VI. HLA Expression

[0216] In one aspect, MHC I and / or MHC II knock-out and / or knock down can be incorporated in the cells for use in “allogeneic” cell therapies, in which cells are harvested from a subject, modified to knock-out or knock-down, e.g., disrupt, B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP gene expression, and then returned to a different subject. Knocking out or knocking down the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes as described herein can: (1) prevent Graft versus Host response; (2) prevent Host versus Graft response; and / or (3) improve cell safety and efficacy. Accordingly, certain embodiments of the present disclosure comprise independently knocking out and / or knocking down one or more genes selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes in an iPSC. In certain embodiments, a presently disclosed method comprises independently knocking out and / or knocking down two genes selected from the group consisting B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes in an iPSC, in particular, B2M and CIITA to achieve class I and II HLA disruption. In certain embodiments, an iPSC or derivative cell thereof of the application can be further modified by introducing an exogenous polynucleotide encoding one or more proteins related to immune evasion, such as non-classical HLA class I proteins (e g., HLA-E and HLA-G). In particular, disruption of the B2M gene eliminates surface expression of all MHC class I molecules, leaving cells vulnerable to lysis by NK cells through the “missing self’ response. Exogenous HLA-E expression can lead to resistance to NK-mediated lysis (Gornalusse et al., Nat Biotechnol. 2017; 35(8): 765-772).

[0217] Incorporating MHC I and / or MHC II knock-out and / or knock down in the cells for use in “allogeneic” cell therapies will allow the cell product candidates to escape recognition and destruction by the host immune system. The reduction in allogeneic reactivity enabled by use of this technology will allow repeat dosing of the CAR- modified cell therapies to improve their therapeutic potential. In combination with the extended killing capability of optimized immune cells derived from single genetically engineered cell cloning, the cells will have the capacity for repeat dosing to maximize durability of response and efficacy. Additionally, this technology may permit dosing in patients with limited or no immune preconditioning regimens.

[0218] Accordingly, in certain embodiments, an iPSC or derivative cell thereof of the application can be further modified by introducing a third exogenous polynucleotide encoding one or more proteins related to immune evasion, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G).

[0219] In certain embodiments, the iPSC or derivative cell thereof comprises a third exogenous polypeptide encoding at least one of a human leukocyte antigen E (HLA-E) and human leukocyte antigen G (HLA-G). In a particular embodiment, the HLA-E comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 65, preferably the amino acid sequence of SEQ ID NO: 65. In a particular embodiment, the HLA-G comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 68, preferably SEQ ID NO: 68.

[0220] In certain embodiments, the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein that is fused to an HLA-E via a linker. In a particular embodiment, the third exogenous polypeptide comprises an amino acid sequence at least sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 66.

[0221] In other embodiments, the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein that is fused to an HLA-G via a linker. In a particular embodiment, the third exogenous polypeptide comprises an amino acid sequence at least sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 69.

[0222] VII. Other Optional Genome Edits

[0223] In one embodiment of the above described cell, the genomic editing at one or more selected sites may comprise insertions of one or more exogenous polynucleotides encoding other additional artificial cell death polypeptides, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins promoting engraftment, trafficking, migration, viability, self-renewal, persistence, and / or survival of the genome-engineered iPSCs or derivative cells thereof.

[0224] In some embodiments, the exogenous polynucleotides for insertion are operatively linked to (1) one or more exogenous promoters comprising CMV, EFla, PGK, CAG, UBC, or other constitutive, inducible, temporal-, tissue-, or cell type-specific promoters; or (2) one or more endogenous promoters comprised in the selected sites comprising AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hll, beta-2 microglobulin, GAPDH, TCR or RUNX1, or other locus meeting the criteria of a genome safe harbor. In some embodiments, the genome-engineered iPSCs generated using the above method comprise one or more different exogenous polynucleotides encoding proteins comprising caspase, thymidine kinase, cytosine deaminase, B-cell CD20, ErbB2 or CD79b wherein when the genome-engineered iPSCs comprise two or more suicide genes, the suicide genes are integrated in different safe harbor locus comprising AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, beta-2 microglobulin, GAPDH, TCR or RUNX1. Other exogenous polynucleotides encoding proteins may include those encoding PET reporters, homeostatic cytokines, and inhibitory checkpoint inhibitory proteins such as PD1, PD-L1, CTLA4, and proteins that target the CD47 / signal regulatory protein alpha (SIRPa) axis.

[0225] In one aspect, the cell may comprise an exogenous polynucleotide encoding a CD 16 protein and / or an NKG2D protein, wherein the CD 16 protein and the NKG2D protein may be operably linked by an autoprotease peptide as disclosed in co-pending patent application PCT7US23 / 68079. Accordingly, in some aspects, the cells of the present disclosure can comprise genetically engineered iPSCs and cells derived therefrom that exogenously express recombinant CD 16 and recombinant NKG2D. The surface receptor CD16 ( FcγRIIIA) affects human natural killer (NK) cells during maturation. NK cells bind the Fc portion of IgG via CD16, and execute antibody-dependent cellular cytotoxicity, which is critical for the effectiveness of several anti-tumor monoclonal antibody therapies. NKG2D is a stimulatory / activating receptor that is mostly expressed on cells of the cytotoxic arm of the immune system including NK cells and subsets of T cells. NKG2D is crucial in diverse aspects of innate and adaptive immune functions. In some embodiments, CD 16 and NKG2D are expressed from in a single polynucleotide construct as it is advantageous to reduce the number of gene edits of a cell.

[0226] . In some embodiments, the polynucleotide construct encoding the CD 16 protein and the NKG2D protein also includes a polynucleotide sequence encoding an autoprotease peptide or self-cleaving peptide. In some embodiments, an exogenous polynucleotide construct encoding the CD 16 protein, the NKG2D protein and the self- cleaving peptide is introduced into the iPSC or derivative cell thereof. The exogenous or isolated polynucleotide construct can be introduced into a gene locus of the iPSC or derivative cell thereof.

[0227] In some embodiments, the exogenous polynucleotide construct comprises the nucleic acid sequence of SEQ ID NO: 84. In some embodiments, the exogenous polynucleotide construct encodes for the amino acid sequence of SEQ ID NO: 85. In some embodiments, the CD 16 protein (which is also referred to as “low affinity immunoglobulin gamma Fc region receptor III-A” or “Fc gamma receptor Illa”) is a wildtype CD 16 protein. In some embodiments, the human wildtype CD 16 protein has the amino acid sequence set forth in NCBI Ref. Seq. No. NP_000560.7 or UniProt No. P08637. In some instance, the coding sequence of human wildtype CD16 is set forth in NCBI Ref. No. NM_000569.8.

[0228] In some embodiments, the CD 16 protein is a CD 16 variant protein. In some instances, the CD 16 variant protein has an amino acid sequence having at least 90%, e.g., 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 wildtype CD 16 such as that of SEQ ID NO: 86. In some instances, the CD16 variant is a high affinity CD16 variant. In other instances, the CD 16 variant is a non-cleavable CD 16 variant. In some instances, the CD 16 variant is a high affinity and non-cleavable CD 16 variant.

[0229] In some embodiments, the CD 16 variant comprises one or more amino acid substitutions selected from the group consisting of Fl 58 V, F176 V, 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 CD 16 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 CD 16 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 CD 16 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 Fl 58V, Fl 76V, S197P, D205A, S219A, T220A, and any combination thereof. In some embodiments, the CD 16 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 CD 16 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 variant CD 16 protein has the sequence of SEQ ID NO: 87. In some embodiments, the nucleic acid sequence encoding the variant CD 16 protein has the sequence of SEQ ID NO: 88. In some embodiments, the wildtype CD16 protein has the sequence of SEQ ID NO: 86. In some embodiments, the NKG2D protein (which is also referred to as NKG2-D type II integral membrane protein, CD314, killer cell lectin-like receptor subfamily KI member 1 or KLRK1) is a wildtype NKG2D protein. In some embodiments, the human wildtype NKG2D protein has the amino acid sequence set forth in NCBI Ref. Seq. Nos. NP_001186734.1 or NP_031386.2 or UniProt No. P26718. In some instance, the coding sequence of human wildtype NKG2D is set forth in NCBI Ref. Nos. NM_001199805.1 or NM_007360.3. In some embodiments, the NKG2D protein is a NKG2D variant protein. In some instances, the NKG2D variant protein has an amino acid sequence having at least 90%, e.g., 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 wildtype NKG2D such as that of SEQ ID NO: 89. In some embodiments, the NKG2D protein has the amino acid sequence of SEQ ID NO: 89. In some embodiments, the nucleic acid sequence encoding the NKG2D protein has sequence of SEQ ID NO: 90.

[0230] As discussed above, provided herein are constructs containing autoprotease peptide sequences including 2A peptides that can induce ribosomal skipping during translation of an polypeptide. 2A peptides function to “cleave” an mRNA transcript by making the ribosome skip the synthesis of a peptide bond at the C-terminus, between the glycine (G) and proline (P) residues, thereby leading to separation between the end of the 2A sequence and the next peptide downstream. 2A peptides include, but are not limited to, a porcine tesehovirus-1 2 A (P2A) peptide, a foot-and-mouth disease virus (FMDV) 2A (F2A) peptide, an Equine Rhinitis A Virus (ERAV) 2A (E2A) peptide, a Thosea asigna virus 2A (T2A) peptide, a cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and a Flacherie Virus 2A (BmIFV2A) peptide.

[0231] An exemplary P2A peptide can include an amino acid sequence having at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 91. In some embodiment, the P2A peptide has the amino acid sequence of SEQ ID NO: 91.

[0232] Another optional genome edit is the insertion of a polynucleotide encoding a a membrane-bound interleukin 12 (IL-12) comprising a first polypeptide comprising an IL- 12 alpha subunit p35, a second polypeptide comprising an IL-12 beta subunit p40 and a transmembrane fused to the terminus of the first and / or second IL-12 subunit polypeptide as disclosed in co-pending patent application PCT / US23 / 68105. In certain embodiments, the polynucleotide encoding the membrane bound IL-12 is fused to a polynucleotide encoding an ADAM 17 protease cleavage site peptide for the activation induced release of the IL-12 through the protease ADAM17. ADAM17 is expressed by activated lymphocytes and is directly involved in the liberation of other immune mediators like TNFa that are similarly presented as a membrane anchored form. When this membrane tethered IL- 12 is expressed on engineered iNK or T cells, it remains cell associated. Upon cell activation and the increased expression of ADAM 17, the protease cleaves the membrane stalk and releases IL- 12 into the extracellular space. This type of regulation ensures that the activities of the IL- 12 are confined to spaces surrounding the tumor where the engineered immune cells engage their targets on the tumor cells that cause their activation. Accordingly, the cell of the present disclosure can further comprise (i) an exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL- 12) comprising a first polypeptide comprising an IL-12 alpha subunit p35 or a polypeptide at least 90% similar thereto, a second polypeptide comprising an IL-12 beta subunit p40 or a polypeptide at least 90% similar thereto, and a transmembrane domain fused to the terminus of the first and / or second IL- 12 subunit polypeptide.

