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WO2024182539A1 - Procédés pour la culture de cellules reprogrammées - Google Patents

Procédés pour la culture de cellules reprogrammées Download PDF

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Publication number
WO2024182539A1
WO2024182539A1 PCT/US2024/017729 US2024017729W WO2024182539A1 WO 2024182539 A1 WO2024182539 A1 WO 2024182539A1 US 2024017729 W US2024017729 W US 2024017729W WO 2024182539 A1 WO2024182539 A1 WO 2024182539A1
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days
cells
cell
aspects
cancer
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PCT/US2024/017729
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English (en)
Inventor
Jessica Fioravanti
Raul Vizcardo SAKODA
Takuya Maeda
Naritaka Tamaoki
Yin Huang
Yasuhiro Yamazaki
Zheng ZHONG
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Lyell Immunopharma, Inc.
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Publication of WO2024182539A1 publication Critical patent/WO2024182539A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the present disclosure relates to methods of culturing cells, e.g., pluripotent, multipotent, and/or immune cells (e.g., T cells, NK cells, and/or TILs).
  • pluripotent, multipotent, and/or immune cells e.g., T cells, NK cells, and/or TILs.
  • the methods disclosed herein promote the formation of redirected T cells with increased sternness and enhanced target cell killing activity.
  • Cells cultured using the methods disclosed herein can be used for various cell therapies, including but not limited to chimeric antigen receptor (CAR) T cell therapy and TCR T cell therapy including neoantigen directed-T cell therapies or TIL therapies.
  • CAR chimeric antigen receptor
  • T cells also termed T lymphocytes
  • T lymphocytes are a type of blood cell with unique properties that develop from stem cells found in bone marrow; T cells protect against infection and fight cancer.
  • T cells are structurally and functionally different from other cell types such as fibroblasts and undergo a complex development that requires positive and negative selection in the thymus and involves somatic gene rearrangement at the T cell receptor gene loci (see e.g., Kurd and Robey, Immunol Rev. 2016 May; 271(1): 114-126).
  • fibroblasts are also involved in the body’s immune response, fibroblasts are cells that synthesize extracellular matrix and collagen, thereby producing the structural framework for animal tissues. Fibroblasts are primarily involved in the process of wound healing.
  • Tumor reactive T cells in addition to exhibiting characteristics of exhaustion and increased differentiation, are subject to the induction of cell senescence pathways.
  • many groups have pursued methods of cellular dedifferentiation or reprogramming to iPSC as a method of reversing T cell exhaustion, differentiation and senescence.
  • Some aspects of the present disclosure are directed to a method of culturing immune cells, comprising (i) contacting immune cells with a reprogramming factor to generate partially reprogrammed cells; (ii) inducing the partially reprogrammed cells to express at least one T cell marker, increase expression of at least one T cell marker, gain a T cell cell phenotype, and/or become T cell lineage-specific cells (“redirected cells”); and (iii) culturing the redirected cells in a medium comprising a phosphoAKT inhibitor.
  • the phosphoAKT inhibitor is selected from phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 751, ipatasertib, and any combination thereof.
  • the phosphoAKT inhibitor comprises phosphoAKT inhibitor VIII.
  • the medium comprises at least about 0.1 pM to at least about 100 pM of the phosphoAKT inhibitor.
  • the medium comprises at least about 0.1 pM, at least about 0.5 pM, at least about 1.0 pM, at least about 1.5 pM, at least about 2.0 pM, at least about 2.5 pM, at least about 3.0 pM, at least about 3.5 pM, at least about 4.0 pM, at least about 4.5 pM, at least about 5.0 pM, at least about 5.5 pM, at least about 6.0 pM, at least about 6.5 pM, at least about 7.0 pM, at least about 7.5 pM, at least about 8.0 pM, at least about 8.5 pM, at least about 9.0 pM, at least about 9.5 pM, at least about 10 pM, at least about 11 pM, at least about 12 pM, at least about 13 pM, at least about 14 pM, at least about 15 pM, at least about 16 pM, at least about 17 pM, at least about
  • the medium comprises at least about 10 pM to at least about 15 pM of the phosphoAKT inhibitor.
  • the medium comprises at least about 10 pM to at least about 15 pM phosphoAKT inhibitor VIII.
  • the medium comprises about 12 pM phosphoAKT inhibitor VIII.
  • Some aspects of the present disclosure are directed to a method of culturing immune cells, comprising (i) contacting immune cells with a reprogramming factor to generate partially reprogrammed cells; (ii) inducing the partially reprogrammed cells to express at least one T cell marker, increase expression of at least one T cell marker, gain a T cell phenotype, and/or become T cell lineage cells (“redirected cells”); and (iii) culturing the redirected cells in a medium comprising metformin.
  • the medium comprises at least about 1 pM to at least about 1000 pM metformin.
  • the medium comprises at least about 1 pM, at least about 10 pM, at least about 20 pM, at least about 30 pM, at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 110 pM, at least about 120 pM, at least about 130 pM, at least about 140 pM, at least about 150 pM, at least about 160 pM, at least about 170 pM, at least about 180 pM, at least about 190 pM, at least about 200 pM, at least about 210 pM, at least about 220 pM, at least about 230 pM, at least about 240 pM, at least about 250 pM, at least about 300 pM, at least about 350 pM, at least about 400 pM, at least about 450
  • the medium comprises about 50 pM to about 250 pM metformin. [0019] In some aspects, the medium comprises about 50 pM to at least about 150 pM metformin.
  • the medium comprises at least about 100 pM metformin.
  • the medium comprises about 150 pM to at least about 250 pM metformin.
  • the medium comprises about 200 pM metformin.
  • Some aspects of the present disclosure are directed to a method of culturing immune cells, comprising (i) contacting immune cells with a reprogramming factor to generate partially reprogrammed cells; (ii) inducing the partially reprogrammed cells to express at least one T cell marker, increase expression of at least one T cell marker, gain a T cell phenotype, and/or become T cell lineage cells (“redirected cells”); and (iii) culturing the redirected cells in a medium comprising an mTOR inhibitor.
  • the mTOR inhibitor comprises a rapalog.
  • the mTOR inhibitor is selected from rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof.
  • the mTOR inhibitor comprises rapamycin.
  • the medium comprises at least about 0.01 pM to at least about 100 pM of the mTOR inhibitor.
  • the medium comprises at least about 0.01 pM, at least about 0.05 pM, at least about 0.1 pM, at least about 0.2 pM, at least about 0.3 pM, at least about 0.4 pM, at least about 0.5 pM, at least about 0.6 pM, at least about 0.7 pM, at least about 0.8 pM, at least about 0.9 pM, at least about 1.0 pM, at least about 1.1 pM, at least about 1.2 pM, at least about 1.3 pM, at least about 1.4 pM, at least about 1.5 pM, at least about 1.6 pM, at least about 1.7 pM, at least about 1.8 pM, at least about 1.9 pM, at least about 2.0 pM, at least about 2.5 pM, at least about 3.0 pM, at least about 3.5 pM, at least about 4.0 pM, at least about 4.5 pM, at least about 0.01 pM, at least
  • the medium comprises about 1 pM to about 10 pM rapamycin.
  • the medium comprises about 1 pM rapamycin.
  • the medium comprises about 10 pM rapamycin.
  • Some aspects of the present disclosure are directed to a method of culturing immune cells, comprising (i) contacting immune cells with a reprogramming factor to generate partially reprogrammed cells; (ii) inducing the partially reprogrammed cells to express at least one T cell marker, increase expression of at least one T cell marker, gain a T cell phenotype, and/or become T cell lineage cells (“redirected cells”); and (iii) culturing the redirected cells in a medium comprising 2DG.
  • the medium comprises at least about 0.1 nM to at least about 100 nM of 2DG.
  • the medium comprises at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 41 nM, at least about 42 nM, at least about 43 nM, at least about 44 nM, at least about 45 nM, at least about 46 nM, at least about 47 nM, at least about 48 nM, at least about 49 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about 65 nM, at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, at least about 95 nM, or at least about 100 nM 2DG.
  • the medium comprises about 40 mM to about 50 mM 2DG.
  • the medium comprises about 45 nM 2DG.
  • the medium further comprises a concentration of potassium higher than 5 mM.
  • the redirected cells are cultured in the medium for at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, or at least about 30 days. [0040] In some aspects, the redirected cells are cultured in the medium for about 7 days to about 21 days.
  • the redirected cells are cultured in the medium for about 5 to about 15 days, about 5 to about 14 days, about 5 to about 13 days, about 5 to about 12 days, about 5 to about 1 days, about 5 to about 10 days, about 6 to about 14 days, about 7 to about 13 days, about 8 to about 12 days, about 9 to about 11 days, or about 10 to about 11 days.
  • the reprogramming factor comprises a molecule that increases expression of an OCT4 protein, a SOX2 protein, a KLF4 protein, a c-MYC protein, a LIN28 protein, a NANOG protein, or any combination thereof.
  • the reprogramming factor comprises (i) a molecule that increases expression of an OCT4 protein, a molecule that increases expression of a SOX2 protein, a molecule that increases expression of a KLF4 protein, and a molecule that increases expression of a c-MYC protein, and (ii) optionally a molecule that increases expression of a SV40 protein.
  • the reprogramming factor comprises a molecule that increases expression of an OCT4 protein, a molecule that increases expression of a SOX2 protein, and a molecule that increases expression of a KLF4 protein.
  • the reprogramming factor comprises a molecule that increases expression of an OCT4 protein, a molecule that increases expression of a SOX2 protein, a molecule that increases expression of a KLF4 protein, a molecule that increases expression of a c-MYC protein, a molecule that increases expression of a LIN28 protein, and a molecule that increases expression of a NANOG protein.
  • the partially reprogrammed cells are reprogrammed for at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days.
  • the partially reprogrammed cells are reprogrammed between about 1 day to about 14 days, about 1 day to about 13 days, about 1 day to about 12 days, about 1 day to about 11 days, about 1 day to about 10 days, about 1 day to about 9 days, about 1 day to about 8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 5 to about 14 days, about 6 to about 14 days, about 7 to about 14 days, about 5 to about 13 days, about 5 to about 12 days, or about 5 to about 11 days.
  • the partially reprogrammed cells do not express the at least one T cell marker expressed by the redirected T cells.
  • the partially reprogrammed cells do not exhibit the T cell phenotype exhibited by the redirected T cells.
  • the partially reprogrammed cells are not T cell lineage cells or are not conventional T cell lineage cells.
  • the redirected T cells further exhibit an increased expression of an additional T cell marker compared to the reprogrammed cells.
  • the redirected T cells exhibit a decreased expression of one or more stem cell markers compared and/or non-T cell lineage markers to the partially reprogrammed cells.
  • the expression of the T cell marker is measured by Flow cytometry, RNA Seq, CITEseq, PCR, RT-PCR, qPCR or other PCR method, Western blot, ELISA, PCR, MSD, CyTof, immunofluorescent tissue staining, or any combination thereof.
  • the redirected T cells express at least two more, at least three more, at least four more, at least five more, at least six more, at least seven more, at least eight more, at least nine more, or at least ten more T cell markers compared to the partially reprogrammed cells.
  • the redirected T cells express at least two less, at least three less, at least four less, at least five less, at least six less, at least seven less, at least eight less, at least nine less, or at least ten less stem cell markers and/or non-T cell lineage markers compared to the partially reprogrammed cells.
  • the T cell marker comprises one or more of TCRa, TCRb, TCRg, TCRd, CD3, CD3gamma, LCK, CD3zeta, IL2Rgamma, IL2RA, IL2RB, IL7RA, CCR7, CD62L, CD27, CD28, TCF7, CD4, CD5, CD7, CD8, CD45, CD69, LEF1, CXCR4, and GZMB.
  • the partially reprogrammed cells are capable of adhering to a culture vessel surface.
  • the partially reprogrammed cells are not induced pluripotent stem (iPS) cells or totipotent cells.
  • iPS induced pluripotent stem
  • the partial reprogramming comprises contacting original cells obtained from a subject with at least one reprogramming factor for a period of time sufficient for partially reprogrammed cells to form.
  • the original cells are immune cells.
  • the concentration of potassium ion is between about 40 mM and about 50 mM.
  • the concentration of potassium ion is between about 50 mM and about 60 mM.
  • the concentration of potassium ion is about 50 mM, 55 mM, or about 60 mM.
  • the potassium ion comprises potassium chloride.
  • the medium is hypotonic or isotonic.
  • the medium is hypertonic.
  • the medium further comprises a sodium ion.
  • the medium comprises potassium ion at a concentration between
  • the medium further comprises one or more cytokines and/or growth factors.
  • the one or more cytokines comprise Tumor Necrosis Factor alpha (TNFa), Interferon-gamma (IFNy), Fibroblast Growth Factor (FGF), Interleukin-2 (IL-2), Interleukin-7 (IL-2), Interleukin-21 (IL-21), Interleukin- 15 (IL-15), or any combination thereof.
  • TNFa Tumor Necrosis Factor alpha
  • IFNy Interferon-gamma
  • FGF Fibroblast Growth Factor
  • IL-2 Interleukin-2
  • IL-7 Interleukin-7
  • IL-21 Interleukin-21
  • IL-15 Interleukin- 15
  • the one or more cytokines comprise IL-2.
