# US 10,370,452 B2 · Effective generation of tumor-targeted T cells derived from pluripotent stem cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,370,452 B2 - Document ID: US10370452B2 - Status: Granted - Kind: B2 - Application number: 14/873,836 - Filed: 2015-10-02 - Published: 2016-01-14 - Granted: 2019-08-06 - Earliest claimed: 2013-04-03 - Assignees: Memorial Sloan Kettering Cancer Center - Applicants: Memorial Sloan-Kettering Cancer Center - Inventors: Maria Themeli; Michel Sadelain; Christopher C. Kloss - Web page: https://patentgrep.com/patent/US10370452B2 ## CPC - C07K 16/30: from tumour cells - A61K 39/0011: {Cancer antigens} - A61K 39/001102: {Receptors, cell surface antigens or cell surface determinants} - A61K 39/001104: {Epidermal growth factor receptors [EGFR]} - A61K 39/001106: {Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ErbB4} - A61K 39/001109: {Vascular endothelial growth factor receptors [VEGFR]} - A61K 39/001112: {CD19 or B4} - A61K 39/001113: {CD22, BL-CAM, siglec-2 or sialic acid- binding Ig-related lectin 2} - A61K 39/001114: {CD74, Ii, MHC class II invariant chain or MHC class II gamma chain} - A61K 39/001117: {Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR] or CD30} - A61K 39/001119: {Receptors for interleukins [IL]} - A61K 39/001124: {CD20} - A61K 39/001126: {CD38 not IgG} - A61K 39/001128: {CD44 not IgG} - A61K 39/001129: {Molecules with a "CD" designation not provided for elsewhere} - A61K 39/001153: {Wilms tumor 1 [WT1]} - A61K 39/001157: {Telomerase or TERT [telomerase reverse transcriptase]} - A61K 39/001166: {Adhesion molecules, e.g. NRCAM, EpCAM or cadherins} - A61K 39/001168: {Mesothelin [MSLN]} - A61K 39/00117: {Mucins, e.g. MUC-1} - A61K 39/001171: {Gangliosides, e.g. GM2, GD2 or GD3} - A61K 39/00118: {from embryonic or fetal origin} - A61K 39/001182: {Carcinoembryonic antigen [CEA]} - A61K 39/001184: {Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE} - A61K 39/001186: {MAGE} - A61K 39/001188: {NY-ESO} - A61K 39/001193: {Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR} - A61K 39/001195: {Prostate specific membrane antigen [PSMA]} - A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells - A61K 40/31: Chimeric antigen receptors [CAR] - A61K 40/4211: {CD19 or B4} - C07K 14/47: from mammals - C07K 14/7051: {T-cell receptor (TcR)-CD3 complex} - C07K 14/70521: {CD28, CD152} - C07K 16/2803: {against the immunoglobulin superfamily} - C12N 5/0638: {Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]} - C12N 5/0646: {Natural killers cells [NK], NKT cells} - A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells} - A61K 2239/48: Blood cells, e.g. leukemia or lymphoma - C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered - C07K 2317/56: variable (Fv) region, i.e. VH and/or VL - C07K 2317/622: Single chain antibody (scFv) - C07K 2319/03: containing a transmembrane segment - C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies - C07K 2319/70: containing domain for protein-protein interaction - C07K 2319/74: containing a fusion for binding to a cell surface receptor - C12N 2506/45: from artificially induced pluripotent stem cells - C12N 2510/00: Genetically modified cells - C12N 2740/13043: viral genome or elements thereof as genetic vector - C12N 2740/16043: viral genome or elements thereof as genetic vector - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention relates to the field of adoptive immunotherapy. The invention provides methods for generating phenotypically defined, functional, and/or expandable T cells from pluripotent stem cells engineered through safe genetic modifications. The engineered cells may provide one or more of: 1) targeting a specific predetermined antigen expressed on the cell surface of a target cell in an HLA independent manner, 2) enhanced survival and functional potential 3) “off-the-shelf” T cells for administration to multiple recipients, eventually across immunogenic barriers, and/or 4) cytotoxic potential and anti-tumor activity. ## Claims ### Claim 1 A population of T cells that are produced by in vitro differentiation of a pluripotent stem cell, wherein (i) the pluripotent stem cell expresses a chimeric antigen receptor (CAR), and (ii) the population of T cells comprises a T cell exhibiting a