[0233] In some other embodiments, the genome-engineered iPSCs generated using the method provided herein comprise in / del at one or more endogenous genes associated with targeting modality, receptors, signaling molecules, transcription factors, drug target candidates, immune response regulation and modulation, or proteins suppressing engraftment, trafficking, migration, viability, self-renewal, persistence, and / or survival of the iPSCs or derivative cells thereof.

[0234] VIII. Targeted Genome Editing at Selected Locus in iPSCs

[0235] According to embodiments of the application, one or more of the exogenous polynucleotides are integrated at one or more loci on the chromosome of an iPSC.

[0236] Genome editing, or genomic editing, or genetic editing, as used interchangeably herein, is a type of genetic engineering in which DNA is inserted, deleted, and / or replaced in the genome of a targeted cell. Targeted genome editing (interchangeable with “targeted genomic editing” or “targeted genetic editing”) enables insertion, deletion, and / or substitution at pre-selected sites in the genome. When an endogenous sequence is deleted or disrupted at the insertion site during targeted editing, an endogenous gene comprising the affected sequence can be knocked-out or knocked-down due to the sequence deletion or disruption. Therefore, targeted editing can also be used to disrupt endogenous gene expression with precision. Similarly used herein is the term “targeted integration,” referring to a process involving insertion of one or more exogenous sequences at pre-selected sites in the genome, with or without deletion of an endogenous sequence at the insertion site.

[0237] Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease-dependent approach. In the nuclease-independent targeted editing approach, homologous recombination is guided by homologous sequences flanking an exogenous polynucleotide to be inserted, through the enzymatic machinery of the host cell.

[0238] Alternatively, targeted editing could be achieved with higher frequency through specific introduction of double strand breaks (DSBs) by specific rare-cutting endonucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms including non-homologous end joining (NHEJ), which occurs in response to DSBs. Without a donor vector containing exogenous genetic material, the NHEJ often leads to random insertions or deletions (in / dels) of a small number of endogenous nucleotides. In comparison, when a donor vector containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome during homology directed repair (HDR) by homologous recombination, resulting in a “targeted integration.”

[0239] Available endonucleases capable of introducing specific and targeted DSBs include, but not limited to, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), RNA-guided CRISPR (Clustered Regular Interspaced Short Palindromic Repeats) systems. Additionally, DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxbl integrases is also a promising tool for targeted integration.

[0240] ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain. By a “zinc finger DNA binding domain” or “ZFBD” it is meant a polypeptide domain that binds DNA in a sequence-specific manner through one or more zinc fingers. A zinc finger is a domain of about 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of a zinc ion.

[0241] Examples of zinc fingers include, but not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A “designed” zinc finger domain is a domain not occurring in nature whose design / composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496. A “selected” zinc finger domain is a domain not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. ZFNs are described in greater detail in U.S. Pat. No. 7,888,121 and U.S. Pat. No. 7,972,854, the complete disclosures of which are incorporated herein by reference. The most recognized example of a ZFN in the art is a fusion of the Fokl nuclease with a zinc finger DNA binding domain.

[0242] A TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain. By “transcription activator-like effector DNA binding domain”, “TAL effector DNA binding domain”, or “TALE DNA binding domain” it is meant the polypeptide domain of TAL effector proteins that is responsible for 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 the plant cell, bind effector-specific DNA sequences via their DNA binding domain, and activate gene transcription at these sequences via their transactivation domains. TAL effector DNA binding domain specificity depends on an effector-variable number of imperfect 34 amino acid repeats, which comprise polymorphisms at select repeat positions called repeat variable-diresidues (RVD). TALENs are described in greater detail in U.S. Patent Application No. 2011 / 0145940, which is herein incorporated by reference. The most recognized example of a TALEN in the art is a fusion polypeptide of the Fokl nuclease to a TAL effector DNA binding domain.

[0243] Another example of a targeted nuclease that finds use in the subject methods is a targeted Spoil nuclease, a polypeptide comprising a Spol 1 polypeptide having nuclease activity fused to a DNA binding domain, e.g. a zinc finger DNA binding domain, a TAL effector DNA binding domain, etc. that has specificity for a DNA sequence of interest. See, for example, U.S. Application No. 61 / 555,857, the disclosure of which is incorporated herein by reference.

[0244] Additional examples of targeted nucleases suitable for the present application include, but not limited to Bxbl, phiC3 1, R4, PhiBTl, and Wp / SPBc / TP901-l, whether used individually or in combination.

[0245] Other non-limiting examples of targeted nucleases include naturally occurring and recombinant nucleases; CRISPR related nucleases from families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr; restriction endonucleases; meganucleases; homing endonucleases, and the like. As an example, CRISPR / Cas9 requires two major components: (1) a 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 comprising PAM and a seeding region near PAM. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cas9 to target selected sequences. These two components can then be delivered to mammalian cells via transfection or transduction. As another example, CRISPR / Cpfl comprises two major components: (1) a CPfl endonuclease and (2) a crRNA. When co-expressed, the two components form a ribobnucleoprotein (RNP) complex that is recruited to a target DNA sequence comprising PAM and a seeding region near PAM. The crRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cpfl to target selected sequences. These two components can then be delivered to mammalian cells via transfection or transduction.

[0246] MAD7 is an engineered Casl2a variant originating from the bacterium Eubacterium rectale that has a preference for 5'-TTTN-3' and 5'-CTTN-3' PAM sites and does not require a tracrRNA. See, for example, PCT Publication No. 2018 / 236548, the disclosure of which is incorporated herein by reference.

[0247] DICE mediated insertion uses a pair of recombinases, for example, phiC31 and Bxbl, to provide unidirectional integration of an exogenous DNA that is tightly restricted to each enzymes’ own small attB and attP recognition sites. Because these target att sites are not naturally present in mammalian genomes, they must be first introduced into the genome, at the desired integration site. See, for example, U.S. Application Publication No. 2015 / 0140665, the disclosure of which is incorporated herein by reference.

[0248] One aspect of the present application provides a construct comprising one or more exogenous polynucleotides for targeted genome integration. In one embodiment, 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 to cells to enable site specific homologous recombination by the cell host enzymatic machinery. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing a ZFN expression cassette comprising a DNA-binding domain specific to a desired integration site to the cell to enable a ZFN-mediated insertion. In yet another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing a TALEN expression cassette comprising a DNA-binding domain specific to a desired integration site to the cell to enable a TALEN-mediated insertion. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, introducing a Cpfl expression cassette, and a gRNA comprising a guide sequence specific to a desired integration site to the cell to enable a Cpfl -mediated insertion. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, introducing a Cas9 expression cassette, and a gRNA comprising a guide sequence specific to a desired integration site to the cell to enable a Cas9-mediated insertion. In still another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more att sites of a pair of DICE recombinases to a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing an expression cassette for DICE recombinases, to enable DICE-mediated targeted integration.

[0249] Sites for targeted integration include, but are not limited to, genomic safe harbors, which are intragenic or extragenic regions of the human genome that, theoretically, are able to accommodate predictable expression of newly integrated DNA without adverse effects on the host cell or organism. In certain embodiments, the genome safe harbor for the targeted integration is one or more loci of genes selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, TCR and RUNX1 genes.

[0250] In other embodiments, the site for targeted integration is selected for deletion or reduced expression of an endogenous gene at the insertion site. As used herein, the term “deletion” with respect to expression of a gene refers to any genetic modification that abolishes the expression of the gene. Examples of “deletion” of expression of a gene include, e.g., a removal or deletion of a DNA sequence of the gene, an insertion of an exogenous polynucleotide sequence at a locus of the gene, and one or more substitutions within the gene, which abolishes the expression of the gene.

[0251] Genes for target deletion include, but are not limited to, genes of major histocompatibility complex (MHC) class I and MHC class II proteins. Multiple MHC class I and class II proteins must be matched for histocompatibility in allogeneic recipients to avoid allogeneic rejection problems. “MHC deficient”, including MHC-class I deficient, or MHC-class II deficient, or both, refers to cells that either lack, or no longer maintain, or have reduced level of surface expression of a complete MHC complex comprising a MHC class I protein heterodimer and / or a MHC class II heterodimer, such that the diminished or reduced level is less than the level naturally detectable by other cells or by synthetic methods. MHC class I deficiency can be achieved by functional deletion of any region of the MHC class I locus (chromosome 6p21), or deletion or reducing the expression level of one or more MHC class-I associated genes including, not being limited to, beta-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene and Tapasin genes. 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 functional deletion or reduction of MHC-II associated genes including, not being limited to, RFXANK, CIITA, RFX5 and RFXAP. CIITA is a transcriptional coactivator, functioning through activation of the transcription factor RFX5 required for class II protein expression. CIITA null cells are MHC-II deficient. In certain embodiments, one or more of the exogenous polynucleotides are integrated at one or more loci of genes selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes to thereby delete or reduce the expression of the gene(s) with the integration. Other genes that may be targeted for deletion include NKG2A, CD38, CD70 and CD33.

[0252] In certain embodiments, the exogenous polynucleotides are integrated at one or more loci on the chromosome of the cell, preferably the one or more loci are of genes selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hl 1, GAPDH, RUNX1, B2M, TAPI, TAP2, Tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD33, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, or TIGIT genes, provided at least one of the one or more loci is of a MHC gene, such as a gene selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes. Preferably, the one or more exogenous polynucleotides are integrated at a locus of an MHC class-I associated gene, such as a beta-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene or Tapasin gene; and at a locus of an MHC-II associated gene, such as a RFXANK, CIITA, RFX5, RFXAP, or CIITA gene; and optionally further at a locus of a safe harbor gene selected from the group consisting of AAVS1, CLYBL, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, TCR and RUNX1 genes. More preferably, the one or more of the exogenous polynucleotides are integrated at the loci of CIITA, AAVS1 and B2M genes.

[0253] In certain embodiments, (i) the first exogenous polynucleotide is integrated at a locus of AAVS1 gene or CLYBL gene; (ii) the second exogenous polypeptide is integrated at a locus of CIITA gene; and (iii) the third exogenous polypeptide is integrated at a locus of B2M gene; wherein integrations of the exogenous polynucleotides delete or reduce expression of CIITA and B2M genes.

[0254] In certain embodiments, (i) the second exogenous polynucleotide comprises the polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 75; and (ii) the third exogenous polynucleotide comprises the polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 67. In certain embodiments, (i) the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75; and (ii) the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67.