  • the medium is capable of increasing the number of redirected cells and/or maintaining a reduced epigenetic age of the redirected cells compared to the original cells. [0074] In some aspects, following culture in the medium, the redirected cells exhibit increased in vivo persistence as compared to culturing in a control media.
  • the redirected cells exhibit increased in vivo target cell killing as compared to culturing in a control media.
  • the original cells are genetically modified prior to reprogramming.
  • the original cells comprise (i) a chimeric antigen receptor (CAR) or an engineered T cell receptor (eTCR), (ii) a nucleic acid molecule encoding a CAR or an eTCR, or (iii) any combination thereof.
  • CAR chimeric antigen receptor
  • eTCR engineered T cell receptor
  • nucleic acid molecule encoding a CAR or an eTCR
  • the partially reprogrammed cells are further genetically modified before the culturing.
  • the partially reprogrammed cells are genetically modified to comprise a nucleic acid molecule encoding a CAR or an eTCR.
  • the redirected cells are further genetically modified before the culturing.
  • the redirected cells are further genetically modified after the culturing.
  • the redirected cells are genetically modified to comprise a nucleic acid molecule encoding a CAR or an eTCR.
  • the CAR or the eTCR comprises an antigen-binding domain that specifically binds a tumor antigen.
  • the CAR or the eTCR comprises an antigen-binding domain that specifically binds an antigen selected from AFP, CD19, TRAC, TCRP, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL- 13Ra2, mesothelin, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, L
  • the redirected cells are cultured in the medium comprising 2DG for about 1 day to about 14 days.
  • the redirected cells are cultured in the medium comprising 2DG for about 1 day to about 13 days, about 1 day to about 12 days, about 1 day to about 11 days, about 1 day to about 10 days, about 1 day to about 9 days, about 1 day to about 8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 14 days, about 2 days to about 13 days, about 2 days to about 12 days, about 2 days to about 11 days, about 2 days to about 10 days, about 2 days to about 9 days, about 2 days to about 8 days, about 2 days to about 7 days, about 2 days to about 6 days, about 2 days to about 5 days, about 2 days to about 4 days, about 2 days to about 3 days, about 3 days to about 14
  • 5 days to about 12 days about 5 days to about 11 days, about 5 days to about 10 days, about 5 days to about 9 days, about 5 days to about 8 days, about 5 days to about 7 days, about 5 days to about
  • the redirected cells are cultured in the medium comprising 2DG for about 7 days to about 14 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 1 day to about 7 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 4 days to about 10 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 5 days to about 9 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 6 days to about 8 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 6 days to about 7 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 7 days to about 8 days.
  • the redirected cells are cultured in the medium comprising 2DG for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days.
  • the redirected cells are cultured in the medium comprising 2DG for about 7 days.
  • the medium comprises about 4 mM 2DG.
  • the redirected cells are administered to a human subject following the culturing.
  • Some aspects of the present disclosure are directed to a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a redirected cell prepared according to a method disclosed herein.
  • Some aspects of the present disclosure are directed to a population of redirected cells prepared by a method disclosed herein.
  • Some aspects of the present disclosure are directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a population of redirected cells disclosed herein and a pharmaceutically acceptable carrier.
  • Some aspects of the present disclosure are directed to a method of treating a disease or a condition in a subject in need thereof comprising administering to the subject a population of cells disclosed herein or a pharmaceutical composition disclosed herein.
  • the disease or condition comprises a cancer, a viral condition or an automimmune disorder.
  • the cancer comprises acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, head and neck cancers (e.g., cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity), cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma
  • the cancer is locally advanced or metastatic.
  • the cancer is relapsed or refractory.
  • FIGs. 1 A-1C show the CD 19-C AR transduction rates in TCT and TRJ and the in vitro functionality of these cells. Representative flow cytometry plots of CD 19-C AR transduction efficiency in TCT and TRJ CD8 + T cells on day 19 are shown. Cells were gated as: Single cells > Lymphocytes > Live cells CD3 + > CD8 + CD4 > CD19 CAR idiotype (FIGs. 1A-1B).
  • FIGs. 2A-2D show that CD19-CAR 4 TRJ cells have less mitochondrial membrane potential after specific antigen recognition.
  • day 22 day 22
  • day 25 day 25
  • cells were stained with 20uM TMRM.
  • Representative histogram flow cytometry plots of TCT (filled black) or TRJ (filled white) (FIG 2A and 2C) or bar graph quantification of mean fluorescence intensity (MFI) of TMRM n 3 donors (FIGs 2B and 2D).
  • MFI mean fluorescence intensity
  • FIGs. 3A-3D show that CD19-CAR 4 TRJ cells have lower expression of Glucose Transporter 1 (Glutl) and similar expression of carnitine palmitoyltransferase 1A (CPTla).
  • Representative flow cytometry plots of Glutl and CPTla expression in TCT and TRJ cells (FIGs. 3A-3B, respectively).
  • CD3 > CD8 4 CD19 CAR idiotype 4 Data in bar graph are shown as the mean ⁇ s.e.m; each symbol represents individual donor. P* ⁇ 0.02 (unpaired t test).
  • FIG. 4 shows that pretreatment of CD19-CAR 4 TRJ with selected small molecules increases expression of CCR7.
  • Data in bar graph are shown as the mean ⁇ s.e.m; each symbol represents technical duplicate.
  • FIG. 5 shows the effect of pretreatment with AKT inhibitor VIII (12mM) of CD19- CAR + Tcrand TRJ.
  • TCT and TRJ control or partially reprogramed CD19-CAR samples
  • TCM tumor necrosis factor
  • FIG. 6 shows the effect of pretreatment with a low dose of Rapamycin (luM) or a high dose of Rapamycin (lOuM) of CD19-CAR Tcr and TRJ.
  • FIG. 7 shows the effect of pretreatment with a low dose of Metformin (lOOuM) or a high dose of Metformin (200uM) of CD19-CAR Tcr and TRJ.
  • TCT and TRJ control or partially reprogramed CD19-CAR samples
  • TCM tumor necrosis factor
  • FIGs. 8A-8B shows the effect of pretreatment with AKT inhibitor VIII (12mM), low dose of Rapamycin (luM), high dose of Rapamycin (lOuM), low dose of Metformin (lOOuM) or a high dose of Metformin (200uM) of CD19-CAR + TCT and TRJ after specific antigen recognition.
  • FIGs. 9A-9B shows the effect of pretreatment with AKT inhibitor VIII (12mM), low dose of Rapamycin (luM), high dose of Rapamycin (lOuM), low dose of Metformin (lOOuM) or a high dose of Metformin (200uM) of CD19-CAR 4 TCT and TRJ after specific antigen recognition.
  • day 35 cells were stained for surface phenotype markers CCR7 (FIG. 9A) or CD62L (FIG. 9B) percentage expression.
  • Floating bar graphs of TCT (filled black) or TRJ (filled white) showing median values of triplicate wells. Data shown after gating on Single cells > Lymphocytes > Live cells CD3 + > CD8 + CD19 CAR idiotype
  • FIG. 10 shows that pretreatment of NY-ESO-1 TRJ with selected small molecules increases expression of CCR7.
  • Percentage of CCR7 4 cells in NY-ESO-1 4 (TCT, black patterns or TRJ, white patterns) after pretreatment with AKT inhibitor VIII (12uM), Rapamycin (Rapa luM), Metformin (lOOuM), Deoxy-Glucose (2-DG, 45nM) or left untreated (TCM) by exchanging every other day the compounds from day 8 to day 18, previous to perform sequential killing assay (FIG. 10).
  • N 2 independent donors. Data in bar graph are shown as the mean ⁇ s.e.m; each symbol represents technical duplicate.
  • FIGs. 11A-11B show the effect of pretreatment with AKT inhibitor VIII (12mM) of NY-ESO-1 4 TCT and TRJ.
  • AKT inhibitor VIII (12mM) of NY-ESO-1 4 TCT and TRJ.
  • TCT and TRJ control or partially reprogramed NY-ESO-1 samples from two independent donors
  • FIGs. 12A-12B show the effect of pretreatment with Rapamycin (luM) of NY- ESO-1 + Tcr and TRJ.
  • Rapamycin lactamycin
  • TCT and TRJ control or partially reprogramed NY-ESO-1 samples from two independent donors
  • FIGs. 13A-13B show the effect of pretreatment with Metformin (lOOuM) of NY- ESO-1 4 Tcr and TRJ.
  • Metformin lactin
  • FIGs. 14A-14B show the effect of pretreatment with 2-DG (45nM) of NY-ESO-1 4 Tcr and TRJ.
  • 2-DG 45nM
  • TCT and TRJ control or partially reprogramed NY-ESO-1 samples from two independent donors
  • FIGs. 15A-15D show the differences between NY-ESO-1 Tcrand TRJ after second round of sequential killing assay in terms of %CD3 (FIG. 15 A), %CCR7 (FIG. 15B), MFI Glutl (FIG. 15C), and MFI of TMRM (FIG. 15D).
  • %CD3 FIG. 15 A
  • %CCR7 FIGG. 15B
  • MFI Glutl FIG. 15C
  • MFIG. 15D MFIG. 15D
  • TMRM MFIG. 15D
  • FIGs. 16A-16F show the metabolic differences at day 13 (FIGs. 16A-16F) between CD4 + and CD8 + TCT and TRJ.
  • CD4 + or CD8 + cells from 3 independent donors were thawed (day -2), and stimulated with TransAct 1/500 for 1 day (day -1) in TCM + 60 lU/ml IL2.
  • days were infected with SeV (4F + SV40), transferred to iMatrix coated plates on dayl, in iPSC conditions.
  • TRJ detached cells, or TCT cells were activated with TransAct 1/500, and grown TCM + IL2.
  • FIGs. 17A-17C show the CD4/CD8 ratio of TCT, TRJ, and TRJ+2DG on Day 28 of the partial reprogramming and redirection process.
  • TRJ+2DG was treated with 4mM 2DG from Day 8 to Day 21 of the process.
  • Representative flow cytometry plots of CD4 + and CD8 + in TCT, TRJ, and TRJ+2DG on day 28 are shown.
  • the CD4 + percentage represents the percentage of CD4 T helper cell and the CD8 + percentage represents the percentage of CD8 cytotoxic T cells.
  • Cells were gated as: Lymphocytes > Single cells > Live cells > CD3 + (FIGs. 17A-17C).
  • FIGs. 18A-18C show the expression of CCR7 and CD45RO of TCT, TRJ, and TRj+2DG on Day 28 of the partial reprogramming and redirection process.
  • Representative flow cytometry plots of CCR7 + CD45RO + in TCT, TRJ, and TRJ+2DG on Day 28 are shown.
  • the percentage of CCR7 + CD45RO + represents the percentage of putative central memory T cells (Tcm).
  • Cells were gated as: Lymphocytes > Single cells > Live cells > CD3 + (FIGs. 18A-18C).
  • FIG. 19 shows the tumor cell killing of TCT, TRJ, and TRJ+2DG in a sequential killing assay.
  • the T cells (TCT, TRJ, and TRJ+2DG) and A375-NLR-CD3svFc were cultured in a E:T ratio 2: 1 (4e5 T cells and 2e5 A375-NLR-CD3svFc cells), in the presence of RPMI 1640 with 10% FBS and 1% pen-strep for 4 sequential rounds, by adding to the next culture a 1 :4 of the previous coculture to a new plate seeded with fresh 2e5 A375-NLR-CD3svFc cells sequentially every 3-4 days.
  • NLR NucLight Red.
  • Crossed diamond Blank sample containing only the cancer cells; open diamond curve: TCT; open circles: TRJ; and filled diamond: TRJ+2DG.
  • FIGs. 20A-20B show the CD4/CD8 ratio of TRJ and TRJ+2DG on Day 19 of the partial reprogramming and redirection process.
  • TRJ+2DG was treated with 4mM 2DG from Day 8 to Day 14.
  • Representative flow cytometry plots of CD4 + and CD8 + in TRJ and TRJ+2DG on Day 19 are shown.
  • Cells were gated as: Lymphocytes > Single cells > Live cells > CD3 + (FIGs. 17A-17C).
  • FIGs. 21 A-21B show the expression of CCR7 and CD45RO of TRJ and TRJ+2DG on Day 19 of the partial reprogramming and redirection process. Representative flow cytometry plots of CCR7 + CD45RO + in TRJ and TRJ+2DG on Day 19 are shown. Cells were gated as: Lymphocytes > Single cells > Live cells > CD3 + (FIGs. 21A-21B).
  • Some aspects of the present disclosure are directed to methods of culturing immune cells, comprising (i) contacting immune cells with a reprogramming factor to generate partially reprogrammed cells; (ii) inducing the partially reprogrammed cells to express at least one T cell marker, increase expression of at least one T cell marker, gain a T cell cell phenotype, and/or become T cell lineage-specific cells (“redirected cells”); and (iii) culturing the redirected cells in a medium comprising an agent that increases the sternness of the redirected cells and/or increases the cell killing ability of the redirected cells.
  • the cell culturing methods of the present disclosure are capable of promoting redirection of reprogrammed cells to a T cell lineage, wherein the resulting T cells have stem-like properties while maintaining or having improved cell killing actitivity.