CD45RA+CD27−CD28−CCR7−CD62L− phenotype. ### Claim 2 (depends on 1) The population of claim 1, wherein T cells of said population target specifically to one antigen, and said antigen specificity is HLA-independent. ### Claim 3 (depends on 1) The population of claim 1, wherein said CAR is encoded by a nucleic acid sequence that is a heterologous sequence. ### Claim 4 (depends on 3) The population of claim 3, wherein said heterologous sequence is integrated into said pluripotent stem cell's genome at a genomic safe harbor site. ### Claim 5 (depends on 2) The population of claim 2, wherein said antigen is a tumor antigen or a pathogen antigen. ### Claim 6 (depends on 5) The population of claim 5, wherein the antigen is selected from the group consisting of carbonic anhydrase IX (CAlX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, an antigen of a cytomegalovirus (CMV) infected cell, epithelial glycoprotein2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb B2,3,4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (LlCAM), melanoma antigen family A, 1 (MAGE-AI), Mucin 16 (Muc-16), Mucin 1 (Muc-1), Mesothelin (MSLN), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF R2), and Wilms tumor protein (WT-1). ### Claim 7 (depends on 1) The population of claim 1, wherein said CAR comprises a single-chain variable fragment (scFv). ### Claim 8 (depends on 1) The population of claim 1, wherein said CAR comprises one or more of a CD3ζ polypeptide, a CD4 polypeptide, a CD8 polypeptide, a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, or a BTLA polypeptide. ### Claim 9 (depends on 8) The population of claim 8, wherein said CAR comprises a CD3ζ polypeptide. ### Claim 10 (depends on 9) The population of claim 9, wherein said CAR further comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a PD-1 polypeptide, a CTLA-4 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, or a BTLA polypeptide. ### Claim 11 (depends on 1) The population of claim 1 wherein said CAR is 1928z. ### Claim 12 (depends on 1) The population of claim 1, wherein T cells of said population comprise a silenced gene selected from the group consisting of a HLA gene transcription factor and a beta-2 microglobulin for an HLA gene. ### Claim 13 (depends on 1) The population of claim 1, wherein T cells of said population comprise a T helper cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a Natural killer T cell, a Mucosal associated invariant T cell, a γδ T cell, or a combination thereof. ### Claim 14 (depends on 1) The population of claim 1, wherein said pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell. ### Claim 15 (depends on 1) The population of claim 1, wherein said pluripotent stem cell is an induced pluripotent stem cell. ### Claim 16 A method of reducing tumor burden in a subject, comprising administering a population of T cells generated from a pluripotent stem cell that expresses a chimeric antigen receptor (CAR) to said subject having tumor, wherein the population of T cells comprises a T cell exhibiting a CD45RA+CD27−CD28−CCR7−CD62L− phenotype. ### Claim 17 (depends on 16) The method of claim 16, wherein said T cell is cytotoxic to said tumor. ### Claim 18 (depends on 16) The method of claim 16, wherein said tumor cell expresses a tumor antigen and said T cells target specifically to said tumor antigen. ### Claim 19 (depends on 18) The method of claim 18, wherein antigen-specificity of said T cell is HLA-independent. ### Claim 20 (depends on 16) The method of claim 16, wherein said pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell. ### Claim 21 (depends on 20) The method of claim 20, wherein said pluripotent stem cell is an induced pluripotent stem cell. ### Claim 22 (depends on 21) The method of claim 21, wherein said pluripotent stem cell is derived from a T cell. ### Claim 23 (depends on 22) The method of claim 22, wherein said pluripotent stem cell expresses one ligand for immunoregulatory T cell receptor, wherein said ligand is selected from the group consisting of PD-L1, CD48 and TNFRSF14. ### Claim 24 (depends on 22) The method of claim 22, wherein said pluripotent stem cell expresses HLA-G. ### Claim 25 (depends on 21) The method of claim 21, wherein said pluripotent stem cell is derived from a viral-specific T cell. ### Claim 