[0255] IX. Derivative Cells

[0256] In another aspect, the present disclosure relates to a cell derived from differentiation of an iPSC, a derivative cell. As described above, the genomic edits introduced into the iPSC are retained in the derivative cell. In certain embodiments of the derivative cell obtained from iPSC differentiation, the derivative cell is a hematopoietic cell, including, but not limited to, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, B cells, antigen presenting cells (APC), monocytes and macrophages. In certain embodiments, the derivative cell is an immune effector cell, such as a NK cell or a T cell.

[0257] In certain embodiments, the application provides a natural killer (NK) cell or a T cell comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding a truncated epithelial growth factor receptor (tEGFR) variant and an interleukin 15 (IL- 15), wherein the tEGFR variant and IL- 15 are operably linked by an autoprotease peptide sequence, such as autoprotease peptide sequence of porcine tesehovirus-1 2A (P2A); and (iii) a deletion or reduced expression of an MHC class I associated gene and an MHC class II associated gene, such as an MHC class-I associated gene selected from the group consisting of a B2M gene, TAP 1 gene, TAP 2 gene and Tapasin gene, and an MHC-II associated gene selected from the group consisting of a RFXANK gene, CIITA gene, RFX5 gene, RFXAP gene, and CIITA gene, preferably the B2M gene and CIITA gene.

[0258] In certain embodiments, the NK cell or T cell further comprises a third exogenous polynucleotide encoding at least one of a human leukocyte antigen E (HLA-E) and a human leukocyte antigen G (HLA-G).

[0259] Also provided is a NK cell or a T cell comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding a truncated epithelial growth factor receptor (tEGFR) variant having the amino acid sequence of SEQ ID NO: 71 , an autoprotease peptide having the amino acid sequence of SEQ ID NO: 73, and interleukin 15 (IL-15) having the amino acid sequence of SEQ ID NO: 72; and (iii) a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66; wherein the first, second and third exogenous polynucleotides are integrated at loci of AAVS1, CIITA and B2M genes, respectively, to thereby delete or reduce expression of CIITA and B2M.

[0260] In certain embodiments, the first exogenous polynucleotide encodes a CAR targeting a tumor antigen; the second exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 75; and the third exogenous polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 67.

[0261] Also provided is a CD34+ hematopoietic progenitor cell (HPC) derived from an induced pluripotent stem cell (iPSC) comprising: (i) a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a second exogenous polynucleotide encoding an inactivated cell surface receptor that comprises a monoclonal antibody- specific epitope and an interleukin 15 (IL-15), wherein the inactivated cell surface receptor and IL- 15 are operably linked by an autoprotease peptide sequence; and (iii) a deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes.

[0262] In certain embodiments, the CD34+ HPC further comprises a third exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G).

[0263] In certain embodiments, the CAR comprises (i) a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to a tumor antigen; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a co-stimulatory domain, such as a co-stimulatory domain comprising a CD28 signaling domain.

[0264] Also provided is a method of manufacturing the derivative cell. The method comprises differentiating the iPSC under conditions for cell differentiation to thereby obtain the derivative cell. An iPSC of the application can be differentiated by any method known in the art. Exemplary methods are described in US8846395, US8945922, US8318491, W02010 / 099539, W02012 / 109208, W02017 / 070333, WO2017 / 179720, W02016 / 010148, WO2018 / 048828, WO2019 / 157597, WO2022 / 120334, WO2022 / 133169, WO2022 / 216624, WO2022 / 216514, and WO2022 / 216524, each of which are herein incorporated by reference in its entirety. The differentiation protocol may use feeder cells or may be feeder-free. As used herein, “feeder cells” or “feeders” are terms describing cells of one type that are co-cultured with cells of a second type to provide an environment in which the cells of the second type can grow, expand, or differentiate, as the feeder cells provide stimulation, growth factors and nutrients for the support of the second cell type.

[0265] In another embodiment of the present disclosure, the iPSC derivative cells are NK cells which are prepared by a method of differentiating an iPSC into an NK cell by subjecting the cells to a differentiation protocol including the addition of recombinant human IL-12p70 for the final 24 hours of culture. By including the IL- 12 in the differentiation protocol, cells that are primed with IL-12 demonstrate more rapid cell killing compared to those that are differentiated in the absence of IL-12. In addition, the cells differentiated using the IL-12 conditions demonstrate improved cancer cell growth inhibition.

[0266] X. Polynucleotides, vectors, and host cells

[0267] (1) Nucleic acids encoding a CAR

[0268] In another general aspect, the present disclosure relates to an isolated nucleic acid encoding a chimeric antigen receptor (CAR) useful for embodiments of the application. It will be appreciated by those skilled in the art that the coding sequence of a CAR can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding CARs of the application can be altered without changing the amino acid sequences of the proteins.

[0269] In another general aspect, the application provides a vector comprising a polynucleotide sequence encoding a CAR useful for embodiments of the application. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. A number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of a CAR in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the application.

[0270] In a particular aspect, the application provides vectors for targeted integration of a CAR useful for embodiments of the application. In certain embodiments, the vector comprises an exogenous polynucleotide having, in the 5’ to 3’ order, (a) a promoter; (b) a polynucleotide sequence encoding a CAR according to an embodiment of the application; and (c) a terminator / polyadenylation signal.

[0271] In certain embodiments, the promoter is a CAG promoter. In certain embodiments, the CAG promoter comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 63. Other promoters can also be used, examples of which include, but are not limited to, EFla, UBC, CMV, SV40, PGK1, and human beta actin.

[0272] In certain embodiments, the terminator / polyadenylation signal is a SV40 signal. In certain embodiments, the SV40 signal comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 64. Other terminator sequences can also be used, examples of which include, but are not limited to, BGH, hGH, and PGK.

[0273] In some embodiments, the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide. As used herein, “left homology arm” and “right homology arm” refers to a pair of nucleic acid sequences that flank an exogenous polynucleotide and facilitate the integration of the exogenous polynucleotide into a specified chromosomal locus. Sequences of the left and right arm homology arms can be designed based on the integration site of interest. In some embodiments, the left or right arm homology arm is homologous to the left or right side sequence of the integration site.

[0274] In certain embodiments, the left homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to one of SEQ ID NOs: 92-103, 130, and 132. In certain embodiments, the right homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to one of SEQ ID NOs: 104-115, 131, and 133.

[0275] Table 9 provides an exemplary list of homology arm sequences and corresponding guide sequences for facilitating integration of an exogenous polynucleotide at various loci.

[0276] Table 9.

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299] (2) Nucleic acids encoding an inactivated cell surface receptor

[0300] In another general aspect, the present disclosure relates to an isolated nucleic acid encoding an inactivated cell surface receptor useful for embodiments of the application. It will be appreciated by those skilled in the art that the coding sequence of an inactivated cell surface receptor can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding an inactivated cell surface receptor of the application can be altered without changing the amino acid sequences of the proteins.

[0301] In certain embodiments, an isolated nucleic acid encodes any inactivated cell surface receptor described herein, such as that comprises a monoclonal antibody-specific epitope, and / or a cytokine, such as an IL-15 or IL-2, wherein the monoclonal antibody- specific epitope and the cytokine are optionally operably linked by an autoprotease peptide sequence.

[0302] In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by an antibody, such as ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab. ofatumumab, panitumumab, or ustekinumab. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by cetuximab. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by trastuzumab. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by bevacizumab. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by avelumab. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by ipilimumab. In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having a truncated epithelial growth factor receptor (tEGFR) variant. Preferably, the inactivated cell surface receptor comprises an epitope specifically recognized by cetuximab, matuzumab, necitumumab or panitumumab, preferably cetuximab.

[0303] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD79b, such as an epitope specifically recognized by polatuzumab vedotin.

[0304] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD20, such as an epitope specifically recognized by rituximab.

[0305] In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of Her 2 receptor, such as an epitope specifically recognized by trastuzumab

[0306] In certain embodiments, the autoprotease peptide sequence is porcine tesehovirus- 1 2A (P2A).

[0307] In certain embodiments, the truncated epithelial growth factor receptor (tEGFR) variant consists of an amino acid sequence having 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: 71.

[0308] In certain embodiments, the monoclonal antibody-specific epitope specifically recognized by polatuzumab vedotin consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 78.

[0309] In certain embodiments, the monoclonal antibody-specific epitope specifically recognized by rituximab consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 80.

[0310] In certain embodiments, the monoclonal antibody-specific epitope specifically recognized by trastuzumab consists of an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 82.

[0311] In certain embodiments, the IL- 15 comprises an amino acid sequence having 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: 72.

[0312] In certain embodiments, the autoprotease peptide has an amino acid sequence having 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: 73.

[0313] In certain embodiments, the polynucleotide sequence encodes a polypeptide comprising an amino acid sequence having 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: 74.

[0314] In a particular embodiment, the isolated nucleic acid encoding the inactivated cell surface receptor comprises a polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 75, preferably the polynucleotide sequence of SEQ ID NO: 75.

[0315] In certain embodiments, the polynucleotide sequence encodes a polypeptide comprising an amino acid sequence having 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: 79.

[0316] In another general aspect, the application provides a vector comprising a polynucleotide sequence encoding an inactivated cell surface receptor useful for embodiments of the application. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. A number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of a inactivated cell surface receptor in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the application.

[0317] In a particular aspect, the application provides a vector for targeted integration of an inactivated cell surface receptor useful for embodiments of the application. In certain embodiments, the vector comprises an exogenous polynucleotide having, in the 5’ to 3’ order, (a) a promoter; (b) a polynucleotide sequence encoding an inactivated cell surface receptor, such as an inactivated cell surface receptor comprising a truncated epithelial growth factor receptor (tEGFR) variant and an interleukin 15 (IL- 15), wherein the tEGFR variant and IL- 15 are operably linked by an autoprotease peptide sequence, such as porcine tesehovirus-1 2A (P2A), and (c) a terminator / polyadenylation signal.

[0318] In certain embodiments, the promoter is a CAG promoter. In certain embodiments, the CAG promoter comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 63. Other promoters can also be used, examples of which include, but are not limited to, EFla, UBC, CMV, SV40, PGK1, and human beta actin.

[0319] In certain embodiments, the terminator / polyadenylation signal is a SV40 signal. In certain embodiments, the SV40 signal comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 64. Other terminator sequences can also be used, examples of which include, but are not limited to BGH, hGH, and PGK.

[0320] In certain embodiments, the polynucleotide sequence encoding an inactivated cell surface receptor comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 75.

[0321] In some embodiments, the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide.

[0322] In certain embodiments, the left homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 130. In certain embodiments, the right homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 131 (3) Nucleic acids encoding an HLA construct

[0323] In another general aspect, the present disclosure relates to an isolated nucleic acid encoding an HLA construct useful for embodiments of the application. It will be appreciated by those skilled in the art that the coding sequence of an HLA construct can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding an HLA construct of the application can be altered without changing the amino acid sequences of the proteins.