  • the culturing methods are capable of increasing in vivo viability, in vivo persistence, in vivo effector function, or any combination thereof.
  • the agent that increases the sternness of the redirected cells and/or increases the cell killing ability of the redirected cells comprises a phospho AKT inhibitor.
  • the agent that increases the sternness of the redirected cells and/or increases the cell killing ability of the redirected cells comprises metformin.
  • the agent that increases the sternness of the redirected cells and/or increases the cell killing ability of the redirected cells comprises an mTOR inhibitor.
  • Some aspects of the present disclosure are directed to methods of culturing immune cells, comprising (i) contacting immune cells with a reprogramming factor to generate partially reprogrammed cells; (ii) inducing the partially reprogrammed cells to express at least one T cell marker, increase expression of at least one T cell marker, gain a T cell cell phenotype, and/or become T cell lineage-specific cells (“redirected cells”); and (iii) culturing the redirected cells in a medium comprising a phosphoAKT inhibitor.
  • Some aspects of the present disclosure are directed to methods of culturing immune cells, comprising (i) contacting immune cells with a reprogramming factor to generate partially reprogrammed cells; (ii) inducing the partially reprogrammed cells to express at least one T cell marker, increase expression of at least one T cell marker, gain a T cell phenotype, and/or become T cell lineage cells (“redirected cells”); and (iii) culturing the redirected cells in a medium comprising metformin.
  • Some aspects of the present disclosure are directed to methods of culturing immune cells, comprising (i) contacting immune cells with a reprogramming factor to generate partially reprogrammed cells; (ii) inducing the partially reprogrammed cells to express at least one T cell marker, increase expression of at least one T cell marker, gain a T cell phenotype, and/or become T cell lineage cells (“redirected cells”); and (iii) culturing the redirected cells in a medium comprising an mTOR inhibitor.
  • Some aspects of the present disclosure are directed to methods of culturing immune cells, comprising (i) contacting immune cells with a reprogramming factor to generate partially reprogrammed cells; (ii) inducing the partially reprogrammed cells to express at least one T cell marker, increase expression of at least one T cell marker, gain a T cell phenotype, and/or become T cell lineage cells (“redirected cells”); and (iii) culturing the redirected cells in a medium comprising 2DG.
  • a or “an” entity refers to one or more of that entity; for example, "a chimeric polypeptide,” is understood to represent one or more chimeric polypeptides.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • “or” is used mean an open list of the components in the list. For example, “wherein X comprises A or B” means X comprises A, X comprises B, X comprises A and B, or X comprises A or B and any other components.
  • the terms "about” or “comprising essentially of' refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “comprising essentially of' can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, “about” or “comprising essentially of' can mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about” or “comprising essentially of' should be assumed to be within an acceptable error range for that particular value or composition.
  • the term "AKT inhibitor” or “AKTi” refers to any agent that blocks, reduces, or otherwise inhibits that activity, expression, or downstream signaling of AKT.
  • phosphoAKT inhibitor refers to an agent that specifically inhibits phosphorylated AKT.
  • AKT is a serine/threonine kinase that is a key component of the PI3K/AKT/mTOR signaling pathway. AKT exerts a pivotal role in cell growth, proliferation, survival, and metabolism.
  • Activated AKT phosphorylates its downstream targets, including tuberos sclerosis complex 2 (TSC2), glycogen synthase kinase-3p (GSK3P), and the forkhead kinase transcription factors (FOXO), eventually promoting cell proliferation, metabolism, and survival.
  • TSC2 tuberos sclerosis complex 2
  • GSK3P glycogen synthase kinase-3p
  • FOXO forkhead kinase transcription factors
  • Any phosphoAKT inhibitor can be used in the methods disclosed herein.
  • the phosphoAKT inhibitor comprises phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 751, ipatasertib, and any combination thereof.
  • the term “metformin” refers to a biguanide drug that has historically been used in the treatment of diabetes, which has been shown to be effective as an antiinflammatory, anti-oxidative, and anti-tumor agent.
  • mTOR inhihibitor refers to any agent that blocks, reduces, or otherwise inhibits that activity, expression, or downstream signaling of mTOR.
  • mTOR or mammalian target of rapamycin
  • mTOR As a core component of mT0RC2, mTOR also functions as a tyrosine protein kinase that promotes the activation of insulin receptors and insulin-like growth factor 1 receptors. mT0RC2 has also been implicated in the control and maintenance of the actin cytoskeleton.
  • the mTOR inhibitor comprises rapamycin.
  • 2DG or "2-deoxy-D-glucose” refers to a chemical compoung comprising glucose, wherein the 2-hydroxyl group of the glucose is replaced by a hrdrogen. Unlike glucose, 2DG is not able to enter glycolysis and contribute to ATP production. 2DG is known to interrupt glycolysis, reduce glycosylation, and inhibit the pentose phosphate pathway.
  • the term “approximately,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some aspects, the term “approximately” refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • day in the context of cell culture methods refers to a time period of about twenty-four hours (i.e., 1 day) before or after a cell culture is contacted with one or more reprogramming factors.
  • reprogramming factors may include, but are not limited to, OCT4, SOX2, KLF4, C-MYC, LIN28, NANOG and SV40 Large T cell antigen.
  • the day of contacting the culture with the reprogramming factors is referred to as day 0.
  • day 1 represents any time between about twenty-four and about forty-eight hours after the cell culture had been contacted by a reprogramming factor.
  • cell engineering refers to the targeted modification of a cell, e.g., a pluripotent cell, a multipotent cell, or an immune cell disclosed herein.
  • the cell engineering comprises viral genetic engineering, non-viral genetic engineering, introduction of receptors to allow for tumor specific targeting (e.g., a TCR, TCRm, and/or a CAR), introduction of one or more endogenous genes that improve T cell function, introduction of one or more synthetic genes that improve T cell function, or any combination thereof.
  • Expansion refers to the process of stimulating or activating the cells and culturing the cells.
  • the expansion process can lead to an increase in the proportion or the total number of desired cells, e.g., an increase in the proportion or total number of redirected T cells, in a population of cultured cells, after the cells are reprogrammed, redirected, and cultured.
  • Expansion does not require that all cell types in a population of cultured cells are increased in number. Rather, in some aspects, only a subset of cells in a population of cultured cells are increased in number during expansion, while the number of other cell types may not change or may decrease.
  • stem cell refers to a cell that retains the ability to renew itself through mitotic cell division and that can differentiate into a diverse range of specialized cell types.
  • Mammalian stem cells can be divided into three broad categories: embryonic stem cells, which are derived from blastocysts, adult stem cells, which are found in adult tissues, and cord blood stem cells, which are found in the umbilical cord. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body by replenishing specialized cells. Totipotent stem cells are produced from the fusion of an egg and sperm cell.
  • Induced pluripotent stem cells are a type of pluripotent stem cell derived from adult cells that have been reprogrammed into an embryonic-like pluripotent state. Induced pluripotent stem cells can be derived, for example, from adult somatic cells such as skin or blood cells.
  • immune cell(s) denotes any type of immune cell, including for example T cells, B cells, monocytes, macrophages, dendritic cells, and the like. In some exemplary aspects, immune cells disclosed herein are T cells.
  • polynucleotide”, “nucleotide”, or “nucleic acid” includes both singlestranded and double-stranded nucleotide polymers.
  • the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
  • Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2’, 3 ’-dideoxyribose, and intemucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphoro-diselenoate, phosphoro- anilothioate, phoshoraniladate and phosphoroamidate.
  • base modifications such as bromouridine and inosine derivatives
  • ribose modifications such as 2’, 3 ’-dideoxyribose
  • intemucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphoro-diselenoate, phosphoro- anilothioate, phoshoraniladate and phosphoroamidate.
  • oligonucleotide refers to a polynucleotide comprising 200 or fewer nucleotides. Oligonucleotides can be single stranded or double stranded, e.g., for use in the construction of a mutant gene. Oligonucleotides can be sense or antisense oligonucleotides. An oligonucleotide can include a label, including a radiolabel, a fluorescent label, a hapten or an antigenic label, for detection assays. Oligonucleotides can be used, for example, as PCR primers, cloning primers or hybridization probes.
  • operably linked means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions.
  • vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus) used to transfer protein coding information into a host cell.
  • expression vector or “expression construct” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control (in conjunction with the host cell) expression of one or more heterologous coding regions operatively linked thereto.
  • An expression construct can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of a coding region operably linked thereto.
  • the term “host cell” refers to a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest.
  • the term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.
  • transformation refers to a change in a cell’s genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA or RNA.
  • a cell is transformed where it is genetically modified from its native state by introducing new genetic material via transfection, transduction, delivery using nanoparticles (e.g., lipid nanoparticles), or other techniques.
  • the transforming DNA can recombine with that of the cell by physically integrating into a chromosome of the cell, or can be maintained transiently as an episomal element without being replicated, or can replicate independently as a plasmid.
  • a cell is considered to have been “stably transformed” when the transforming DNA is replicated with the division of the cell.
  • transfection refers to the uptake of foreign or exogenous genetic material (DNA or RNA) by a cell.
  • a number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology, 1973, 52:456; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2001, supra; Davis et al., Basic Methods in Molecular Biology, 1986, Elsevier; Chu et al., 1981, Gene, 13: 197.
  • transduction refers to the process whereby foreign DNA or RNA is introduced into a cell via viral vector. See, e.g., Jones et al., Genetics: Principles and Analysis, 1998, Boston: Jones & Bartlett Publ.
  • polypeptide or “protein” refer to a macromolecule having the amino acid sequence of a protein, including deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence.
  • polypeptide and protein specifically encompass antigen-binding molecules, antibodies, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of antigen-binding protein.
  • polypeptide fragment refers to a polypeptide that has an amino-terminal deletion, a carboxyl- terminal deletion, and/or an internal deletion as compared with the full-length native protein. Such fragments can also contain modified amino acids as compared with the native protein.
  • Useful polypeptide fragments include immunologically functional fragments of antigen-binding molecules.
  • isolated means (i) free of at least some other proteins with which it would normally be found, (ii) is essentially free of other proteins from the same source, e.g., from the same species, (iii) separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (iv) operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (v) does not occur in nature.
  • terapéuticaally effective amount refers to the amount of immune cells (e.g., T cells) or other therapeutic agent determined to produce a therapeutic response in a mammal. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art.
  • patient and “subject” are used interchangeably and include human and non-human animal subjects as well as those with formally diagnosed disorders, those without formally recognized disorders, those receiving medical attention, those at risk of developing the disorders, etc.
  • treat and “treatment” includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses aspects in which one reduces symptoms or underlying risk factors.
  • prevent does not require the 100% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound or method.
  • Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
  • the term “substantially” or “essentially” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the terms “essentially the same” or “substantially the same” refer to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is about the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the terms “substantially free of’ and “essentially free of’ are used interchangeably, and when used to describe a composition, such as a cell population or culture media, refer to a composition that is free of a specified substance, such as, 95% free, 96% free, 97% free, 98% free, 99% free of the specified substance, or is undetectable as measured by conventional means. Similar meaning can be applied to the term “absence of,” where referring to the absence of a particular substance or component of a composition.
  • the term “appreciable” refers to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length or an event that is readily detectable by one or more standard methods.
  • the terms “not-appreciable” and “not appreciable” and equivalents refer to a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length or an event that is not readily detectable or undetectable by standard methods.
  • an event is not appreciable if it occurs less than 5%, 4%, 3%, 2%, 1%, 0.1%, 0.001%, or less of the time.
  • association with denotes a relationship between two events, entities and/or phenomena. Two events, entities and/or phenomena are “associated” with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other.
  • memory T cells refers to T cells that have previously encountered and responded to their cognate antigen (e.g., in vivo, in vitro, or ex vivo) or which have been stimulated, e.g., with an anti-CD3 antibody (e.g., in vitro or ex vivo).
  • Immune cells having a "memory-like" phenotype upon secondary exposure, such memory T cells can reproduce to mount a faster and stronger immune response than during the primary exposure.
  • memory T cells comprise central memory T cells (TCM cells), effector memory T cells (TEM cells), tissue resident memory T cells (TRM cells), stem cell-like memory T cells (TSCM cells), or any combination thereof.
  • central memory T cells or "TCM cells” refers to memory T cells that express CD45RO, CCR7, and CD62L.
  • effector-like refers to tumor cell killing capacity and cytokine polyfunctionality, e.g., ability of a cell to produce inflammatory cytokines and/or cytotoxic molecules.
  • an effector-like cell can be measured by specific markers expressed by the cell.
  • those effector-like markers can be one or more of pSTAT5+, STAT5+, pSTAT3+, and STAT3+.
  • the effector-like marker comprises a STAT target selected from the group consisting of AKT1, AKT2, AKT3, BCL2L1, CBL, CBLB, CBLC, CCND1, CCND2, CCND3, CISH, CLCF1, CNTF, CNTFR, CREBBP, CRLF2, CSF2, CSF2RA, CSF2RB, CSF3, CSF3R, CSH1, CTF1, EP300, EPO, EPOR, GH1, GH2, GHR, GRB2, IFNA1, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA2, IFNA21, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNAR1, IFNAR2, IFNB1, IFNE, IFNG, IFNGR1, IFNGR2, IFNK, IFNL1, IFNL2, IFNL3, IFNLR1, IFNW1, IL10, IL10RA, IL10RB
  • the effector-like cells can be identified by a transcriptome analysis.