26 (depends on 25) The method of claim 25, wherein the viral-specific T cell is a EBV-specific T-cell or a CMV-specific T-cell. ### Claim 27 (depends on 21) The method of claim 21, wherein said pluripotent stem cell is derived from a T cell that does not express a rearranged T-cell receptor (TCR). ### Claim 28 (depends on 18) The method of claim 18, wherein said tumor antigen is selected from the group consisting of carbonic anhydrase IX (CAlX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, an antigen of a cytomegalovirus (CMV) infected cell, epithelial glycoprotein2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb B2,3,4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ-light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (LlCAM), melanoma antigen family A, 1 (MAGE-AI), Mucin 16 (Muc-16), Mucin 1 (Muc-1), Mesothelin (MSLN), NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF R2), and Wilms tumor protein (WT-1). ### Claim 29 (depends on 16) The method of claim 16, wherein said T cell does not induce graft vs. host disease in said subject. ### Claim 30 (depends on 16) The method of claim 16, wherein said CAR comprises a single-chain variable fragment (scFv). ### Claim 31 (depends on 16) The method of claim 16, wherein said CAR comprises one or more of a CD3ζ polypeptide, a CD4 polypeptide, a CD8 polypeptide, a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, and a BTLA polypeptide. ### Claim 32 (depends on 16) The method of claim 16, wherein said CAR comprises a CD3t polypeptide. ### Claim 33 (depends on 32) The method of claim 32, wherein said CAR further comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a PD-1 polypeptide, a CTLA-4 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, or a BTLA polypeptide. ### Claim 34 (depends on 16) The method of claim 16, wherein said CAR is 1928z. ### Claim 35 (depends on 16) The method of claim 16, wherein said method reduces the number of tumor cells. ### Claim 36 (depends on 16) The method of claim 16, wherein said method reduces tumor size. ### Claim 37 (depends on 16) The method of claim 16, wherein said method eradicates the tumor in the subject. ### Claim 38 (depends on 16) The method of claim 16, wherein said T cell is selected from the group consisting of T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, Natural killer T cells, Mucosal associated invariant T cells, γδ T cells, and a combination thereof. ### Claim 39 (depends on 16) The method of claim 16, wherein said T cell comprises a silenced gene selected from the group consisting of a HLA gene transcription factor and a beta-2 microglobulin for an HLA gene. ### Claim 40 (depends on 16) The method of claim 16, wherein said T cell expresses Foxp3. ### Claim 41 (depends on 16) The method of claim 16, wherein said subject is a human. ### Claim 42 A method of increasing survival of a subject having neoplasia, comprising administering a population of T cells generated from a pluripotent stem cell that expresses a chimeric antigen receptor (CAR) to said subject diagnosed with neoplasia, wherein the population of T cells comprises a T cell exhibiting a CD45RA+CD27−CD28−CCR7−CD62L− phenotype. ### Claim 43 (depends on 42) The method of claim 42, wherein said T cell is cytotoxic to said neoplasia. ### Claim 44 (depends on 42) The method of claim 42, wherein said T cell specifically targets an antigen of said neoplasia and said antigen specificity is HLA-independent. ### Claim 45 (depends on 42) The method of claim 42, wherein said pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell. ### Claim 46 (depends on 45) The method of claim 45, wherein said pluripotent stem cell is an induced pluripotent stem cell. ### Claim 47 (depends on 46) The method of claim 46, wherein said pluripotent stem cell is derived from a T cell. ### Claim 48 (depends on 47) The method of claim 47, wherein said pluripotent stem cell expresses one ligand for immunoregulatory T cell receptor, wherein said ligand is selected from the group consisting of PD-L1, CD48 and TNFRSF14. ### Claim 49 (depends on 47) The method of claim 47, wherein said pluripotent stem cell expresses HLA-G. ### Claim 50 (depends on 46) The method of claim 46, wherein said pluripotent stem cell is derived from a viral-specific T cell. ### Claim 51 (depends on 50) The method of claim 50, wherein the viral-specific T cell is a EBV-specific T-cell or a CMV-specific T-cell. ### Claim 52 (depends on 47) The method of claim 47, wherein said pluripotent stem cell is derived from a T cell that does not express a rearranged T-cell receptor (TCR). ### Claim 53 (depends on 42) The method of claim 42, wherein said neoplasia is selected from the group consisting of blood cancer, B cell leukemia, multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin's lymphoma, ovarian cancer, prostate cancer, pancreatic cancer, lung cancer, breast cancer, sarcoma, and acute myeloid leukemia (AML). ### Claim 54 (depends on 42) The method of claim 42, wherein said CAR comprises a single-chain variable fragment (scFv). ### Claim 55 (depends on 42) The method of claim 42, wherein said CAR comprises one or more of a CD3ζ polypeptide, a CD4 polypeptide, a CD8 polypeptide, a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, and a BTLA polypeptide. ### Claim 56 (depends on 42) The method of claim 42, wherein said CAR comprises a CD3t polypeptide. ### Claim 57 (depends on 56) The method of claim 56, wherein said CAR further comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a PD-1 polypeptide, a CTLA-4 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, or a BTLA polypeptide. ### Claim 58 (depends on 42) The method of claim 42, wherein said CAR is 1928z. ### Claim 59 (depends on 42) The method of claim 42, wherein said T cell is selected from the group consisting of T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, Natural killer T cells, Mucosal associated invariant T cells, γδ T cells, and a combination thereof. ### Claim 60 (depends on 42) The method of claim 42, wherein said T cell comprises a silenced gene selected from the group consisting of a HLA gene transcription factor and a beta-2 microglobulin for an HLA gene. ### Claim 61 (depends on 42) The method of claim 42, wherein said T cell expresses Foxp3. ### Claim 62 (depends on 42) The method of claim 42, wherein said subject is a human. ### Claim 63 (depends on 1) A pharmaceutical composition comprising an effective amount of said population of T cells of claim 1 in a pharmaceutically acceptable excipient. ### Claim 64 (depends on 1) A kit for treatment of a neoplasia, pathogen infection, an autoimmune disorder, or an allogeneic transplant, the kit comprising a population of T cells of claim 1. ### Claim 65 (depends on 64) The kit of claim 64, wherein T cells of said population target specifically to one antigen, and said antigen specificity is HLA-independent. ### Claim 66 (depends on 64) The kit of claim 64, wherein said kit further comprises written instructions for using said population for said treatment of a subject diagnosed with a neoplasia, a pathogen infection, an autoimmune disorder, or an allogeneic transplant. ### Claim 67 (depends on 1) The population of claim 1, wherein said pluripotent stem cell further comprises a rearranged T-cell receptor (TCR) locus. ### Claim 68 (depends on 67) The population of claim 67, wherein the rearranged T-cell receptor (TCR) locus is comprised in said T cells produced from said pluripotent stem cell. ### Claim 69 (depends on 1) The population of claim 1, wherein said pluripotent stem cell is derived from reprogramming an isolated T cell. ### Claim 70 (depends on 1) The population of claim 1, wherein said pluripotent stem cell further comprises at least one genetic manipulation. ### Claim 71 (depends on 1) The population of claim 1, wherein said pluripotent stem cell has reduced or undetectable cell surface expression of (i) MHC molecules; (ii) HLA molecules; or (iii) HLA class I molecules. ### Claim 72 (depends on 1) The population of claim 1, wherein said pluripotent stem cell comprises reduced or knocked-out expression in at least one of: (i) Rag gene; (ii) CIITA gene; and (iii) beta-2 microglobulin gene; and/or has expression in Foxp3 gene. ### Claim 73 (depends on 1) The population of claim 1, wherein said pluripotent stem cell further comprises at least one of: a costimulatory signal, a suicide gene, an inducible cytokine, an imaging gene, and a combination thereof. ### Claim 74 (depends on 73) The population of claim 73, wherein the at least one costimulatory signal is (i) co-expressed with the CAR as a costimulatory ligand protein; or (ii) is comprised within an intracellular domain of the CAR. ### Claim 75 (depends on 74) The population of claim 74, wherein the at least one costimulatory signal is co-expressed with the CAR as a costimulatory ligand protein. ### Claim 76 (depends on 75) The population of claim 75, wherein the costimulatory ligand protein is selected from the group consisting of CD80, CD86, CD70, OX40L, 4-1BBL, CD48, TNFRSF14, and PD-L1. ### Claim 77 (depends on 74) The population of claim 74, wherein the at least one costimulatory signal is comprised within the CAR intracellular domain. ### Claim 78 (depends on 77) The population of claim 77, wherein the costimulatory signal is selected from from the group consisting of a CD28 polypeptide, a 4-IBB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, and a CTLA-4 polypeptide. ### Claim 79 (depends on 68) The population of claim 68, wherein said pluripotent stem cell does not express the TCR. ### Claim 80 (depends on 1) The population of claim 1, wherein T cells of said population express the CAR. ### Claim 81 (depends on 80) The population of claim 80, wherein T cells of said population are expandable under stimulation using an antigen specific to the CAR. ### Claim 82 (depends on 80) The population of claim 80, wherein T cells of said population further comprise at least one genetic manipulation. ### Claim 83 (depends on 80) The population of claim 80, wherein T cells of said population have reduced or undetectable cell surface expression of (i) WIC molecules; (ii) HLA molecules; or (iii) HLA class I molecules. ### Claim 84 (depends on 80) The population of claim 80, wherein T cells of said population further have reduced or knocked-out expression in at least one of: (i) Rag gene; (ii) CIITA gene; and (iii) beta-2 microglobulin gene; and/or has expression in Foxp3 gene. ### Claim 85 (depends on 80) The population of claim 80, wherein T cells of said population further comprise at least one of: a costimulatory ligand, a suicide gene, an inducible cytokine, an imaging gene, and a combination thereof. ### Claim 86 (depends on 85) The population of claim 85, wherein the costimulatory signal is (i) co-expressed with the CAR as a costimulatory ligand protein; or (ii) is comprised within the CAR protein. ### Claim 87 (depends on 68) The population of claim 68, wherein T cells of said population express the TCR. ### Claim 88 (depends on 68) The population of claim 68, wherein T cells of said population have disrupted or silenced expression of TCR. ### Claim 89 (depends on 1) The population of claim 1, wherein the expressed CAR comprised in the pluripotent stem cell improves T cell differentiation and expansion. ### Claim 90 (depends on 3) The population of claim 3, wherein the CAR encoded by the heterologous nucleic acid sequence comprises an extracellular domain, a transmembrane domain, an intracellular domain, and optionally one or both of (i) a spacer region linking the extracellular domain and the transmembrane domain; and (ii) an inducible promoter; and wherein the extracellular domain comprises an antigen binding region for a predetermined antigen. ### Claim 91 (depends on 90) The population of claim 90, wherein the predetermined antigen is a tumor antigen, a pathogen antigen, or a CD antigen. ### Claim 92 (depends on 90) The population of claim 90, wherein the extracellular domain further comprises a signal peptide. ### Claim 93 (depends on 90) The population of claim 90, wherein the intracellular domain comprises one or more costimulatory receptors. ### Claim 94 (depends on 1) The population of claim 1, wherein T cells of said population are capable of at least one of: (i) in vitro and in vivo expansion; (ii) in vitro and in vivo persistence; (iii) HLA-independent stimulation or activation; (iv) in vitro and in vivo cytotoxicity and cytokine production; and (v) known TCR specificity. ### Claim 95 (depends on 69) The population of claim 69, wherein said isolated T cell is genetically modified. ### Claim 96 (depends on 64) The kit of claim 64, wherein the population of T cells are produced by in vitro differentiation of a pluripotent stem cell, and wherein the pluripotent stem cell is derived from reprogramming an isolated T cell.