[0324] In certain embodiments, the isolated nucleic acid encodes an HLA construct comprising a signal peptide, such as an HLA-G signal peptide, operably linked to an HLA coding sequence, such as a coding sequence of a mature B2M, and / or a mature HLA-E. In some embodiments, the HLA coding sequence encodes the HLA-G and B2M, which are operably linked by a 4X GGGGS linker, and / or the B2M and HLA-E, which are operably linked by a 3X GGGGS linker. In a particular embodiment, the isolated nucleic acid encoding the HLA construct comprises a polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 67, preferably the polynucleotide sequence of SEQ ID NO: 67. In another embodiment, the isolated nucleic acid encoding the HLA construct comprises a polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 70, preferably the polynucleotide sequence of SEQ ID NO: 70.

[0325] In another general aspect, the application provides a vector comprising a polynucleotide sequence encoding a HLA construct useful for embodiments of the application. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. A number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of a HLA construct in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the application.

[0326] In a particular aspect, the application provides vectors for targeted integration of a HLA construct useful for embodiments of the application. In certain embodiments, the vector comprises an exogenous polynucleotide having, in the 5’ to 3’ order, (a) a promoter; (b) a polynucleotide sequence encoding an HLA construct; and (c) a terminator / polyadenylation signal.

[0327] In certain embodiments, the promoter is a CAG promoter. In certain embodiments, the CAG promoter comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 63. Other promoters can also be used, examples of which include, but are not limited to, EFla, UBC, CMV, SV40, PGK1, and human beta actin.

[0328] In certain embodiments, the terminator / polyadenylation signal is a SV40 signal. In certain embodiments, the SV40 signal comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 64. Other terminator sequences can also be used, examples of which include, but are not limited to BGH, hGH, and PGK.

[0329] In certain embodiments, a polynucleotide sequence encoding a HLA construct comprises a signal peptide, such as a HLA-G signal peptide, a mature B2M, and a mature HLA-E, wherein the HLA-G and B2M are operably linked by a 4X GGGGS linker (SEQ ID NO: 31) and the B2M transgene and HLA-E are operably linked by a 3X GGGGS linker (SEQ ID NO: 25). In particular embodiments, the HLA construct comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 67, preferably the polynucleotide sequence of SEQ ID NO: 67. In another embodiment, the HLA construct comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 70, preferably the polynucleotide sequence of SEQ ID NO: 70.

[0330] In some embodiments, the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide. In certain embodiments, the left homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 132. In certain embodiments, the right homology arm comprises the polynucleotide sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%, identical to SEQ ID NO: 133.

[0331] (4) Host cells

[0332] In another general aspect, the application provides a host cell comprising a vector of the application and / or an isolated nucleic acid encoding a construct of the application. Any host cell known to those skilled in the art in view of the present disclosure can be used for recombinant expression of exogenous polynucleotides of the application. According to particular embodiments, the recombinant expression vector is transformed into host cells by conventional methods such as chemical transfection, heat shock, or electroporation, where it is stably integrated into the host cell genome such that the recombinant nucleic acid is effectively expressed.

[0333] Examples of host cells include, for example, recombinant cells containing a vector or isolated nucleic acid of the application useful for the production of a vector or construct of interest; or an engineered iPSC or derivative cell thereof containing one or more isolated nucleic acids of the application, preferably integrated at one or more chromosomal loci. A host cell of an isolated nucleic acid of the application can also be an immune effector cell, such as a T cell or NK cell, comprising the one or more isolated nucleic acids of the application. The immune effector cell can be obtained by differentiation of an engineered iPSC of the application. Any suitable method in the art can be used for the differentiation in view of the present disclosure. The immune effector cell can also be obtained transfecting an immune effector cell with one or more isolated nucleic acids of the application.

[0334] XI. Cell Compositions

[0335] In another general aspect, the application provides a composition comprising an isolated polynucleotide of the application, a host cell and / or an iPSC or derivative cell thereof of the application. In certain embodiments, the composition further comprises one or more therapeutic agents selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), siRNA, oligonucleotide, mononuclear blood cells, , a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD).

[0336] In certain embodiments, the composition is a pharmaceutical composition comprising an isolated polynucleotide of the application, a host cell and / or an iPSC or derivative cell thereof of the application and a pharmaceutically acceptable carrier. The term “pharmaceutical composition” as used herein means a product comprising an isolated polynucleotide of the application, an isolated polypeptide of the application, a host cell of the application, and / or an iPSC or derivative cell thereof of the application together with a pharmaceutically acceptable carrier. Polynucleotides, polypeptides, host cells, and / or iPSCs or derivative cells thereof of the application and compositions comprising them are also useful in the manufacture of a medicament for therapeutic applications mentioned herein.

[0337] As used herein, the term “carrier” refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic material that does not interfere with the effectiveness of a composition described herein or the biological activity of a composition described herein. According to particular embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in a polynucleotide, polypeptide, host cell, and / or iPSC or derivative cell thereof can be used.

[0338] The formulation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, e.g., Remington: The Science and Practice of Pharmacy (e.g. 21st edition (2005), and any later editions). Non-limiting examples of additional ingredients include: buffers, diluents, solvents, tonicity regulating agents, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carrier may be used in formulating the pharmaceutical compositions of the application.

[0339] XII. Methods of Use of Engineered Cells

[0340] Primary cancer cells can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that normally manifests as a solid tumour, a "clinically detectable" tumour is one that is detectable on the basis of tumour mass; e.g., by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation on physical examination, and / or which is detectable because of the expression of one or more cancer- specific antigens in a sample obtainable from a patient.

[0341] Cancer conditions may be characterized by the abnormal proliferation of malignant cancer cells and may include leukemias, such as AML, CML, ALL and CLL, lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma, and solid cancers such as sarcomas, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer, ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer, cancer of the gall bladder and biliary tracts, thyroid cancer, thymus cancer, cancer of bone, and cerebral cancer, as well as cancer of unknown primary (CUP).

[0342] Cancer cells within an individual may be immunologically distinct from normal somatic cells in the individual (i.e. the cancerous tumour may be immunogenic). For example, the cancer cells may be capable of eliciting a systemic immune response in the individual against one or more antigens expressed by the cancer cells. The tumour antigens that elicit the immune response may be specific to cancer cells or may be shared by one or more normal cells in the individual. The cancer cells of an individual suitable for treatment as described herein may express the antigen and / or may be of correct HLA type to bind the antigen receptor expressed by the T cells.

[0343] An individual suitable for treatment as described above may be a mammal. In preferred embodiments, the individual is a human. In other preferred embodiments, non- human mammals, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, primate, porcine, canine, or rabbit animals) may be employed.

[0344] In some embodiments, the individual may have minimal residual disease (MRD) after an initial cancer treatment. In some embodiments, the individual may have no minimal residual disease after one or more cancer treatments or repeated dosing.

[0345] In some aspects, the present disclosure provides methods for treating a subject with a therapeutic dose of a composition comprising iPSCs or a derivative cell thereof (e.g., a genetically engineered allogenic NK or T cell). In some embodiments, such methods can comprise a step of administering a therapeutically effective dose of the composition. In some cases, a single therapeutically effective dose is administered, In some cases, two or more therapeutically effective doses are administered in series. In some embodiments, engineered cells of the present disclosure may be administered in a single dose, and such administration may be by injection, e.g., intravenous injection. In some embodiments, engineered cell of the present disclosure may be administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing may be once a month, once every two weeks, once a week, or once every other day. Administration of engineered cells of the present disclosure may continue as long as necessary. Suitable administration of a therapeutically effective doses can comprise administration of a single dose, or can comprise administration of sequential doses daily, semi-weekly, weekly, once every two weeks, once a month, annually, etc. In some cases, a therapeutically effective dose is administered as two or more doses of escalating concentration (i.e., increasing doses), where (i) all of the doses are therapeutic doses, or where (ii) a sub-therapeutic dose (or two or more sub-therapeutic doses) is initially given and therapeutic doses are achieved by said escalation. As one non-limiting example to illustrate escalating concentration (i.e., increasing doses), a therapeutically effective dose can be administered weekly, beginning with a sub-therapeutic dose, and each subsequent dose can be increased by a particular increment, or by variable increments, until a therapeutic dose is reached, at which point administration may cease or may continue. As another non-limiting example to illustrate escalating concentration, a therapeutically effective dose can be administered weekly, beginning with a therapeutic dose, and each subsequent dose can be increased by a particular increment, or by variable increments, until a therapeutic dose is reached, at which point administration may cease or may continue. In some embodiments, administration of a therapeutically effective dose can be a continuous infusion and the dose can altered (e.g., escalated) over time. Dosage and frequency may vary depending on the half-life of the cell composition in the patient, and / or mode of administration.

[0346] An individual with cancer may display at least one identifiable sign, symptom, or laboratory finding that is sufficient to make a diagnosis of cancer in accordance with clinical standards known in the art. Examples of such clinical standards can be found in textbooks of medicine such as Harrison’s Principles of Internal Medicine, 15th Ed., Fauci AS et al., eds., McGraw-Hill, New York, 2001. In some instances, a diagnosis of a cancer in an individual may include identification of a particular cell type (e.g. a cancer cell) in a sample of a body fluid or tissue obtained from the individual.

[0347] An anti-tumor effect is a biological effect which can be manifested by a reduction in the rate of tumor growth, decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies, also T cells which may be obtained according to the methods of the present disclosure , as described herein in prevention of the occurrence of tumors in the first place.

[0348] Treatment may be any treatment and / or therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, cure or remission (whether partial or total) of the condition, preventing, delaying, abating or arresting one or more symptoms and / or signs of the condition or prolonging survival of a subject or patient beyond that expected in the absence of treatment.

[0349] Treatment may also be prophylactic (i.e. prophylaxis). For example, an individual susceptible to or at risk of the occurrence or re-occurrence of cancer may be treated as described herein. Such treatment may prevent or delay the occurrence or re-occurrence of cancer in the individual.

[0350] In particular, treatment may include inhibiting cancer growth, including complete cancer remission, and / or inhibiting cancer metastasis. Cancer growth generally refers to any one of a number of indices that indicate change within the cancer to a more developed form. Thus, indices for measuring an inhibition of cancer growth include a decrease in cancer cell survival, a decrease in tumor volume or morphology (for example, as determined using computed tomographic (CT), sonography, or other imaging method), a delayed tumor growth, a destruction of tumor vasculature, improved performance in delayed hypersensitivity skin test, an increase in the activity of T cells, and a decrease in levels of tumor-specific antigens. Administration of T cells modified as described herein may improve the capacity of the individual to resist cancer growth, in particular growth of a cancer already present the subject and / or decrease the propensity for cancer growth in the individual.