  • the effector-like marker comprises a marker disclosed in Kaech et al., Cell 777:837-51 (2002); Tripathi et al., J. Immunology 755:2116-24 (2010); and/or Johnnidis et al., Science Im.
  • tissue resident memory T cells refers to memory T cells that do not circulate and remain resident in peripheral tissues, such as skin, lung, and gastrointestinal tract. In some aspects, tissue resident memory T cells are also effector memory T cells.
  • TN cells refers to T cells that express CD45RA, CCR7, and CD62L, but which do not express CD95.
  • TN cells represent the most undifferentiated cell in the conventional T cell lineage. The interaction between a TN cell and an antigen presenting cell (APC) induces differentiation of the TN cell towards an activated TEFF cell and an immune response.
  • APC antigen presenting cell
  • sternness refers to a cell (e.g., a T cell, an NK cell, or a TIL) that expresses markers consistent with a more naive phenotype.
  • a less differentiated T cell can express one or more marker characteristic of a naive T (TN) cell or a stem cell memory T (TSCM) cell.
  • TN naive T
  • TSCM stem cell memory T
  • a “less-differentiated” or “stem-like” T cell expresses CD45RA, CCR7, and CD62L.
  • a "less-differentiated” or “stem-like” T cell expresses CD45RA, CCR7, CD62L, and TCF7. In some aspect, a "less-differentiated” or “stem-like” T cell does not express CD45RO or is CD45ROiow.
  • the methods disclosed herein promote immune cells (e.g., T cells) having a less-differentiated phenotype. Without being bound by any particular mechanism, in some aspects, the methods disclosed herein block, inhibit, or limit differentiation of less-differentiated immune cells (e.g., T cells), resulting in an increased number of stem-like cells in culture.
  • Sternness is characterized by the capacity to self-renew, the multipotency, and the persistence of proliferative potential.
  • sternness is characterized by a particular gene signature, e.g., a combined pattern of expression across a multitude of genes.
  • the stem-like cells can be identified by a transcriptome analysis, e.g., using sternness gene signatures disclosed herein.
  • the gene signature comprises one or more genes selected from ACTN1, DSC1, TSHZ2, MYB, LEF1, TIMD4, MAL, KRT73, SESN3, CDCA7L, LOC283174, TCF7, SLC16A10, LASS6, UBE2E2, IL7R, GCNT4, TAF4B, SULT1B1, SELP, KRT72, STXBP1, TCEA3, FCGBP, CXCR5, GPA33, NELL2, APBA2, SELL, VIPR1, FAM153B, PPFIBP2, FCER1G, GJB6, 0CM2, GCET2, LRRN1, IL6ST, LRRC16A, IGSF9B, EFHA2, LOC129293, APP, PKIA, ZC3H12D, CHMP7, KIAA0748, SLC22A17, FLJ13197, NRCAM, C5orfl3, GIPC3, WNT7A, FAM117B, BEND5, L
  • the gene signature comprises one or more gene selected from NOG, TIMD4, MYB, UBE2E2, FCER1G, HAVCR1, FCGBP, PPFIBP2, TPST1, ACTN1, IGF1R, KRT72, SLC16A10, GJB6, LRRN1, PRAGMIN, GIPC3, FLNB, ARRB1, SLC7A8, NUCB2, LRRC7, MYO15B, MAL, AEBP1, SDK2, BZW2, GAL3ST4, PITPNM2, ZNF496, FAM117B, C16orf74, TDRD6, TSPAN32, C18orf22, C3orf44, LOC129293, ZC3H12D, MLXIP, C7orfl0, STXBP1, KCNQ1, FLJ13197, LDLRAP1, RAB43, RIN3, SLC22A17, AGBL3, TCEA3, NCRNA00185, FAM153B, FAM153C, VIPR1, MMP
  • nuclear cell refers to a cell found in blood that has a single, round nucleus.
  • totipotent refers to the ability of a cell to give rise to any cell type found in an embryo as well as extra-embryonic (placenta) cells.
  • pluripotent refers to the ability of a cell to form all lineages of the body or soma (i.e., the embryo proper but not the placenta).
  • an embryonic stem cell is a type of pluripotent stem cell that is available to form cells from each of the three germ layers: the ectoderm, the mesoderm and the endoderm. Pluripotency can be determined in part, by assessing the pluripotency characteristics of the cells.
  • Pluripotency characteristics may include, but are not limited to: (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal; (iii) ability to differentiate to all three somatic lineages (ectoderm, mesoderm and endoderm); (iv) teratoma formation consisting of the three somatic lineages; and (v) formation of embryoid bodies consisting of cells from the three somatic lineages; (vi) expression of one or more pluripotent stem cell markers including, but not limited to SSEA1 (mouse only), SSEA3/4, SSEA5, TRA1-60/81, TRA2-54 (ALP), TRA1-85, GCTM-2, TG343, TRA2-49, CD340, CD326, Podoplanin, and TG30 (CD9); (vii) expression of certain other markers associated with somatic stem cells or early differentiated cells from embryonic stem cells, including, but not limited to, integrin a6pi, CD29, CD133/promin
  • multipotenf refers to the ability of a cell to develop into a limited number of cell types in a particular lineage.
  • non-pluripotent cell refers to any cell that does not possess full pluripotency, such as incompletely or partially pluripotent stem cells, multipotent cells, oligopotent cells, unipotent cells (e.g., progenitor cells), and terminally differentiated cells.
  • introducing refers to a process that comprises contacting a cell with a polynucleotide, polypeptide, or small molecule.
  • An introducing step may also comprise microinjection of polynucleotides or polypeptides into the cell, use of liposomes to deliver polynucleotides or polypeptides into the cell, or fusion of polynucleotides or polypeptides to cell permeable moi eties to introduce them into a cell.
  • An aspect of the disclosure provides a method of preparing an isolated or purified population of immune cells (e.g., T cells) in vitro.
  • the isolated or purified population of immune cells e.g., T cells
  • “Differentiation” is the process by which a cell loses its potency and capacity for self-renewal and ultimately becomes a mature and discrete cell type within a discrete lineage (see e.g., Crompton 2014, Trends in Immunol. 35: 178-185).
  • Dedifferentiation refers to a process by which cells become less specialized.
  • dedifferentiation is the loss of specialization characteristics that are within the normal development path or within the same cell lineage.
  • the dedifferentiation is within the same cell lineage (hierarchical dedifferentiation).
  • dedifferentiation may not be within the same cell lineage.
  • partial reprogramming of T cells as described herein results in cells that dedifferentiate by gaining expression of CD9 and/or CD90 and/or SSEA4, markers not usually associated with the conventional T cell lineage in humans, and which cells begin to lose expression of T cell markers CD3 and/or CD4 and/or CD8.
  • Such dedifferentiation is not along the standard T cell hierarchical lineage. These cells can return to conventional T cells following stimulation with T cell activation agents. CD90 expression is only associated with rare populations of human T cells such as cortical thymocytes and a subset of Thl7.
  • "Rejuvenation" is a process in which cellular function lost by cell aging is restored. Cell age can be described as the sum of the chronological age of the donor plus an alpha factor determined by the combination of several factors inducing stress in the cell microenvironment. These factors can be observed in vivo (e.g. heavy smoking, chronic illness including autoimmune disease, infection, obesity, metabolic problems and possibly depression) and might be artificially induced in vitro (e.g.
  • epigenetic age means the age of a cell as determined using known epigenetic clocks, e.g., the Horvath Clock, the Hannum Clock or the Levine Clock (see, e.g., Horvath et al., 2018, Aging 10, 1758-1775; Hannum et al., 2013, Mol. Cell. 49(2), 359- 367; Levine et al., 2018, Aging, 10(4): 573-592).
  • the eAge is the age of a cell as determined using the Horvath Clock method of measuring methylation status at 353 CpGs (Horvath and Raj, 2018 Nature Reviews Genetics, 19:371-385).
  • T cells that are differentiated from iPS cells in absence of an autologous thymic education, lack true T cell function and possess abnormalities including immature phenotype, non MHC dependent killing, improper CD8aP dimerization, dysregulation of gene expression and failure to produce a developmentally homogeneous population of T cells.
  • the reprogrammed cells generated using the methods described herein provide unexpected and advantageous properties over T cells generated using methods known in the art.
  • high potassium media or its abbreviation “HPM” refers to media comprising a higher concentration of potassium ion than a conventional medium (e.g., greater than about 5 mM potassium ion). In some aspects, the high potassium media is not hypertonic.
  • the high potassium media can be any media supplemented with potassium or any additional elements disclosed herein, e.g. sodium, glucose, calcium, and/or cytokines.
  • the media can be any media for culturing immune cells, e.g., T cells, NK cells, and/or TILs.
  • the basal media is selected from a balanced salt solution (e.g., PBS, DPBS, HBSS, EBSS), Dulbecco's Modified Eagle's Medium (DMEM), Click’s medium, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), F-10, F-12, RPMI 1640, Glasgow Minimal Essential Medium (GMEM), alpha Minimal Essential Medium (alpha MEM), Iscove's Modified Dulbecco's Medium (IMDM), M199, OPTMIZERTM CTSTM T-Cell Expansion Basal Medium (ThermoFisher), OPTMIZERTM Complete, IMMUNOCULTTM XF (STEMCELLTM Technologies), IMMUNOCULTTM XF, AIM V, TEXMACSTM medium, TRANSACTTM TIL expansion medium, TIL rapid expansion protocol medium, and any combination thereof.
  • a balanced salt solution e.g., PBS, DPBS, HBSS, EBSS
  • DMEM
  • the basal medium is serum free.
  • the basal medium further comprises immune cell serum replacement (ICSR).
  • ICSR immune cell serum replacement
  • the basal medium comprises OPTMIZERTM Complete supplemented with ICSR, AIM V supplemented with ICSR, IMMUNOCULTTM XF supplemented with ICSR, RPMI supplemented with ICSR, TEXMACSTM supplemented with ICSR, or any combination thereof.
  • suitable basal media include Click's medium, OPTIMIZER® (CTS®) medium, STEMLINE® T cell expansion medium (Sigma-Aldrich), AIM V® medium (CTS®), TEXMACS® medium (Miltenyi Biotech), IMMUNOCULT® medium (Stem Cell Technologies), PRIME-XV® T-Cell Expansion XSFM (Irvine Scientific), Iscoves medium, and/or RPMI-1640 medium.
  • the basal media comprises NaCl free CTSTM OpTimizerTM.
  • cytokine refers to small, secreted proteins released by cells that have a specific effect on the interactions and communications between cells.
  • Non-limiting examples of cytokines include interleukins (e.g., interleukin (IL)-l, IL-2, IL-4, IL-7, IL-9, IL- 13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-10, IL-20, IL-14, IL-16, IL-17, IL-21 and IL-23), interferons (IFN; e.g., IFN-a, IFN-P, and IFN-y), tumor necrosis factor (TNF) family members, and transforming growth factor (TGF) family members.
  • IFN interferons
  • TGF tumor necrosis factor
  • cytokine is an interleukin.
  • the cytokine is selected from IL-2, IL-7, IL-15, IL-21 and any combination thereof.
  • IL-2 (UniProtKB - P60568) is produced by T cells in response to antigenic or mitogenic stimulation. IL-2 is known to stimulate T cell proliferation and other activities crucial to regulation of the immune response.
  • IL-7 (UniProtKB - Pl 3232) is a hematopoietic growth factor capable of stimulating the proliferation of lymphoid progenitors.
  • IL-7 is believed to play a role in proliferation during certain stages of B-cell maturation.
  • IL-15 (UniProtKB - P40933), like IL-2, is a cytokine that stimulates the proliferation of T-lymphocytes.
  • IL-21 (UniProtKB - Q9HBE4) is a cytokine with immunoregulatory activity. IL-21 is thought to promote the transition between innate and adaptive immunity and to induce the production of IgGl and IgG3 in B-cells.
  • IL-21 may also play a role in proliferation and maturation of natural killer (NK) cells in synergy with IL- 15, and IL-21 may regulate proliferation of mature B- and T-cells in response to activating stimuli.
  • NK natural killer
  • IL-15 also stimulates interferon gamma production in T-cells and NK cells, and IL-21 may also inhibit dendritic cell activation and maturation during a T-cell-mediated immune response.
  • the term "tonicity” refers to the measure of the effective osmotic pressure gradient across a cell membrane.
  • the tonicity of a medium is defined by the sum of the potassium concentration and the NaCl concentration, multiplied by two. Tonicity can be expressed in terms of the osmolality of the solution, e.g., the media.
  • a solution, e.g., medium is considered “isotonic” when the concentration of solutes in the media is equivalent to the concentration of solutes inside the cell.
  • an isotonic medium has an osmolality of about 280 mOsm/L.
  • a solution e.g., a medium
  • a hypotonic solution has a tonicity of less than 280 mOsm/L.
  • a hypotonic medium has a tonicity from at least about 220 mOsm/L to less than about 280 mOsm/L.
  • a hypotonic medium has a tonicity from at least about 230 mOsm/L to less than about 280 mOsm/L.