[0351] This application provides a method of treating a disease or a condition in a subject in need thereof. The methods comprise administering to the subject in need thereof a therapeutically effective amount of cells of the application and / or a composition of the application. In certain embodiments, the disease or condition is cancer. The cancer can, for example, be a solid or a liquid cancer. The cancer, can, for example, be selected from the group consisting of a lung cancer, a gastric cancer, a colon cancer, a liver cancer, a renal cell carcinoma, a bladder urothelial carcinoma, a metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, an endometrial cancer, a prostate cancer, a thyroid cancer, a glioma, a glioblastoma, and other solid tumors, and a non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma / disease (HD), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a chronic myelogenous leukemia (CML), a multiple myeloma (MM), an acute myeloid leukemia (AML), and other liquid tumors. In a preferred embodiment, the cancer is a non-Hodgkin’s lymphoma (NHL).

[0352] According to embodiments of the application, the composition comprises a therapeutically effective amount of an isolated polynucleotide, an isolated polypeptide, a host cell, and / or an iPSC or derivative cell thereof. As used herein, the term “therapeutically effective amount” refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject. A therapeutically effective amount can be determined empirically and in a routine manner, in relation to the stated purpose.

[0353] As used herein with reference to a cell of the application and / or a pharmaceutical composition of the application a therapeutically effective amount means an amount of the cells and / or the pharmaceutical composition that modulates an immune response in a subject in need thereof.

[0354] According to particular embodiments, a therapeutically effective amount refers to the amount of therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition to be treated or a symptom associated therewith; (ii) reduce the duration of the disease, disorder or condition to be treated, or a symptom associated therewith; (iii) prevent the progression of the disease, disorder or condition to be treated, or a symptom associated therewith; (iv) cause regression of the disease, disorder or condition to be treated, or a symptom associated therewith; (v) prevent the development or onset of the disease, disorder or condition to be treated, or a symptom associated therewith; (vi) prevent the recurrence of the disease, disorder or condition to be treated, or a symptom associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (viii) reduce hospitalization length of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (ix) increase the survival of a subject with the disease, disorder or condition to be treated, or a symptom associated therewith; (xi) inhibit or reduce the disease, disorder or condition to be treated, or a symptom associated therewith in a subject; and / or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy. The therapeutically effective amount or dosage can vary according to various factors, such as the disease, disorder or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, e.g., age, body weight, health), whether the subject is a human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.

[0355] According to particular embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.

[0356] The cells of the application and / or the pharmaceutical compositions of the application can be administered in any convenient manner known to those skilled in the art. For example, the cells of the application can be administered to the subject by aerosol inhalation, injection, ingestion, transfusion, implantation, and / or transplantation. The compositions comprising the cells of the application can be administered transarterially, subcutaneously, intradermaly, intratumorally, intranodally, intramedullary, intramuscularly, intrapleurally, by intravenous (i.v.) injection, or intraperitoneally. In certain embodiments, the cells of the application can be administered with or without lymphodepletion of the subject.

[0357] The pharmaceutical compositions comprising cells of the application can be provided in sterile liquid preparations, typically isotonic aqueous solutions with cell suspensions, or optionally as emulsions, dispersions, or the like, which are typically buffered to a selected pH. The compositions can comprise carriers, for example, water, saline, phosphate buffered saline, and the like, suitable for the integrity and viability of the cells, and for administration of a cell composition.

[0358] Sterile injectable solutions can be prepared by incorporating cells of the application in a suitable amount of the appropriate solvent with various other ingredients, as desired. Such compositions can include a pharmaceutically acceptable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like, that are suitable for use with a cell composition and for administration to a subject, such as a human. Suitable buffers for providing a cell composition are well known in the art. Any vehicle, diluent, or additive used is compatible with preserving the integrity and viability of the cells of the application.

[0359] The cells of the application and / or the pharmaceutical compositions of the application can be administered in any physiologically acceptable vehicle. A cell population comprising cells of the application can comprise a purified population of cells. Those skilled in the art can readily determine the cells in a cell population using various well known methods. The ranges in purity in cell populations comprising genetically modified cells of the application can be from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 100%. Dosages can be readily adjusted by those skilled in the art, for example, a decrease in purity could require an increase in dosage.

[0360] The cells of the application are generally administered as a dose based on cells per kilogram (cells / kg) of body weight of the subject to which the cells and / or pharmaceutical compositions comprising the cells are administered. Generally, the cell doses are in the range of about 104to about 1010cells / kg of body weight, for example, about 105to about 109, about 105to about 108, about 105to about 107, or about 105to about 106, depending on the mode and location of administration. In general, in the case of systemic administration, a higher dose is used than in regional administration, where the immune cells of the application are administered in the region of a tumor and / or cancer. Exemplary dose ranges include, but are not limited to, 1 x 104to 1 x 108, 2 x 104to 1 x 108, 3 x 104to 1 x 108, 4 x 104to 1 x 108, 5 x 104to 6 x 108, 7 x 104to 1 x 108, 8 x

[0361] 104to 1 x 108, 9 x 104to 1 x 108, 1 x 105to 1 x 108, 1 x 105to 9 x 107, 1 x 105to 8 x 107,

[0362] 1 x 105to 7 x 107, 1 x 105to 6 x 107, 1 x 105to 5 x 107, 1 x 105to 4 x 107, 1 x 105to 4 x 107, 1 x 105to 3 x 107, 1 x 105to 2 x 107, 1 x 105to 1 x 107, 1 x 105to 9 x 106, 1 x 105to 8 x 106, 1 x 105to 7 x 106, 1 x 105to 6 x 106, 1 x 105to 5 x 106, 1 x 105to 4 x 106, 1 x 105to 4 x 106, l x 105to 3 x 106, 1 x 105to 2 x 106, 1 x 103to 1 x 106, 2 x 105to 9 x 107, 2 x

[0363] 105to 8 x 107, 2 x 105to 7 x 107, 2 x 105to 6 x 107, 2 x 105to 5 x 107, 2 x 105to 4 x 107,

[0364] 2 x 105to 4 x 107, 2 x 105to 3 x 107, 2 x 105to 2 x 107, 2 x 105to 1 x 107, 2 x 105to 9 x 106, 2 x 105to 8 x 106, 2 x 105to 7 x 106, 2 x 105to 6 x 106, 2 x 105to 5 x 106, 2 x 105to 4 x 106, 2 x 105to 4 x 106, 2 x 105to 3 x 106, 2 x 105to 2 x 106, 2 x 105to 1 x 106, 3 x 105to 3 x 106cells / kg, and the like. Additionally, the dose can be adjusted to account for whether a single dose is being administered or whether multiple doses are being administered. The precise determination of what would be considered an effective dose can be based on factors individual to each subject.

[0365] As used herein, the terms “treat,” “treating,” and “treatment” are all intended to refer to an amelioration or reversal of at least one measurable physical parameter related to a cancer, which is not necessarily discernible in the subject, but can be discernible in the subject. The terms “treat,” “treating,” and “treatment,” can also refer to causing regression, preventing the progression, or at least slowing down the progression of the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an alleviation, prevention of the development or onset, or reduction in the duration of one or more symptoms associated with the disease, disorder, or condition, such as a tumor or more preferably a cancer. In a particular embodiment, “treat,” “treating,” and “treatment” refer to prevention of the recurrence of the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an increase in the survival of a subject having the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to elimination of the disease, disorder, or condition in the subject.

[0366] The cells of the application and / or the pharmaceutical compositions of the application can be administered in combination with one or more additional therapeutic agents. In certain embodiments the one or more therapeutic agents are selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), siRNA, oligonucleotide, mononuclear blood cells, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD). In certain embodiments, the one or more therapeutic agents comprise an antibody. In certain embodiments, the one or more therapeutic agents comprise one or more antibodies independently selected from the group consisting of ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, avelumab, ofatumumab, panitumumab, and ustekinumab. In certain embodiments, the one or more therapeutic agents comprise cetuximab. In certain embodiments, the one or more therapeutic agents comprise trastuzumab. In certain embodiments, the one or more therapeutic agents comprise bevacizumab. In certain embodiments, the one or more therapeutic agents comprise avelumab. In certain embodiments, the one or more therapeutic agents comprise ipilimumab.

[0367] EXAMPLES

[0368] Abbreviations

[0369] C-X-C Chemokine

[0370] CXCR4

[0371] Receptor Type 4

[0372] HDR Homology Directed Repair

[0373] WT Wild type ug Microgram mb Milliliter iNK iPSC-derived NK Cell

[0374] Tumor Growth

[0375] TGI

[0376] Inhibition

[0377] CAR Chimeric antigen receptor

[0378] Example 1. Engineering of CXCR4 into iPSCs and Derivative Cells

[0379] Purpose

[0380] Transgene expression vectors were generated to overexpress the CXCR4 wt protein when inserted by HDR into the CLYBL locus. These vectors were engineered into iPSC611 cells for differentiation into iNK cells to determine effects on in vivo biodistribution and function.

[0381] Materials gBlocks were ordered from IDT for wt CXCR4 having a sequence according SEQ

[0382] ID NO: 134

[0383] Table 10.

[0384]

[0385] These sequences were cloned into the p 1616 vector into the Sfbl site downstream of the CAG promoter and upstream of the SV40 polyA site. This vector possessed an operon for geneticin selection driven by a mini-TK promoter and homology arms for insertion into the CLYBL locus using CLYBL gRNA2.

[0386] Homology directed repair using the guide RNAs to CLBYL and CPF1 RNA- guided endonuclease was performed to insert the CXCR4 gene into the CLYBL locus. After performing HDR, the cell line was passaged in geneticin at a final concentration of 500 ug / ml of culture media to enforce selection of the integrated CXCR4 sequence. After selection, cells were analyzed for transgene expression by flow cytometry using PE- labeled anti-human CXCR4 antibody. NK cell differentiation was performed as described in PCT Publication No. WO2022120334A1, which is hereby incorporated by reference herein in its entirety for all purposes. Results Mouse Leukemia Xenograft Model for Evaluating iNK Efficacy

[0387] Mice were injected intravenously with 0.1e6 luciferase-tagged NALM-6 on Day 0. On Day 1, 10e6 CXCR4-negative iNK cells (iPSC611) or CXCR4-positive iNK cells (iPSC 1613) were injected intravenously. Tumor burden was monitored twice weekly using bioluminescence imaging. At study termination (Day 14), tumor growth inhibition (TGI) was calculated. As shown in FIG. 2, CXCR4-negative iNK cells (iPSC611) elicited 63.6% TGI, and CXCR4-positive iNK cells (iPSC 1613) elicited significantly better TGI at 97.1% (p=0.0029).

[0388] Flow Cytometry of NK cells in Mouse Blood and Bone Marrow to evaluate NK persistence and Migration

[0389] Mice were injected intravenously with 0.1 e6 luciferase-tagged NALM-6 on Day 0. On Day 1, 10e6 CXCR4-negative iNK cells (iPSC611) or CXCR4-positive iNK cells (iPSC 1613) were injected intravenously into tumor-bearing and non-tumor-bearing mice. On Day 14, whole blood and bone marrow aspirates were sampled and analyzed via flow cytometry for the presence of iNK cells. As shown in FIG. 3 A, there was no significant difference between iNK treatment or the presence of tumor in the blood. As shown in FIG. 3B, in the bone marrow, iNK cells were present only in mice treated with CXCR4- positive iNK cells (iPSC1613, both tumor-bearing and non-tumor-bearing mice). No iNK were detected in the bone marrow of mice treated with CXCR4-negative iNK cells (iPSC611).

[0390] Example 2. Development of cells expressing CXCR4

[0391] An iPSC line engineered to express a CAR against a tumor cell antigen was further engineered, through homology-directed-repair (HDR) into the CLYBL safe harbor site, to express the human wild type CXCR4 protein. The iPSC lines were differentiated to iNK cells, and evaluated for CXCR4 expression through the process. At iPSC stage (following engineering), HPC stage, Day 14 (DI 4) of iNK differentiation, and Day 21 (D21) of iNK differentiation, cells were analyzed for CXCR4 levels by flow cytometry (FIG. 4). Briefly, cells were collected, centrifuged, and stained with antibody detecting human CXCR4 (PE mouse anti-human CD184 [CXCR4] mAb). FACS was conducted on the BD Symphony flow cytometer, and results depicted as % CXCR4 positive cells of live cells. CXCR4 was found to be expressed in 42.4% of the engineered Day 21 iNK cells (IPSC1613) versus 2.4% of the iNK cells not engineered to express CXCR4 (iPSC611). The iPSC lines were differentiated at larger scale for use in functional assays, and CXCR4 expression was evaluated as previously indicated. At scale, the CXCR4-engineered iNK cells maintained 47.7% expression of CXCR4 (FIG.

[0392] 5) iNK cells differentiated from iPSC611 (CAR+ / CXCR4-), iPSC1613 (CAR+ / CXCR4+), and a CAR-CXCR4- iT cell line (iTCAR‘CXCR4‘) were tested for activity in a cytotoxicity assay against NALM-6 CD19+ tumor cell line as shown FIG. 6. 2xl05effector cells were co-cultured with 2xl05NucLight Red (NLR) labeled NALM-6 tumor cells in flatbottom 96-well tissue culture treated plates and placed in an Incucyte live cell imager. Whole well images were taken every 3 hours for 72 hours total collecting phase contrast and NLR images. Raw data was normalized to NLR count of tumor cell alone condition.

[0393] To generate CXCR4-expressing iNK lines for evaluation in a second tumor model, iPS cells expressing a dual-targeting CAR to two tumor targets (IPSC2984) was transduced with a lentivirus containing sequence encoding for WT CXCR4 upon integration into the cell line genome. Hematopoietic progenitor cells (HPCs) derived from IPSC2984 were transduced with the WT CXCR4 lentivirus and then differentiated 14 days to iNK cells. CXCR4 expression was evaluated as previously described, and the lentivirus- transduced cells determined to be 54.5% CXCR4 positive (versus 3.29% for non-transduced cells) (FIG. 7).

[0394] To assess the functionality of the CXCR4 transgene in iPSC2984, CXCR4 positive cells (CAR-iNKCXCR4+) and CXCR4 negative cells (CAR-iNKCXCR4’) were assessed in a transwell migration assay and compared to Jurkat (CXCR4+) and M0LM13 (CXCR4-) tumor cells as positive and negative controls as shown in FIGs. 8A-B. Transwell plates were coated with rat tail collagen at a concentration of 50 pg / mL for 2 hours. After coating, rat tail collagen solution was removed, and plates were allowed to dry for 30 minutes. In the lower chamber of the assay plate, 1 mL of assay media with or without 400 ng / mL C-X-C motif chemokine 12 (CXCL12) was added. The insert of the assay plate was loaded with 200 pl. of cells at a concentration of 1x106cells / mL in assay media. Assay plates were transferred to a tissue culture incubator at 37°C and incubated for 4 hours. After 4 hours cells were removed from the lower chamber of the transwell plate and stained for live / dead (near-IR), CD56 (BV786), and CXCR4 (BV421). Samples were collected on a BD Symphony A3 cytometer using absolute flow count beads to quantify the number of total cells in the well. Raw cell counts are displayed in FIG. 8A. Chemotactic index was calculated as the number of cells in experimental wells (400 ng CXCL12) divided by the average of cells in the baseline wells (0 ng CXCL12) and is shown in FIG. 8B.

[0395] CAR-iNKCXCR4+and CAR-iNKCXCR4-were tested for functionality in a cytotoxicity assay against NucLight Red-labeled antigen-positive and antigen-knock-out tumor cell lines as shown in FIGS. 9A-B. 2xl05target cells were co-cultured with either 2xl05or 4xl05effector cells for effector to target ratios (E:T) of 1 : 1 (FIG. 9A) and 2: 1 (FIG. 9B), respectively. Target cell growth was measured by Total Integrated Intensity readings from Incucyte Live-Cell Imaging system every 3 hours for 72 hours total. Figure curves represent target cell growth curves when co-cultured with effector cells, normalized to target cell growth curves when target cells are cultured alone.

[0396] CAR-iNK cells with (CAR-iNKCXCR4+) and without (CAR-iNKCXCR4’) CXCR4 engineering were subject to in vivo functional studies. As shown in FIG. 10, CAR-iNK cells were injected to tumor bearing female NSG mice, and the resulting cytotoxic effect was assessed over 9 days. M0LM13-Luc cells were implanted intravenously (IV) at IxlO5cells / mouse 4 days prior to intravenous injection of CAR-iNK at 1x107cells / mouse. Day 9 blood and tissue sampling were conducted to evaluate CAR-iNK presence by FACS and histology. FIG. 11 shows the efficacy results of in vivo efficacy screening of CAR-iNK cells with (CAR-iNKCXCR4+) and without (CAR-iNKCXCR4-) CXCR4 engineering using the disseminated M0LM13-Luc xenograft model in NSG mice. M0LM13-Luc cells were implanted intravenously (IV) at IxlO5cells / mouse 4 days prior to intravenous injection of CAR-iNK at IxlO7cells / mouse. Tumor burden was measured using bioluminescent imaging (BLI). Tumor growth inhibition (TGI) was calculated on Day 8, the final day of measurements for the tumor control group. Significant TGI compared to tumor control was observed only for CAR-iNKCXCR4+(82% TGI, p=0.0061). On Day 9, mice were humanely euthanized and sampled for blood, bone marrow, and tissue analysis by FACS and histology. Blood was collected into lithium heparin coated tubes, and bone marrow aspirate was collected and kept in serum-free RPMI until FACS analysis. Briefly, samples were centrifuged to pellet cells and medium discarded. Samples underwent RBC lysis and were resuspended for FACS antibody staining. Samples were stained with Near-IR Live / Dead stain (Thermo cat# L34976A) and FC block (Innovex# NB309) added to samples. Samples were then stained with the following antibodies: FITC (CD33), BV421 (CD45), BV786 (CD56), APC (CD184 / CXCR4), incubated, and FACS conducted on the BD Symphony A3 cytometer. FIG. 12 shows bone marrow presence of CAR-iNK in the presence and absence of tumor, assessed by FACS. Both tumor-bearing and non-tumor-bearing mice that received CAR- iNKCXCR4‘ did not have iNK present in the bone marrow. Significant numbers of iNK were present in the bone marrow of both tumor-bearing (p<0.0001) and non-tumor- bearing mice (p=0.0018) that received CAR-iNKCXCR4compared with tumor-bearing and non-tumor-bearing mice (respectively) that received CAR-iNKCXCR4‘. Femur, spleen, liver, and lung were collected from mice, and fixed in 10% NBF, then transferred to 70% ethanol. Tissues were then processed, paraffin-embedded, and cut for immunohistochemical (IHC) staining. Immunohistochemistry of normal NSG mouse bone marrow stained using CXCL12 antibody (SDF1 / CXCL12 [D8G6H] Rabbit mAb, 1 : 100 dilution) is shown in FIG. 13. Femur, spleen, liver, and lung were stained with CD3 antibody (NCL-L-CD3-565 [LN10] Mouse mAb) to detect iNK. CAR-iNKCXCR4‘ and CAR-iNKCXCR4+tissue infiltration of femur, spleen, liver, and lung is shown in FIG. 14.

[0397] Example 3. Flow Cytometry of iT Cells with Endogenous or Engineered CXCR4 Transgene expression vectors were generated to overexpress the CXCR4 wt protein when inserted by HDR into the CLYBL locus. These vectors were engineered into iPSCs for differentiation into iT cells to determine effects on in vivo biodistribution and function.

[0398] Materials gBlocks were ordered from IDT for wt CXCR4 having a sequence according SEQ ID NO: 134.

[0399] These sequences were cloned into the p 1616 vector into the Sfbl site downstream of the CAG promoter and upstream of the SV40 polyA site. This vector possessed an operon for geneticin selection driven by a mini-TK promoter and homology arms for insertion into the CLYBL locus using CLYBL gRNA2.

[0400] Homology directed repair using the guide RNAs to CLBYL and CPF1 RNA- guided endonuclease was performed to insert the CXCR4 gene into the CLYBL locus. After performing HDR, the cell line was passaged in geneticin at a final concentration of 500 ug / ml of culture media to enforce selection of the integrated CXCR4 sequence. After selection, cells were analyzed for transgene expression by flow cytometry using PE- labeled anti-human CXCR4 antibody. T cell differentiation was performed as described in PCT Publication No. WO2022216514A1, which is hereby incorporated by reference herein in its entirety for all purposes.

[0401] Results

[0402] Chemotaxis of i Cells In Vitro iPSCs were engineered with the following genetic edits, and differentiated into iT cells: IL15 / IL15Ra at the NKG2A locus, HLA-E / G at the B2M locus, CD16 at the CD70 locus, CIITA knockout, and an anti-tumor antigen chimeric antigen receptor at the CD33 locus.

[0403] As shown in FIG. 16, iT Cells with either endogenous levels of CXCR4 expression or enhanced CXCR4 expression driven by transgene expression were tested for chemotaxis to the chemokine SDF-1 in a transwell assay. The SDF-1 chemotaxis assay is a tool for evaluating the migratory potential of engineered T cells that express the SDF-1 receptor CXCR4. This assay involved creating an SDF-1 concentration gradient using a series of dilutions, and then measuring the migration of the iT cells towards these gradients in a transwell system. The iT cells were first labeled and added to the upper chamber of the transwell, while the various SDF-1 dilutions were placed in the lower chambers. After incubation, the number of iT cells that had migrated to the lower chambers through a membrane barrier in response to the chemokine gradient was quantified. iT cells were tested for migration through a membrane barrier either in the presence or absence of the chemokine. Chemotaxis was measured as the amount of cells in the bottom of the well, that have migrated through the membrane barrier.