  • a hypotonic medium has a tonicity from at least about 240 mOsm/L to less than about 280 mOsm/L. In some aspects, a hypotonic medium described herein has a tonicity of about 250 mOsm/L.
  • a solution e.g., a medium
  • a hypertonic solution has an osmolality of greater than 300 mOsm/L. In some aspects, a hypertonic medium described herein has an osmolality of about 320 mOsm/L.
  • the tonicity of the solution, e.g., medium is adjusted by increasing or decreasing the concentration of one or more solute selected from potassium ions, sodium ions, glucose, and any combination thereof.
  • the tonicity of the solution, e.g., medium is adjusted by increasing or decreasing the concentration of potassium ions and NaCl.
  • the tonicity of a medium can be maintained by offsetting the increase of one solute with a decrease in a second solute. For example, increasing the concentration of potassium ion in a medium without changing the concentration of sodium ions can increase the tonicity of the medium. However, if the concentration of potassium ions is increased and the concentration of sodium ions is decreased, the tonicity of the original medium can be maintained.
  • cell culture media includes any media for culturing immune cells, e.g., T cells.
  • cell culture media comprises a balanced salt solution (e.g., PBS, DPBS, HBSS, EBSS), Dulbecco's Modified Eagle's Medium (DMEM), Click’s medium, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), F-10, F-12, RPMI 1640, Glasgow Minimal Essential Medium (GMEM), alpha Minimal Essential Medium (alpha MEM), Iscove's Modified Dulbecco's Medium (IMDM), Ml 99, OPTMIZERTM Pro, OPTMIZERTM CTSTM T- Cell Expansion Basal Medium (ThermoFisher), OPTMIZERTM, OPTMIZERTM Complete, IMMUNOCULTTM XF (STEMCELLTM Technologies), AIM VTM, TEXMACSTM medium, PRIME-XV® T cell CDM, X-VIVO
  • DMEM Dulbec
  • cell culture media is serum free.
  • cell culture media comprises PRIME-XV® T cell CDM.
  • cell culture media comprises OPTMIZERTM.
  • cell culture media comprises OPTMIZERTM Pro.
  • cell culture media further comprises immune cell serum replacement (ICSR).
  • ISR immune cell serum replacement
  • potassium As used herein, the terms “potassium,” “potassium ion,” “potassium cation,” and “K+” are used interchangeably to refer to elemental potassium. Elemental potassium exists in solution as a positive ion. However, it would be readily apparent to a person of ordinary skill in the art that standard means of preparing a solution comprising potassium ion include diluting a potassium containing salt (e.g., KC1) into a solution. As such, a solution, e.g., a medium, comprising a molar (M) concentration of potassium ion, can be described as comprising an equal molar (M) concentration of a salt comprising potassium.
  • a potassium containing salt e.g., KC1
  • T cell receptor refers to a heterodimer composed of two different transmembrane polypeptide chains: an a chain and a P chain, or a y chain and a 8 chain, each comprising a constant region, which anchors the chain inside the T-cell surface membrane, and a variable region, which recognizes and binds to the antigen presented by MHCs.
  • the TCR complex is associated with six polypeptides forming two heterodimers, CD3ys and CD35s, and one homodimer CD3 which together forms the CD3 complex.
  • T-cell receptor- engineered T-cell therapy utilizes the modification of T cells that retain these complexes to specifically target the antigens expressed by particular tumor cells.
  • TCR includes naturally occurring TCRs and engineered TCRs.
  • an “engineered TCR” or “engineered T-cell receptor” refers to a T- cell receptor (TCR) engineered to specifically bind with a desired affinity to a major histocompatibility complex (MHC)/peptide target antigen that is selected, cloned, and/or subsequently introduced into a population of immune cells, e.g., T cells, NK cells, and/or TILs.
  • TCR T- cell receptor
  • MHC major histocompatibility complex
  • a single chain TCR is a single chain TCR.
  • a "TCR mimic” or a “TCRm” refers to a type of antibody or antigen-binding fragment thereof that recognize epitopes comprising both the peptide and the MHC-I molecule, similar to the recognition of such complexes by the TCR on T cells.
  • the term “higher than” means greater than but not equal to.
  • “higher than 4 mM” means any amount that is more than 4 mM, but which does not include 4 mM.
  • administering refers to the physical introduction of a therapeutic agent or a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems.
  • the different routes of administration for a therapeutic agent described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, intratracheal, pulmonary, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation.
  • a therapeutic agent described herein e.g., a redirected T cell cultured as described herein
  • a non-parenteral route such as a topical, epidermal, or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • the term "antigen” refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten.
  • the term “cognate antigen” refers to an antigen which an immune cell (e.g., T cell) recognizes and thereby, induces the activation of the immune cell (e.g., triggering intracellular signals that induce effector functions, such as cytokine production, and/or for proliferation of the cell).
  • a "cancer” refers a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. "Cancer” as used herein refers to primary, metastatic and recurrent cancers.
  • hematological malignancy refers to mammalian cancers and tumors of the hematopoietic and lymphoid tissues.
  • Non-limiting examples of hematological malignancies include those affecting tissues of the blood, bone marrow, lymph nodes, and lymphatic system, including acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CIVIL), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphomas.
  • Hematological malignancies are also referred to as "liquid tumors.”
  • Liquid tumor cancers include, but are not limited to, leukemias, myelomas, and lymphomas, as well as other hematological malignancies.
  • a "solid tumor,” as used herein, refers to an abnormal mass of tissue. Solid tumors may be benign or malignant. Nonlimiting examples of solid tumors include sarcomas, carcinomas, and lymphomas, such as cancers of the lung, breast, prostate, colon, rectum, and bladder.
  • the tissue structure of a solid tumor includes interdependent tissue compartments including the parenchyma (cancer cells) and the supporting stromal cells in which the cancer cells are dispersed, and which may provide a supporting microenvironment.
  • the cancer is selected from adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bone metastasis, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma of the skin, malignant mes
  • the cancer is selected from chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblast
  • the cancer is selected from acra-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, metastatic melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma.
  • the cancer is selected from acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma
  • the cancer is selected from Leukemia, Hodgkin's Disease, NonHodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, papillary thyroid cancer, neuroblastoma, neuroendocrine cancer, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, prostate cancer, Mullerian cancer, ovarian cancer, peritoneal cancer, fallopian tube cancer, or uterine papillary serous carcinoma.
  • immune response refers to a biological response within a vertebrate against foreign agents, which response protects the organism against these agents and diseases caused by them.
  • An immune response is mediated by the action of a cell of the immune system (e.g., a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • a cell of the immune system e.g., a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutr
  • An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell or a Th cell, such as a CD4 + or CD8 + T cell, or the inhibition of a Treg cell.
  • a T cell e.g., an effector T cell or a Th cell, such as a CD4 + or CD8 + T cell, or the inhibition of a Treg cell.
  • T cell and “T lymphocytes” are interchangeable and refer to any lymphocytes produced or processed by the thymus gland.
  • a T cell is a CD4+ T cell.
  • a T cell is a CD8+ T cell.
  • a T cell is a NKT cell.
  • anti-tumor immune response refers to an immune response against a tumor antigen.
  • a “subject” includes any human or nonhuman animal.
  • nonhuman animal includes, but is not limited to, vertebrates such as nonhuman primates, sheep, dogs, and rodents such as mice, rats and guinea pigs.
  • the subject is a human.
  • subject and patient are used interchangeably herein.
  • the phrase "subject in need thereof includes subjects, such as mammalian subjects, that would benefit, e.g., from administration of T cells cultured as described herein to control tumor growth.
  • an effective amount refers to an amount of an agent (e.g., a T cell or NK cell cultured as described herein) that provides the desired biological, therapeutic, and/or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation.
  • an effective amount is an amount sufficient to delay tumor development.
  • an effective amount is an amount sufficient to prevent or delay tumor recurrence.
  • An effective amount can be administered in one or more administrations.
  • the effective amount of the composition can, for example, (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow to some extent and can stop cancer cell infiltration into peripheral organs; (iv) inhibit (ie., slow to some extent and can stop tumor metastasis); (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.
  • a "therapeutically effective amount” is the amount of a composition disclosed herein (e.g., T cells cultured as described herein), which is clinically proven to effect a significant decrease in a disease (e.g., a cancer) or slowing of progression (regression) of a disease (e.g., cancer, such as an advanced solid tumor).
  • a therapeutic agent of the present disclosure e.g., T cells cultured as described herein
  • the ability of a therapeutic agent of the present disclosure e.g., T cells cultured as described herein
  • to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
  • ERTAIN effectiveness refers to the ability of a composition disclosed herein (e.g., cells cultured as described herein) to promote cancer regression in the patient.
  • Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ, and/or organism level (adverse effects) resulting from administration of a composition disclosed herein (e.g., cells cultured as described herein).
  • chimeric antigen receptor and "CAR,” as used herein, refer to a recombinant fusion protein that has an antigen-specific extracellular domain coupled to an intracellular domain that directs the cell to perform a specialized function upon binding of an antigen to the extracellular domain.
  • chimeric antigen receptors have the ability to bind MHC-independent antigen and transduce activation signals via their intracellular domain.
  • a chimeric antigen receptor may bind an MHC-dependent antigen (e.g., a CAR comprising a TCR mimic) and transduce activation signals via their intracellular domain.
  • the antigen-specific extracellular domain of a chimeric antigen receptor recognizes and specifically binds an antigen, typically a surface-expressed antigen of a malignancy.
  • An antigen-specific extracellular domain specifically binds an antigen when, for example, it binds the antigen with an affinity constant or affinity of interaction (KD) between about 0.1 pM to about 10 pM, for example, about 0.1 pM to about 1 pM or about 0.1 pM to about 100 nM.
  • KD affinity constant or affinity of interaction
  • An antigen-specific extracellular domain suitable for use in a CAR of the present disclosure can be any antigen-binding polypeptide, a wide variety of which are known in the art.
  • the antigen-binding domain is a single chain Fv (scFv).
  • Other antibody-based recognition domains such as cAb VHH (camelid antibody variable domains) and humanized versions thereof, IgNAR VH (shark antibody variable domains) and humanized versions thereof, sdAb VH (single domain antibody variable domains), and "camelized” antibody variable domains are also suitable for use in a CAR of the present disclosure.
  • T cell receptor (TCR) based recognition domains such as single chain TCR (scTv, i.e., single chain two-domain TCR containing V.alpha.V.beta.) are also suitable for use in a TCR of the present disclosure.
  • Some aspects of the present disclosure relate in part to methods of generating redirected T cells by a process of partial reprogramming and redirection to return the reprogrammed cells to the redirected T cells.
  • cell reprogramming comprises the expression or induction of the expression of one or more reprogramming factors in the cells.
  • reprogramming comprises contacting origin cells, i.e., a starting cell population, with at least one reprogramming factor or an agent that induces expression of at least one reprogramming factor.
  • partial reprogramming and “partially reprogrammed” refer to a process of reprogramming an origin cell without reaching a totipotent stem cell state or a pluripotent stem cell state (iPS cell).
  • partial preprogramming is any reprogramming that is not complete reprogramming.
  • “partial” or “incomplete” or “transient” reprogramming is reprogramming that is not complete reprogramming, e.g., as compared to a cell that has been completely reprogrammed to an iPS cell.
  • partial reprogramming is achieved by reprogramming origin cells for a duration less than the duration for complete reprogramming.
  • partial reprogramming is reprogramming carried out from about 1 to about 20 days. In some aspects, partial reprogramming is reprogramming carried out for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about 25 days. [0212] In some aspects, the reprogramming process starts with an origin cell and then results in the formation of “partially reprogrammed cells” that express one or more markers from a cell lineage that is different from the origin cell and that at least partially lose expression of one or more origin cell lineage-specific markers.
  • the reprogramming process results in the formation of partially reprogrammed cells that express one or more markers from a cell lineage that is different from the origin cell and that substantially or completely lose expression of origin cell lineage-specific markers. In some aspects, the reprogramming process results in the formation of partially reprogrammed cells that express one or more stem cell related markers compared to an origin cell. In some aspects, partially reprogrammed cells coexpress lineagespecific markers as well as non-lineage specific markers. In some aspects, the partially reprogrammed cells retain the ability to regain the identity of the origin cell without complex differentiation processes or without involving differentiation factors that are necessary in triggering cell differentiation.
  • the origin cell is an immune cell collected or obtained from a subject. In some aspects, the origin cell is an immune cell that has been engineered to express certain receptors on the surface. In some aspects, the origin cell is an immune cell that has been expanded in vitro. In some aspects, the immune cell comprises a peripheral blood mononuclear cell (PBMC). In some aspects, the immune cell comprises a T cell, a tumor infiltrating lymphocyte (TIL), a B cell, a natural killer (NK) cell, an NK/T cell, a monocyte, a macrophage, or a dendritic cell, or any combination thereof. In some aspects, the immune cell comprises a T cell (or a population of T cells).
  • TIL tumor infiltrating lymphocyte
  • B cell a tumor infiltrating lymphocyte
  • NK natural killer
  • the immune cell comprises a T cell (or a population of T cells).
  • the immune cell comprises a TIL (or a population of TILs).