[0404] Detection of Bone Engineered iT Cells in Bone Marrow by Fluorescence-activated Cell Sorting (FACS) and Immunohistochemistry (IHC)

[0405] NSG mice with established disseminated M0LM13 xenografts were intravenously injected with 7.5x10^6 CXCR4 transgene-positive or -negative iPSC- derived gamma delta iT cells per mouse. Nine days following injection, femurs were sampled for flow cytometry detection of iT cells. FIG. 17 shows the presence of iT cells in the bone marrow of mice, as measured using FACS. iT cells with CXCR4 transgene were found in greater numbers in the bone marrow than iT cells without the CXCR4 transgene.

[0406] FIGs. 18A-C show (A) the presence of iT cells in the bone marrow of mice, as measured using IHC; (B) anti-CD3 stained IHC image of bone marrow tissue from mice that were administered CXCR4 transgene-positive iPSC-derived gamma delta iT cells; and (C) anti-CD3 stained IHC image of bone marrow tissue from mice that were administered CXCR4 transgene-negative iPSC-derived gamma delta iT cells. Briefly, NSG mice with established disseminated M0LM13 xenografts were intravenously injected with 7.5x10^6 CXCR4 transgene-positive or -negative iPSC-derived gamma delta iT cells per mouse. Nine days following injection, femurs were sampled for immunohistochemical detection of T cells using a pan CD3 antibody (LN10). iT cells with CXCR4 transgene were found in greater numbers in the bone marrow than iT cells without the CXCR4 transgene.

[0407] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present description.

Claims

CLAIMS claimed:

1. An induced pluripotent stem cell (iPSC) or a derivative cell thereof comprising: at least one exogenous polynucleotide encoding:(i) one or more chimeric antigen receptors (CARs) comprising an antigen binding domain targeting at least one tumor antigen, and at least one intracellular domain; and(ii) C-X-C chemokine receptor type 4 (CXCR4) or a fragment or variant thereof; and optionally, at least one of:(i) a deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5, RFXAP genes,(ii) an exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G),(iii) an exogenous polynucleotide encoding a natural killer (NK) cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD 16)) and / or an NKG2D protein,(iv) a deletion or reduced expression of one or more of NKG2A CD70, CD38 or CD33 genes,(v) an exogeneous polynucleotide encoding a cytokine,(vi) an exogenous polynucleotide encoding a safety switch;(vii) an exogeneous polynucleotide encoding a PSMA cell tracer; and(viii) an exogeneous polynucleotide encoding a membrane bound IL- 12 polypeptide.

2. The iPSC or the derivative cell according to claim 1 , wherein the at least one tumor antigen comprises a first tumor antigen and a second tumor antigen, and wherein:(i) the one or more CARs consists of a dual-targeting CAR comprising a first antigen-binding domain targeting the first tumor antigen and a second antigen binding domain targeting the second tumor antigen; or(ii) the one or more CARs comprises a plurality of CARs including a first CAR and a second CAR, wherein the first CAR comprises the first antigen binding domain targeting the first tumor antigen, and wherein the second CAR comprises a second antigen binding domain targeting the second tumor antigen.

3. The iPSC or the derivative cell according to claim 1 or 2, wherein the first tumor antigen is selected from Table 2.

4. The iPSC or the derivative cell according to claim 2 or 3, wherein the second tumor antigen is selected from Table 2.

5. The iPSC or the derivative cell according to any one of claims 1-4, wherein the first antigen binding domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 2, 4, and 158-193.

6. The iPSC or the derivative cell according to any one of claims 1-4 wherein the first antigen binding domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 194-229.

7. The iPSC or the derivative cell according to any one of claims 2-6, wherein the second antigen binding domain comprises amino acids having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 2, 4, and 158-193.

8. The iPSC or the derivative cell according to any one of claims 2-6, wherein the second antigen binding domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 194-229.

9. The iPSC or the derivative cell according to any one of claims Error! Reference source not found.-8, wherein in the CAR, at least one of:(i) a signal peptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 or 143;(ii) the at least one extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 2, 4, and 158- 193;(iii) a hinge comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 21 and 22;(iv) a transmembrane domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 23 and 24;(v) the intracellular domain comprises a signaling and / or a co-stimulatory domain (i) comprising amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 6, 8-20, and 156, or (ii) encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 152-155.

10. The iPSC or the derivative cell according to claim 9, wherein in the CAR, at least one of:(i) the signal peptide comprises amino acids having the sequence of SEQ ID NO: 1 or 143;(ii) the at least one extracellular domain comprises amino acids having the sequence of one of SEQ ID NOs: 2, 4, and 158-193;(iii) the hinge comprises amino acids having the sequence of SEQ ID NO: 21 or 22;(iv) the transmembrane domain comprises amino acids having the sequence of SEQ ID NO: 23 or 24;(v) the intracellular domain comprises a signaling domain and / or a co- stimulatory domain (i) comprising amino acids having the sequence of one or more of SEQ ID NOs: 6, 8-20, and 156, or (ii) encoded by a polynucleotide having the sequence of one or more of SEQ ID NOs: 152-155.

11. The iPSC or the derivative cell thereof according to any one of claims 1-10, wherein the CXCR4 or a fragment or variant thereof is selected from the group consisting of wild-type CXCR4 and a mutated variant of CXCR4.

12. The iPSC or the derivative cell thereof according to claim 11 comprising the wild- type CXCR4, wherein the wild-type CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 134.

13. The iPSC or the derivative cell thereof according to claim 11 comprising the wild- type CXCR4, wherein the wild-type CXCR4 is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 142.

14. The iPSC or the derivative cell thereof according to claim 11 comprising the mutated variant of CXCR4, wherein the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 134 with a deletion of the C- terminal domain between 10 and 20 amino acid residues.

15. The iPSC or the derivative cell thereof according to claim 11 comprising the mutated variant of CXCR4, wherein the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 134 with one or more mutations selected from the group consisting of R334X, G336X, E343X, S341fs, S339fs342X, S338X, E343K, T328X, R144A, E179A, and E262A.

16. The iPSC or the derivative cell thereof according to claim 11 comprising the mutated variant of CXCR4, wherein the mutated variant of CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 134-141.

17. The iPSC or the derivative cell thereof according to any one of claims 1-16, wherein the cytokine comprises an interleukin 15 (IL- 15) or a membrane bound IL-15, where all or a functional portion of the IL-15 protein is fused to all or a portion of a transmembrane protein that anchors the expressed IL- 15 as a cell membrane-bound polypeptide (mbIL15)18. The iPSC or the derivative cell thereof according to claim 0, wherein the IL-15 comprises an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope and an IL- 15, and wherein the inactivated cell surface receptor and the IL-15 are operably linked by an autoprotease peptide.

19. The iPSC or the derivative cell thereof according to claim 0, wherein the IL-15 comprises (i) a fusion polypeptide comprising an IL-15 and an IL-15 receptor alpha (IL-15Ra), or (ii) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 145.

20. The iPSC or the derivative cell thereof according to any one of claims 0-19, wherein the IL-15 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 72.

21. The iPSC or the derivative cell according to any one of claims 1-20, comprising the deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes.

22. The iPSC or the derivative cell according to any one of claims 1-21, further comprising an exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G).

23. The iPSC or the derivative cell thereof according to any one of claims 1-22, wherein the CD 16 is a CD 16 variant protein.

24. The iPSC or the derivative cell thereof according to claim 23, wherein the CD16 variant protein is a high affinity CD 16 variant.

25. The iPSC or the derivative cell thereof according to claim 23 or 24, wherein the CD16 variant protein is a non-cleavable CD16 variant.

26. The iPSC or the derivative cell thereof according to any one of claims 23-25, wherein the CD 16 variant protein comprises wild-type CD 16 comprising amino acids having the sequence of SEQ ID NO: 86 and one or more amino acid substitutions selected from the group consisting of Fl 58 V, Fl 76V, S197P, D205A, S219A, T220A.

27. The iPSC or the derivative cell thereof according to any one of claims 23-26, wherein the CD 16 variant protein comprises amino acids having at least 90%sequence identity to any one of SEQ ID NOs: 85 and 86.

28. The iPSC or the derivative cell thereof according to any one of claims 1-27 comprising an exogenous polynucleotide encoding the CD 16 protein and the NKG2D protein, wherein the CD 16 protein and the NKG2D protein are operably linked by an autoprotease peptide.

29. The iPSC or the derivative cell thereof according to claim 28, wherein the NKG2D protein is a wildtype NKG2D protein.

30. The iPSC or the derivative cell thereof according to claim 28, wherein the NKG2D protein comprises amino acids having at least 90% sequence identity to SEQ ID NO: 89.

31. The iPSC or the derivative cell thereof according to any one of claims 28-30, wherein the autoprotease peptide is selected from the group consisting of a porcine tesehovirus-1 2A (P2A) peptide, a foot-and-mouth disease virus 2A (F2A) peptide, an Equine Rhinitis A Virus (ERAV) 2A (E2A) peptide, a Thosea asigna virus 2A (T2A) peptide, a cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and a Flacherie Virus 2A (BmIFV2A) peptide.

32. The iPSC or the derivative cell thereof according to claim 31, wherein the autoprotease peptide is a P2A peptide comprising amino acids having at least 90% sequence identity to SEQ ID NO: 91.

33. The iPSC or the derivative cell thereof according to any one of claims 28-32, wherein the exogenous polynucleotide encoding the CD 16 protein and the NKG2D protein comprises a nucleic acid having at least 90% sequence identity to SEQ ID NO: 91.

34. The iPSC or the derivative cell thereof according to any one of claims 1-33 comprising (i) an exogenous polynucleotide encoding a membrane-bound interleukin 12 (IL-12) comprising a first polypeptide comprising an IL-12 alpha subunit p35 or a polypeptide at least 90% similar thereto, a second polypeptide comprising an IL-12 beta subunit p40 or a polypeptide at least 90% similar thereto, and a transmembrane domain fused to the terminus of the first and / or second IL- 12 subunit polypeptide.

35. The iPSC or the derivative cell according to any one of claims 1-34, wherein the exogenous polynucleotide(s) are integrated into the chromosome of the cell at one or more loci selected from the group consisting of AAVS1, B2M, CBL-B, CCR5, CD33, CD38, CD70, CIITA, CIS, CISH, CLYBL, Collagen, CTLA4 , GAPDH, HTRP, LAG3, NKG2A, NKG2D, NLRC5, PD1, RFX5, RFXANK, RFXAP, ROSA26, RUNX1, SOCS2, TAP2, Tapasin, TAPBP, TAPI, TCR a or b constant region, TIGIT, TIM3, and TRAC genes, provided at least one of the exogenous polynucleotides is integrated at a locus of a gene selected from the group consisting of AAVS1, B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes to thereby result in a deletion or reduced expression of the gene.