  • the origin T cells comprise T cells selected from TCRaP cells; TCRyScells; CD4+CD8aP+ double positive cells, CD4+ single positive cells (such as Thl, Th2, Thl7, Treg), CD137+ cells, naive T cells, central memory T cells, effector memory T cells, or any combination thereof.
  • the origin T cells are activated or stimulated before contact with the reprogramming factors using the T cell activating or stimulating agents as described elsewhere herein. In some aspecats, the origin T cells are activated or stimulated about 1 to about 3 days before contacting with reprogramming factors.
  • the origin T cells are activated before contact with the reprogramming factors and are then enriched by selection for cells expressing CD137, PD1, or LAG3, or a combination thereof and then partially reprogrammed as described herein.
  • polyclonal antigen-specific (e.g., tumor-specific) T cells from cancer patients can be partially reprogrammed.
  • the T cells are selected, sorted, or otherwise enriched for following stimulation with an antigen, such as tumor antigen, tumor organoid, autologous tumor cells or tumor cell line.
  • the T cells are enriched, selected for or sorted by selecting for cells expressing one or more cell surface markers, such as 4- 1BB (CD137), PD1, LAG3, CD45, CD39, TIGIT, TIM3, CD69, 0X40, CD28, CD25, CD49d, and CTLA4.
  • the T cells are enriched, selected for or sorted before activation with any of the T cell activating or stimulating agents described herein.
  • CD45+ T cells are selected for or enriched prior to activation with any of the activation agents described herein.
  • partial T cell reprogramming is a reprogramming process carried out until T cell reactivation is initiated.
  • partial T cell reprogramming is a reprogramming process that is carried out at least until the T cells begin to lose expression of one or more T cell markers and/or begin to express markers associated with a non-T cell lineage.
  • partial T cell reprogramming is a process of reprogramming carried out for a period of time up to the time that the T cells can no longer be returned to a cell expressing CD3, CD4 and/or CD8 by reactivation with a T cell activating or stimulating agent.
  • the partially reprogrammed cells are reprogrammed for at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days.
  • the partially partially reprogrammed cells are reprogrammed between about 1 day to about 14 days, about 1 day to about 13 days, about 1 day to about 12 days, about 1 day to about 11 days, about 1 day to about 10 days, about 1 day to about 9 days, about 1 day to about 8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 5 to about 14 days, about 6 to about 14 days, about 7 to about 14 days, about 5 to about 13 days, about 5 to about 12 days, or about 5 to about 11 days.
  • the cells are contacted with the T cell activating agent about 5 days after the cells are contacted with the one or more reprogramming factors. In some aspects, the cells are contacted with the T cell activating agent about 6 days after the cells are contacted with the one or more reprogramming factors. In some aspects, the cells are contacted with the T cell activating agent about 7 days after the cells are contacted with the one or more reprogramming factors. In some aspects, the cells are contacted with the T cell activating agent about 8 days after the cells are contacted with the one or more reprogramming factors. In some aspects, the cells are contacted with the T cell activating agent about 9 days after the cells are contacted with the one or more reprogramming factors. In some aspects, the cells are contacted with the T cell activating agent about 10 days after the cells are contacted with the one or more reprogramming factors.
  • the period of time that cells are contacted with the one or more reprogramming factors can vary depending on the amount or level of expression of the reprogramming factors.
  • T cells are transduced with a viral vector expressing high levels of one or more of the reprogramming factors, a shorter time period can be required to achieve the desired partial reprogramming.
  • T cells are transduced with a vector expressing low levels of one or more of the reprogramming factors and a longer time period can be required to achieve the desired partial reprogramming.
  • an expression vector, such as a viral vector, encoding the one or more reprogramming factors can comprise an inducible promoter where the expression of the partial reprogramming factors can be tuned to a desired expression level.
  • the partially reprogrammed cells are reprogrammed for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at leaset about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days.
  • the partially reprogrammed cells are reprogrammed for between about 1 day and about 20 days.
  • the partially reprogrammed cells are reprogrammed for between about 1 day and about 14 days.
  • the partially reprogrammed cells are reprogrammed for between about 1 day and about 13 days. In some aspects, the partially reprogrammed cells are reprogrammed for between about 1 day and about 12 days. In some aspects, the partially reprogrammed cells are reprogrammed for between about 1 day and about 11 days. In some aspects, the partially reprogrammed cells are reprogrammed for between about 1 day and about 10 days. In some aspects, the partially reprogrammed cells are reprogrammed for between about 1 day and about 9 days. In some aspects, the partially reprogrammed cells are reprogrammed for between about 1 day and about 8 days.
  • the partially reprogrammed cells are reprogrammed for between about 1 day and about 7 days. In some aspects, the partially reprogrammed cells are reprogrammed for between about 1 day and about 6 days. In some aspects, the partially reprogrammed cells are reprogrammed for between about 5 days and about 14 days. In some aspects, the partially reprogrammed cells are reprogrammed for between about 6 days and about 14 days. In some aspects, the partially reprogrammed cells are reprogrammed for between about 7 days and about 14 days. In some aspects, the partially reprogrammed cells are reprogrammed for between about 5 days and about 13 days. In some aspects, the partially reprogrammed cells are reprogrammed for between about 5 days and about 12 days. In some aspects, the partially reprogrammed cells are reprogrammed for between about 5 days and about 11 days.
  • the partially reprogrammed cells are not induced pluripotent stem (iPS) cells or totipotent cells.
  • iPS induced pluripotent stem
  • transient means for a period of time sufficient to achieve partial reprogramming of one or more T cells, (e.g., a period of time sufficient for the partially reprogrammed cells to redirect back to T cells, but not long enough to transform the one or origin cells into iPS cells or totipotent cells).
  • transiently contacting with one or more reprogramming factors means contacting with one or more reprogramming factors for a period of time which is sufficient for the partially reprogrammed cells to redirect, for at least a portion of the partially reprogrammed cells to express one or more markers associated with stem cells, for at least a portion of the partially reprogrammed cells to lose or have reduced expression of one or more T cell lineage markers, for at least a portion of the partially reprogrammed cells to express one or more markers associated with stem cells and to lose or have reduced expression of one or more T cell lineage markers, or for the one or more T cells to form at least one colony attached to a culture vessel surface, but not a period of time long enough to permit the one or more T cells to transform into iPS cells or totipotent cells.
  • transiently contacting with one or more reprogramming factors means contacting with an agent that induces expression of one or more reprogramming factors.
  • transiently contacting with one or more reprogramming factors means contacting with one or more reprogramming factors for a period of time sufficient for at least a portion of the partially reprogrammed cells to lose or have reduced expression of one or more T cell lineage markers.
  • theT cell lineage markers include, but are not limited to, CD3, CD8, CD352, TCRa, TCRb, TCRg, TCRd, CD3gamma, LCK, CD3zeta, IL2Rgamma, IL2A, IL2B, IL7RA, CCR7, CD62L, CD27, CD28, TCF7, CD4, CD5, CD7, CD45, CD69, LEF1, CXCR4, GZMB, CD31, or any combination thereof.
  • the one or more T cell lineage markers is undetectable using detection methods known in the art, such as FACS.
  • transiently contacting with one or more reprogramming factors means contacting with one or more reprogramming factors for a period of time sufficient for at least a portion of the partially reprogrammed cells to have reduced or undectable expression of CD3. In some aspects, transiently contacting with one or more reprogramming factors means contacting with one or more reprogramming factors for a period of time sufficient for at least a portion of the partially reprogrammed cells to have reduced or undectable expression of CD8. In some aspects, transiently contacting with one or more reprogramming factors means contacting with one or more reprogramming factors for a period of time sufficient for at least a portion of the partially reprogrammed cells to have reduced or undectable expression of CD4.
  • At least about 5% of the partially reprogrammed cells have reduced or undectable expression of one or more of CD3, CD8, or CD4, following the transient contact. In some aspects, at least about 10% of the partially reprogrammed cells have reduced or undectable expression of one or more of CD3, CD8, or CD4, following the transient contact. In some aspects, at least about 20% of the partially reprogrammed cells have reduced or undectable expression of one or more of CD3, CD8, or CD4, following the transient contact. In some aspects, at least about 30% of the partially reprogrammed cells have reduced or undectable expression of one or more of CD3, CD8, or CD4, following the transient contact.
  • At least about 40% of the partially reprogrammed cells have reduced or undectable expression of one or more of CD3, CD8, or CD4, following the transient contact. In some aspects, at least about 50% of the partially reprogrammed cells have reduced or undectable expression of one or more of CD3, CD8, or CD4, following the transient contact. In some aspects, at least about 60% of the partially reprogrammed cells have reduced or undectable expression of one or more of CD3, CD8, or CD4, following the transient contact. In some aspects, at least about 70% of the partially reprogrammed cells have reduced or undectable expression of one or more of CD3, CD8, or CD4, following the transient contact.
  • At least about 80% of the partially reprogrammed cells have reduced or undectable expression of one or more of CD3, CD8, or CD4, following the transient contact. In some aspects, at least about 90% of the partially reprogrammed cells have reduced or undectable expression of one or more of CD3, CD8, or CD4, following the transient contact.
  • transiently contacting with one or more reprogramming factors means contacting with one or more reprogramming factors for a period of time sufficient for at least a portion of the partially reprogrammed cells to express one or more of integrin a6pi, SSEA4, CD9, and/or CD90. In some aspects, transiently contacting with one or more reprogramming factors means contacting with one or more reprogramming factors for a period of time sufficient for at least a portion of the partially reprogrammed cells to express integrin a6pi.
  • transiently contacting with one or more reprogramming factors means contacting with one or more reprogramming factors for a period of time sufficient for at least a portion of the partially reprogrammed cells to express SSEA4. In some aspects, transiently contacting with one or more reprogramming factors means contacting with one or more reprogramming factors for a period of time sufficient for at least a portion of the partially reprogrammed cells to express CD9. In some aspects, transiently contacting with one or more reprogramming factors means contacting with one or more reprogramming factors for a period of time sufficient for at least a portion of the partially reprogrammed cells to express CD90.
  • At least about 5% of the partially reprogrammed cells express one or more of integrin a6pi, SSEA4, CD9, and/or CD90, following the transient contact. In some aspects, at least about 10% of the partially reprogrammed cells express one or more of integrin a6pi, SSEA4, CD9, and/or CD90, following the transient contact. In some aspects, at least about 20% of the partially reprogrammed cells express one or more of integrin a6pi, SSEA4, CD9, and/or CD90, following the transient contact.
  • At least about 30% of the partially reprogrammed cells express one or more of integrin a6pi, SSEA4, CD9, and/or CD90, following the transient contact. In some aspects, at least about 40% of the partially reprogrammed cells express one or more of integrin a6pi, SSEA4, CD9, and/or CD90, following the transient contact. In some aspects, at least about 50% of the partially reprogrammed cells express one or more of integrin a6pi, SSEA4, CD9, and/or CD90, following the transient contact.
  • At least about 60% of the partially reprogrammed cells express one or more of integrin a6pi, SSEA4, CD9, and/or CD90, following the transient contact. In some aspects, at least about 70% of the partially reprogrammed cells express one or more of integrin a6pi, SSEA4, CD9, and/or CD90, following the transient contact. In some aspects, at least about 80% of the partially reprogrammed cells express one or more of integrin a6pi, SSEA4, CD9, and/or CD90, following the transient contact. In some aspects, at least about 90% of the partially reprogrammed cells express one or more of integrin a6pi, SSEA4, CD9, and/or CD90, following the transient contact.
  • transiently contacting with one or more reprogramming factors means contacting with one or more reprogramming factors for a period of time sufficient for at least a portion of the partially reprogrammed cells to express one or more of integrin a6pi, SSEA4, CD9, and/or CD90 and to lose or have reduced expression of one or more T cell lineage markers, such as one or more of CD3, CD8, CD352, TCRa, TCRb, TCRg, TCRd, CD3gamma, LCK, CD3zeta, IL2Rgamma, IL2A, IL2B, IL7RA, CCR7, CD62L, CD27, CD28, TCF7, CD4, CD5, CD7, CD45, CD69, LEF1, CXCR4, GZMB and CD31.
  • T cell lineage markers such as one or more of CD3, CD8, CD352, TCRa, TCRb, TCRg, TCRd, CD3gamma, LCK, CD3zeta,
  • transiently contacting with one or more reprogramming factors means contacting with one or more reprogramming factors for a period of time sufficient for the partially reprogrammed cells to have low or no detectable expression of CD3 and express one or more of SSEA4, CD9, and CD90.
  • At least about 10% of the partially reprogrammed cells have lost or reduced expression of one or more T cell lineage marker, following the transient contact. In some aspects, at least about 20% of the partially reprogrammed cells have lost or reduced expression of one or more T cell lineage marker, following the transient contact. In some aspects, at least about 30% of the partially reprogrammed cells have lost or reduced expression of one or more T cell lineage marker, following the transient contact. In some aspects, at least about 40% of the partially reprogrammed cells have lost or reduced expression of one or more T cell lineage marker, following the transient contact. In some aspects, at least about 50% of the partially reprogrammed cells have lost or reduced expression of one or more T cell lineage marker, following the transient contact.
  • At least about 60% of the partially reprogrammed cells have lost or reduced expression of one or more T cell lineage marker, following the transient contact. In some aspects, at least about 70% of the partially reprogrammed cells have lost or reduced expression of one or more T cell lineage marker, following the transient contact. In some aspects, at least about 80% of the partially reprogrammed cells have lost or reduced expression of one or more T cell lineage marker, following the transient contact. In some aspects, at least about 90% of the partially reprogrammed cells have lost or reduced expression of one or more T cell lineage marker, following the transient contact.
  • a portion of the partially reprogrammed cells no longer have detectable expression of the one or more T cell lineage marker, following the transient contact. In some aspects, a portion of the partially reprogrammed cells have reduced expression of the one or more T cell lineage marker, following the transient contact, as compared to an immune cell that was not contacted with the one or more reprogramming factors.
  • the expression of the one or more T cell lineage marker, following the transient contact is less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors.
  • the expression of the one or more T cell lineage marker, following the transient contact is less than about 95% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors. In some aspects, the expression of the one or more T cell lineage marker, following the transient contact, is less than about 90% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors. In some aspects, the expression of the one or more T cell lineage marker, following the transient contact, is less than about 85% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors.
  • the expression of the one or more T cell lineage marker, following the transient contact is less than about 80% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors. In some aspects, the expression of the one or more T cell lineage marker, following the transient contact, is less than about 75% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors. In some aspects, the expression of the one or more T cell lineage marker, following the transient contact, is less than about 70% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors.
  • the expression of the one or more T cell lineage marker, following the transient contact is less than about 65% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors. In some aspects, the expression of the one or more T cell lineage marker, following the transient contact, is less than about 60% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors. In some aspects, the expression of the one or more T cell lineage marker, following the transient contact, is less than about 55% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors.
  • the expression of the one or more T cell lineage marker, following the transient contact is less than about 50% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors. In some aspects, the expression of the one or more T cell lineage marker, following the transient contact, is less than about 45% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors. In some aspects, the expression of the one or more T cell lineage marker, following the transient contact, is less than about 40% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors.
  • the expression of the one or more T cell lineage marker, following the transient contact is less than about 35% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors. In some aspects, the expression of the one or more T cell lineage marker, following the transient contact, is less than about 30% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors. In some aspects, the expression of the one or more T cell lineage marker, following the transient contact, is less than about 25% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors.
  • the expression of the one or more T cell lineage marker, following the transient contact is less than about 20% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors. In some aspects, the expression of the one or more T cell lineage marker, following the transient contact, is less than about 20% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors. In some aspects, the expression of the one or more T cell lineage marker, following the transient contact, is less than about 15% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors.
  • the expression of the one or more T cell lineage marker, following the transient contact is less than about 10% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors. In some aspects, the expression of the one or more T cell lineage marker, following the transient contact, is less than about 5% that of the expression of the same T cell lineage marker by an immune cell that was not contacted with the one or more reprogramming factors.
  • transiently contacting with one or more reprogramming factors means contacting with one or more reprogramming factors for a period of time sufficient for the partially reprogrammed immune cells to retain the ability to regain immune cell identity after contact with an immune cell activating or stimulating agent.
  • “transiently contacting” can mean contacting for a period of time of up to about 1 day, or up to about 2 days, or up to about 3 days, or up to about 4 days, or up to about 5 days, or up to about 6 days, or up to about 7 days, or up to about 8 days, or up to about 9 days, or up to about 10 days, or up to about 11 days, or up to about 12 days, or up to about 13 days, or up to about 14 days or up to about 15 days, or up to about 16 days, or up to about 17 days, or up to about 18 days, or up to about 19 days, or up to about 20 days, or up to about 21 days, or up to about 22 days, or up to about 23 days, or up to about 24 days or up to about 25 days or up to about 26 days, or up to about 27 days or up to about 28 days, or up to about 29 days, or up to about 30 days.
  • transiently expressing or “transient expression” or the like means expression or causing expression of one or more reprogramming factors so as to achieve partial reprogramming of one or more T cells (i.e., where transformation to an iPS cell or totipotent cell is not achieved).
  • the partially reprogrammed cells are capable of adhering to a culture vessel surface.
  • the partially reprogrammed cells express integrin a6pi, SSEA4, CD9, and/or CD90. In some aspects, the partially reprogrammed cells express low levels of or have no detectable expression of CD3. In some aspects, the expression of CD3 by the partially reprogrammed cells is less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% the expression of CD3 by an immune cell not contacted with the one or more reprogamming factors.
  • the expression of CD3 by the partially reprogrammed cells is less than about 25% the expression of CD3 by an immune cell not contacted with the one or more reprogamming factors. In some aspects, the expression of CD3 by the partially reprogrammed cells is less than about 20% the expression of CD3 by an immune cell not contacted with the one or more reprogamming factors. In some aspects, the expression of CD3 by the partially reprogrammed cells is less than about 15% the expression of CD3 by an immune cell not contacted with the one or more reprogamming factors. In some aspects, the expression of CD3 by the partially reprogrammed cells is less than about 10% the expression of CD3 by an immune cell not contacted with the one or more reprogamming factors.
  • the expression of CD3 by the partially reprogrammed cells is less than about 5% the expression of CD3 by an immune cell not contacted with the one or more reprogamming factors. In some aspects, the expression of CD3 by the partially reprogrammed cells is less than about 1% the expression of CD3 by an immune cell not contacted with the one or more reprogamming factors. In some aspects, the partially reprogrammed cells express CD3 and SSEA4. In some aspects, the partially reprogrammed cells express CD3 and CD9, CD3 and CD90, or CD3, CD9 and CD90. In some aspects, the partially reprogrammed cells express CD3, SSEA4, CD9 and CD90.
  • partially reprogrammed immune cells retain the ability to regain immune cell identity after contact with an immune cell activating or stimulating agent.
  • regaining immune cell identity refers to a partially reprogrammed immune cell that reexpresses one or more immune cell markers, has downregulated or lost expression of one or more stem-cell or non-immune cell lineage markers, and/or has regained one or more immune cell functions.
  • the reprogramming results in the partial or full loss of one or more immune cell markers, immune cell identity, immune cell phenotype, and/or immune cell lineage.
  • partially reprogrammed T cells retain the ability to return to the T cell lineage.
  • partially reprogrammed T cells retain the ability to regain a T cell phenotype without the need for differentiation factors or thymic organoids. In some aspects, partially reprogrammed T cells retain the ability to return to the T cell lineage after contact with a T cell activating or stimulating agent. In some aspects, returning to the T cell lineage refers to a partially reprogrammed T cell that reexpresses one or more T cell markers, has downregulated or lost expression of one or more stem-cell or non-T cell lineage markers, and/or has regained one or more T cell functions.
  • the reprogramming results in the partial or full loss of one or more T cell markers, T cell function, T cell phenotype, and/or T cell lineage.
  • the cells after contacting T cells with one or more reprogramming factors, the cells begin to express one or more of SSEA4, CD9, CD90, CD164, CD63, CD71, CD326, TRA-1-81 and TRA-1-60-R and begin to lose expression of one or more of CD3, CD8, CD352, TCRa, TCRb, TCRg, TCRd, CD3gamma, LCK, CD3zeta, IL2Rgamma, IL2A, IL2B, IL7RA, CCR7, CD62L, CD27, CD28, TCF7, CD4, CD5, CD7, CD45, CD69, LEF1, CXCR4, GZMB and CD31.
  • the reprogrammed cells are partially reprogrammed. In some aspects, the partially reprogrammed cells are reprogrammed for at least about 3 days, at least about
  • the partially reprogrammed cells are reprogrammed for at least about 3 days. In some aspects, the partially reprogrammed cells are reprogrammed for at least about 4 days. In some aspects, the partially reprogrammed cells are reprogrammed for at least about 5 days. In some aspects, the partially reprogrammed cells are reprogrammed for at least about 6 days. In some aspects, the partially reprogrammed cells are reprogrammed for at least about 7 days.
  • the partially reprogrammed cells are reprogrammed for at least about 8 days. In some aspects, the partially reprogrammed cells are reprogrammed for at least about 9 days. In some aspects, the partially reprogrammed cells are reprogrammed for at least about 10 days. In some aspects, the partially reprogrammed cells are reprogrammed for at least about 11 days. In some aspects, the partially reprogrammed cells are reprogrammed for at least about 12 days. In some aspects, the partially reprogrammed cells are reprogrammed for at least about 13 days. In some aspects, the partially reprogrammed cells are reprogrammed for at least about 14 days.
  • the partially reprogrammed cells are reprogrammed between about 1 day and about 14 days, about 1 day and about 13 days, about 1 day and about 12 days, about 1 day and about 11 days, about 1 day and about 10 days, about 1 day and about 9 days, about 1 day and about 8 days, about 1 day and about 7 days, about 1 day and about 6 days, about 5 days and about 14 days, about 6 days and about 14 days, about 7 days and about 14 days, about 5 days and about 13 days, about 5 days and about 12 days, or about 5 days and about 11 days.
  • the partially reprogrammed cells are reprogrammed between about 1 day and about 14 days.
  • the partially reprogrammed cells are reprogrammed between about 1 day and about 13 days. In some aspects, the partially reprogrammed cells are reprogrammed between about 1 day and about 12 days. In some aspects, the partially reprogrammed cells are reprogrammed between about 1 day and about 11 days. In some aspects, the partially reprogrammed cells are reprogrammed between about 1 day and about 10 days. In some aspects, the partially reprogrammed cells are reprogrammed between about 1 day and about 9 days. In some aspects, the partially reprogrammed cells are reprogrammed between about 1 day and about 8 days. In some aspects, the partially reprogrammed cells are reprogrammed between about 1 day and about 7 days.
  • the partially reprogrammed cells are reprogrammed between about 1 day and about 6 days. In some aspects, the partially reprogrammed cells are reprogrammed between about 1 day and about 5 days. In some aspects, the partially reprogrammed cells are reprogrammed between about 1 day and about 6 days. In some aspects, the partially reprogrammed cells are reprogrammed between about 5 days and about 14 days. In some aspects, the partially reprogrammed cells are reprogrammed between about 6 days and about 14 days. In some aspects, the partially reprogrammed cells are reprogrammed between about 7 days and about 14 days. In some aspects, the partially reprogrammed cells are reprogrammed between about 5 days and about 13 days. In some aspects, the partially reprogrammed cells are reprogrammed between about 5 days and about 12 days. In some aspects, the partially reprogrammed cells are reprogrammed between about 5 days and about 11 days.
  • the partially reprogrammed cells are not induced pluripotent stem (iPS) cells or totipotent cells.
  • iPS induced pluripotent stem
  • the partially reprogrammed cells do not express the at least one T cell marker expressed by the redirected T cells.
  • the expression of the at least one T cell marker can be reduced to the point that the marker is no longer detectable on the surface of the cell.
  • the at least one T cell marker is no longer detected on the cell surface, i.e., the T cell marker is no longer detectable on the cell surface.
  • the partially reprogrammed cells do not exhibit the T cell phenotype exhibited by the redirected T cells.
  • the partially reprogrammed cells are not T cell lineage.
  • endogenous reprogramming factor expression is induced using one or more chemical compounds (see e.g., Guan et al., 2022 Nature DOI 10.1038/s41586-022-04593-5).
  • one or more reprogramming factors is transiently expressed in the immune cells to generate reprogrammed cells.
  • the one or more reprogramming factors can include one or more or all of KLF4, OCT4, SOX2 and C-MYC; and optionally SV40, LIN28 and/or NANOG; or can additionally include one or more other reprogramming factors.
  • the reprogramming factors may be expressed in the isolated T cells using a gene editing technology (e.g.
  • the gene editing technology delivers an inducible set of reprogramming factors to be expressed in the isolated T cells.
  • the nucleic acid(s) encoding the reprogramming factors is/are carried in one or more recombinant expression vectors.
  • the recombinant expression vector(s) can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the recombinant expression vector(s) can comprise naturally-occurring or non-naturally-occurring intemucleotide linkages, or both types of linkages.
  • the vector(s) may contain regulatory nucleic acid sequences which provide for expression of the stem cell-associated genes.
  • the partial reprogramming of the methods herein are carried out using media optimized for cell reprogramming, such as but not limited to STEMFIT media (Amsbio, Abington, UK), mTESR2 (Stem Cell Technologies), and ESSENTIAL 6 or 8 (Life Technologies); STEMPRO hESC SFM (Gibco); TESR, clone-R, dMEM Fl 2, KSR.
  • media for cell reprogramming may be supplemented with one or more growth factors and/or cytokines. 3. Reprogramming Factors
  • reprogramming factors refers to any protein, polypeptide, amino acid, mRNA, DNA or small molecule capable of erasing and/or reestablishing epigenetic modifications acquired during mammalian cell development or in cell culture. Reprogramming factors can alter the differentiational state of a cell.
  • Such reprogramming factors can include, but are not limited to the transcription factors, e.g., a molecule that increases expression of an OCT4 protein, a molecule that increases expression of a SOX3 protein, a molecule that increases expression of a KLF4 protein and a molecule that increases expression of a C-MYC protein, discovered by Yamanaka and colleagues (see, e.g., Takahashi and Yamanaka, 2006, Cell, 136, 364-377) which are referred to herein as “OSKM” or the “Yamanaka factors.”