36. The iPSC or the derivative cell according to claim 35, wherein the exogenous polynucleotide(s) are integrated into the chromosome of the cell at one or more loci selected from the group consisting of AAVS1, B2M, CIITA, CD33, and CLYBL.

37. The iPSC or the derivative cell according to any one of claims 1-36 having a deletion or reduced expression of one or both of the B2M and the CIITA genes.

38. The iPSC or the derivative cell thereof according to claim 37, having a deletion or reduced expression of both of the B2M and the CIITA genes.

39. The derivative cell of any one of claims 1 -38, wherein the derivative cell is a T- cell or an NK cell.

40. The derivative cell of claim 39, wherein the derivative cell is a T-cell.

41. The iPSC of any one of claims 1-38, wherein the iPSC is reprogrammed from whole peripheral blood mononuclear cells (PBMCs).

42. The iPSC of any one of claim 1-38, which is derived from a re-programmed T- cell.

43. The iPSC of any one of claims 1-38, 41, and 42, the derivative cell of any one of claims 1-40, further comprising an exogenous polynucleotide encoding a safety switch.

44. The iPSC or the derivative cell thereof of claim 43, wherein the safety switch comprises an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope.

45. The iPSC or the derivative cell according to claim 44, wherein the monoclonal antibody specific epitope is selected from a group of epitopes specifically recognized by ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, polatuzumab vedotin, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, tremelimumab, ofatumumab, panitumumab, and ustekinumab.

46. The iPSC or the derivative cell according to claim 44, wherein the inactivated cell surface receptor is a truncated epithelial growth factor (tEGFR) variant.

47. The iPSC or the derivative cell according to claim 46, wherein the tEGFR variant consists of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 71.

48. The iPSC or the derivative cell thereof according to any one of claims 1-47, wherein the safety switch comprises (i) an intracellular domain comprising a herpes simplex virus thymidine kinase (HSV-TK) or (ii) an inducible Caspase 9 (iCasp9).

49. The iPSC or the derivative cell thereof according to any one of claims 1-48 comprising the exogeneous polynucleotide encoding the PSMA cell tracer, wherein the PSMA cell tracer comprises an extracellular domain comprising a PSMA extracellular domain or fragment thereof.

50. The iPSC or the derivative cell thereof according to claim 49, comprising a combined artificial cell death / reporter system polypeptide comprising an intracellular domain comprising a herpes simplex virus thymidine kinase (HSV- TK) and a linker, a transmembrane region, and an extracellular domain comprising the PSMA extracellular domain or fragment thereof.

51. The iPSC or the derivative cell thereof according to any one of claims 48-50, wherein (i) the HSV-TK comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 146 or 147, or (ii) the iCasp9 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 148 or 149.

52. The iPSC or the derivative cell thereof according to claim 50, wherein the combined artificial cell death / reporter system polypeptide comprises the HSV-TK fused to a truncated variant PSMA polypeptide via the linker.

53. The iPSC or the derivative cell thereof according to claim 52, wherein the truncated variant PSMA polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 150.

54. The iPSC or the derivative cell thereof according to any one of claims 50-53, wherein the linker comprises an autoprotease peptide selected from the group consisting of a P2A peptide, a T2A peptide, an E2A peptide, and a F2A peptide.

55. The iPSC or the derivative cell thereof according to any one of claims 50-54, wherein the artificial cell death / reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 151.

56. The iPSC or the derivative cell thereof according to claim 55, 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 NOs: 234-236.

57. The iPSC or the derivative cell thereof according to any one of claims 50-56, wherein the artificial cell death / reporter system polypeptide is encoded by nucleic acids 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: 237-239.

58. The iPSC or the derivative cell according to any one of claims 1-57, wherein the HLA-E comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 66, or the HLA-G comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 69.

59. The iPSC or the derivative cell according to any one of claims 2-58, wherein:(i) the exogenous polynucleotide encodes the one or more CARs comprising the antigen binding domain targeting the at least one tumor antigen selected from Table 2;(ii) the exogenous polynucleotide encoding CXCR4 (a) comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 142, or (b) encodes a CXCR4 polypeptide comprising amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 134-141;(iii) the exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G) comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 67 and 70;(iv) the exogenous polynucleotide encoding an NK cell receptor immunoglobulin gamma Fc region receptor III (FcyRIII, cluster of differentiation 16 (CD16)) and / or an NKG2D protein comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 84, 88, and 90;(v) the exogeneous polynucleotide encoding a cytokine comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 240;(vi) the exogenous polynucleotide encoding a safety switch comprises a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NO: 237-239; and / or(vii) the exogeneous polynucleotide encoding a PSMA cell tracer comprises a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 150.

60. The iPSC or the derivative cell thereof according to any one of claims 1-59, wherein:(i) the exogenous polynucleotide encodes the one or more CARs comprising the antigen binding domain targeting the at least one tumor antigen selected from Table 2;(ii) the exogenous polynucleotide encoding the CXCR4 (a) comprises polynucleotides having the sequence of SEQ ID NO: 142, or (b) encodes a CXCR4 polypeptide comprising amino acids having the sequence of one of SEQ ID NOs: 134-141;(iii) the exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and / or human leukocyte antigen G (HLA-G) comprises polynucleotides having the sequence SEQ ID NO: 67 and 70;(iv) the exogenous polynucleotide encoding an FcyRIII receptor and / or an NKG2D protein comprises polynucleotides having the sequence of SEQ ID NO: 84, 88, and 90;(v) the exogeneous polynucleotide encoding the cytokine comprises polynucleotides having the sequence of SEQ ID NO: 240; and / or(vi) the exogenous polynucleotide encoding the safety switch comprises polynucleotides having the sequence of one of SEQ ID NOs: 237-239; and / or(vii) the exogenous polynucleotide encoding the PSMA cell tracer comprises polynucleotides having the sequence of one of SEQ ID NOs: 150.61 . The iPSC or the derivative cell thereof according to claim 59 or 60, wherein each of the exogenous polynucleotides are integrated into the chromosome of the cell at a locus independently selected from the group consisting of AAVS1, B2M, CBL-B, CCR5, CD33, CD38, CD70, CIITA, CIS, CISH, CLYBL, Collagen, CTLA4 , GAPDH, HTRP, LAG3, NKG2A, NKG2D, NLRC5, PD1, RFX5, RFXANK, RFXAP, ROSA26, RUNX1, SOCS2, TAP2, Tapasin, TAPBP, TAPI, TCR a or b constant region, TIGIT, TIM3, and TRAC.

62. The iPSC or the derivative cell thereof according to claim 61, wherein:(i) the exogenous polynucleotide encoding the one or more CARs is integrated at a locus of the AAVS1 gene, the CD33 gene, or the CLYBL gene;(ii) the exogenous polynucleotide encoding the CXCR4 is integrated at a locus of the CLYBL gene(iii) the exogenous polynucleotide encoding a HLA-E)and / or HLA-G is integrated at a locus of the B2M gene;(iv) the exogenous polynucleotide encoding an FcyRIII receptor and / or an NKG2D protein is integrated at a locus of the CD70 gene;(v) the exogeneous polynucleotide encoding the cytokine is integrated at the locus of the NKG2A gene;(vi) the iPSC or the derivative cell thereof comprises a deletion or reduced expression of the CIITA gene; and, optionally(vii) the exogenous polynucleotide encoding the safety switch or the PSMA is integrated at the locus of the CIITA gene.

63. The derivative cell of any one of claims 1-62, wherein the derivative cell is a natural killer (NK) cell or a T cell.

64. The derivative cell according to claim 63, wherein the derivative cell is a natural killer (NK) cell.

65. The derivative cell according to claim 63, wherein the derivative cell is a T cell.

66. The derivative cell according to claim 65, wherein the T cell is (i) a gamma delta T cell, or (ii) an alpha beta T cell.

67. The derivative cell according to claim 65, wherein the T cell is a gamma delta Vy9 / V31 T cell.

68. A composition comprising the iPSC according to any one of claims 1-38 and 41- 62 or the derivative cell according to any one of claims 1-38 and 43-67.

69. The composition according to claim 68, further comprising or being used in combination with, one or more therapeutic agents selected from the group consisting of a small-molecule therapeutic agent, a biologic, a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), a siRNA, an oligonucleotide, mononuclear blood cells, a vector comprising one or more polynucleotides of interest, an antibody, a chemotherapeutic agent, a radioactive moiety, and an immunomodulatory drug (IMiD).

70. A method of treating cancer in a subject in need thereof, comprising administering the derivative cell according to any one of claims 1-40 and 43-67, or the composition according to claim 68 or 69 to a subject in need thereof.

71. The method of treatment according to claim 70, wherein the cancer is selected from the group consisting of leukemias, such as AML, CML, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), and chronic lymphocytic leukemia (CLL), lymphomas, such as Hodgkin lymphoma, non- Hodgkin lymphoma, multiple myeloma, and follicular lymphoma, and solid cancers such as sarcomas, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer, ovary cancer, prostate cancer, lung cancer, colorectalcancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer, cancer of the gall bladder and biliary tracts, thyroid cancer, thymus cancer, cancer of bone, and cerebral cancer, as well as cancer of unknown primary (CUP).

72. The method of treatment according to claim 71, wherein the cancer is a B-cell malignancy, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), or non-Hodgkin lymphoma, follicular lymphoma.

73. The method of treatment according to any one of claims 70-72, wherein the subject has minimal residual disease (MRD) after an initial cancer treatment.

74. The method of treatment according to any one of claims 70-72, wherein the subject has no minimal residual disease (MRD) after one or more cancer treatments or repeated dosing.

75. A method of manufacturing the derivative cell according to any one of claims 1- 38 and 43-67 comprising differentiating the iPSC according to any one of claims 1-38 and 41-62 under conditions for cell differentiation to thereby obtain the derivative cell.

76. The method according to claim 75, wherein the iPSC is obtained by genomic engineering an unmodified iPSC, wherein the genomic engineering comprises targeted editing.

77. The method according to claim 75, wherein the targeted editing comprises deletion, insertion, or in / del carried out by CRISPR, ZFN, TALEN, homing nuclease, homology recombination, or any other functional variation of these methods.

78. A method of differentiating an induced pluripotent stem cell (iPSC) into an NK cell, comprising subjecting the iPSCs to a differentiation protocol including culturing the cells in a medium containing a recombinant human IL- 12 for the final 24 hours of culturing under the differentiation protocol.

79. The method according to claim 78, wherein the recombinant IL- 12 comprises IL12p70.

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