  • the reprogramming factors also incude a molecule that increases expression of a LIN28 protein or a molecule that increases expression of a NANOG protein.
  • Reprogramming factors refers to other factors that might alter the differentiation state of a cell, or that might enhance or alter the efficiency of cell reprogramming. Such factors are known in the art. Exemplary reprogramming factors are described in Feng et al. 2009, Cell Stem Cell Review, 4:301-313.
  • one or more of the reprogramming factors that come into contact with the origin cells are chemical compounds that increase expression of an OCT4 protein, a KLF4 protein, a SOX2 protein, a C-MYC protein, a LIN28 protein, a NANOG protein, and/or an SV40 large T antigen or other reprogramming factors contemplated herein.
  • endogenous reprogramming factor expression is induced using one or more chemical compounds (see e.g., Guan et al., 2022 Nature DOI 10.1038/s41586-022-04593-5).
  • one or more of the reprogramming factors that come into contact with the origin cells are proteins that increase expression of an OCT4 protein, a KLF4 protein, a SOX2 protein, a C-MYC protein, a LIN28 protein, a NANOG protein, and/or an SV40 large T antigen.
  • one or more of the reprogramming factors that come into contact with the origin cells are polynucleotides (e.g., DNA, RNA, mRNA, and/or expression vector) that encode an OCT4 protein, a KLF4 protein, a SOX2 protein, a C-MYC protein, a LIN28 protein, a NANOG protein, and/or an SV40 large T antigen.
  • polynucleotides e.g., DNA, RNA, mRNA, and/or expression vector
  • the at least one reprogramming factor comprises a polynucleotide encoding an OCT4 protein, a polynucleotide encoding a SOX2 protein, a polynucleotide encoding a KLF4 protein, a polynucleotide encoding a c-MYC protein, a polynucleotide encoding a LIN28 protein, and/or a polynucleotide encoding a NANOG protein and optionally a polynucleotide encoding an SV40 protein.
  • the at least one reprogramming factor comprises a polynucleotide encoding an OCT4 protein, a polynucleotide encoding a SOX2 protein, a polynucleotide encoding a KLF4 protein, and a polynucleotide encoding a c-MYC protein.
  • the reprogramming factor is at least one of a molecule that increases expression of a KLF4 protein, a molecule that increases expression of a OCT4 protein, a molecule that increases expression of a SOX2 protein, a molecule that increases expression of a C- MYC protein, a molecule that increases expression of a LIN28 protein or a molecule that increases expression of a NANOG protein.
  • the origin cells are contacted with a molecule that increases expression of an OCT4 protein. In some aspects, the origin cells are contacted with a molecule that increases expression of an OCT4 protein and a molecule that increases expression of a SOX2 protein.
  • the isolated origin cells are contacted with a molecule that increases expression of an OCT4 protein, a molecule that increases expression of a SOX2 protein and a molecule that increases expression of a C-MYC protein.
  • the origin cells are contacted with a molecule that increases expression of an OCT4 protein, a molecule that increases expression of a SOX2 protein and a molecule that increases expression of a KLF4 protein.
  • the origin cells are contacted with a molecule that increases expression of an OCT4 protein, a molecule that increases expression of a SOX2 protein and a molecule that increases expression of a KLF4 protein, and either a molecule that increases expression of a C- MYC protein or a molecule that increases expression of a SV40 Large T cell antigen.
  • the origin cells are contacted with a molecule that increases expression of an OCT4 protein, a molecule that increases expression of a SOX2 protein, a molecule that increases expression of a KLF4 protein, a molecule that increases expression of a C-MYC protein and a molecule that increases expression of a SV40 Large T cell antigen.
  • the origin cells are contacted with a molecule that increases expression of an OCT4 protein, a molecule that increases expression of a SOX2 protein, a molecule that increases expression of a KLF4 protein, a molecule that increases expression of a C-MYC protein, and a molecule that increases expression of a LIN28 protein.
  • the origin cells are contacted with a molecule that increases expression of an OCT4 protein, a molecule that increases expression of a SOX2 protein, a molecule that increases expression of a KLF4 protein, a molecule that increases expression of a C-MYC protein, a molecule that increases expression of a LIN28 protein and a molecule that increases expression of a SV40 Large T cell antigen.
  • the origin cells are contacted with a molecule that increases expression of an OCT4 protein, a molecule that increases expression of a SOX2 protein, a molecule that increases expression of a KLF4 protein, a molecule that increases expression of a C-MYC protein, a molecule that increases expression of a LIN28 protein, and a molecule that increases expression of a NANOG protein.
  • the origin cells are contacted with a molecule that increases expression of an OCT4 protein, a molecule that increases expression of a SOX2 protein, a molecule that increases expression of a KLF4 protein, a molecule that increases expression of a C-MYC protein, a molecule that increases expression of a LIN28 protein, a molecule that increases expression of a NANOG protein and a molecule that increases expression of a SV40 Large T cell antigen.
  • Additional reprogramming factors can be used in some aspects to partially reprogram the isolated immune cells (e.g., T cells).
  • Such factors include, but are not limited to, Esrrb, Pax5 shRNA, C/EBPa, p53 siRNA, UTF1, DNMT shRNA, Wnt3a, GLIS1, DLX4, CDH1, SV40 Large T cell antigen (LT(T)) and hTERT.
  • reprogramming factors upregulate or downregulate certain miRNAs involved in reprogramming.
  • a reprogramming factor upregulates or downregulates expression of one or more of the genes upstream or downstream of one or more of the Yamanaka Factors.
  • the one or more reprogramming factors include, but are not limited to, histone methyltransferase inhibitors, L-type calcium channel agonists, G9a methyltransferase inhibitors, DNA methyltransferase inhibitors, histone deacetylase inhibitors, MEK inhibitors, GSK3 inhibitors or TGF-B inhibitors. Any factor that modulates the upstream or downstream molecular pathway of the reprogramming transcription factors is contemplated for use in the partial reprogramming methods herein.
  • Illustrative inhibitors include, but are not limited to BIX01294, BayK 8644, CHIR 99021, Forskolin, and RepSox.
  • serine/threonine-protein kinase B-Raf (BRAF) inhibitors may be used in the reprogramming methods herein (see, e.g., US2017114323).
  • BRAF serine/threonine-protein kinase B-Raf
  • EGFR epidermal growth factor receptor
  • VEGFR1 vascular endothelial growth factor 1
  • FGFR1 fibroblast growth factor receptor 1
  • one or more reprogramming factors are transiently expressed in the immune cells to generate reprogrammed cells.
  • the one or more reprogramming factors can include one or more or all of a molecule that increases expression of a KLF4 protein, a molecule that increases expression of an OCT4 protein, a molecule that increases expression of a SOX2 protein and a molecule that increases expression of a C-MYC protein; or can additionally include one or more other reprogramming factors.
  • the reprogramming factors may be expressed in the isolated T cells using a gene editing technology (e.g. TALENS, CRISPR/cas) known in the art.
  • the gene editing technology delivers an inducible set of reprogramming factors to be expressed in the isolated T cells.
  • the nucleic acid(s) encoding the stem cell-associated gene(s) is/are carried in one or more recombinant expression vectors.
  • the recombinant expression vector(s) can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the recombinant expression vector(s) can comprise naturally-occurring or non- naturally-occurring internucleotide linkages, or both types of linkages.
  • the vector(s) may contain regulatory nucleic acid sequences which provide for expression of the stem cell-associated genes.
  • OCT4 is known as POU domain, class 5, transcription factor 1, or also known as Octamer-binding protein 3 (Oct-3), Octamer-binding protein 4 (Oct-4), or Octamer-binding transcription factor 3 (OTF-3). Its gene name is known as POU5F1, OCT3. OCT4, or OTF3.
  • OCT4 is a transcription factor that binds to the octamer motif (5'-ATTTGCAT-3'). It forms a trimeric complex with SOX2 or SOX15 on DNA and controls the expression of a number of genes involved in embryonic development such as YES1, FGF4, UTF1 and ZFP206. OCT4 is known to be critical for early embryogenesis and for embryonic stem cell pluripotency.
  • UNIPROT accessions are known as A6NCS1, A6NLL8, D2IYK4, P31359, or QI 5167.
  • OCT 4 Nucleotide Sequence ID: NM_002701.6 Length: 1409 (Coding sequence: positions 63 to 1145 length 1083) (SEQ ID NO: 1)
  • the OCT4 useful for the present method comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 2.
  • the OCT4 useful for the present method comprises an amino acid sequence set forth in SEQ ID NO: 2.
  • the nucleotide sequence encoding a OCT4 protein useful for the present method comprises a nucleotide sequence having at least about 75%, at least about 76%, at least about 77%, at least 78%, at least about 79%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 1. 3B. S0X2
  • SOX2 is known as Transcription factor SOX-2. Its gene name is known as SOX2.
  • SOX2 is a transcription factor that forms a trimeric complex with OCT4 on DNA and controls the expression of a number of genes involved in embryonic development such as YES1, FGF4, UTF1 and ZFP206 (By similarity). It binds to the proximal enhancer region of NANOG (By similarity). SOX2 is critical for early embryogenesis and for embryonic stem cell pluripotency (PubMed: 18035408).
  • UNIPROT accessions are known as Q14537.
  • NM_003106.4 Length 2512, Coding sequence 437 to 1390, 954bp (SEQ ID NO: 5) (exemplary nucleotides that can be changed are highlighted and underlined)
  • the SOX2 useful for the present method comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 6.
  • the SOX2 useful for the present method comprises an amino acid sequence set forth in SEQ ID NO: 6.
  • the nucleotide sequence encoding a SOX2 protein useful for the present method comprises a nucleotide sequence having at least about 75%, at least about 76%, at least about 77%, at least 78%, at least about 79%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 5.
  • KLF4 is known as Krueppel-like factor 4, Epithelial zinc finger protein EZF, or Gut-enriched krueppel-like factor. Its gene name is known as KLF4, EZF, or GKLF.
  • KLF4 is a transcription factor; can act both as activator and as repressor. It binds the 5'-CACCC-3' core sequence and binds to the promoter region of its own gene and can activate its own transcription. KLF4 can regulate the expression of key transcription factors during embryonic development, plays an important role in maintaining embryonic stem cells, and in preventing their differentiation. KLF4 is required for establishing the barrier function of the skin and for postnatal maturation and maintenance of the ocular surface and is involved in the differentiation of epithelial cells and may also function in skeletal and kidney development. KLF4 contributes to the down-regulation of p53/TP53 transcription. Primary UNIPROT accession number for KLF4 is known as 043474, Secondary UNIPROT accessions are known as B2R8S4, B3KT79, L0R3I6, L0R4N5, or P78338
  • KLF4 aa Sequence ID: NP 004226.3 Length: 479aa (SEQ ID NO: 8)
  • the KLF4 useful for the present method comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 8.
  • the nucleotide sequence encoding a KLF4 protein useful for the present method comprises a nucleotide sequence having at least about 75%, at least about 76%, at least about 77%, at least 78%, at least about 79%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 7.
  • C-MYC is known as c-Myc-binding protein or Associate of Myc 1 (AMY-1). Its gene name is known sMYCBP o AMY 1.
  • C-MYC can control the transcriptional activity of MYC. It can also stimulate the activation of E box-dependent transcription by MYC.
  • cMYC polynucleotide 1320bp; NM_002467.6 Transcript vl - 3721bp (residues 409-1728) (SEQ ID NO: 9)
  • the c-MYC useful for the present method comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ
  • the nucleotide sequence encoding a c-MYC protein useful for the present method comprises a nucleotide sequence having at least about 75%, at least about 76%, at least about 77%, at least 78%, at least about 79%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about
  • LIN28A is known as Protein lin-28 homolog A (Lin-28A) or Zinc finger CCHC domain-containing protein 1. Its gene name is known as LIN28A, CSDD1 , LIN28, or ZCCHC1.
  • LIN28A is an RNA-binding protein that inhibits processing of pre-let-7 miRNAs and regulates translation of mRNAs that control developmental timing, pluripotency and metabolism. It may recognize a common structural G-quartet (G4) feature in its miRNA and mRNA targets (Probable). It is also known as 'translational enhancer' that drives specific mRNAs to polysomes and increases the efficiency of protein synthesis. Its association with the translational machinery and target mRNAs results in an increased number of initiation events per molecule of mRNA and, indirectly, in mRNA stabilization. Binds IGF2 mRNA, MYODI mRNA, ARBP/36B4 ribosomal protein mRNA and its own mRNA.
  • hLIN28_wild sequence (SEQ ID NO: 11) - 99% identical to Homo sapiens lin-28 homolog A (LIN28A), mRNA Sequence ID: NM_024674.6 Length: 3975 - residues 76 to 705.
  • the LIN28A useful for the present method comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 12.
  • the nucleotide sequence encoding a LIN28A protein useful for the present method comprises a nucleotide sequence having at least about 75%, at least about 76%, at least about 77%, at least 78%, at least about 79%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 11.
  • NANOG is known as Homeobox protein NANOG or Homeobox transcription factor Nanog (hNanog). Its gene name is NANOG.
  • NANOG is a transcription regulator involved in inner cell mass and embryonic stem
  • NANOG Primary UNIPROT accession number for NANOG is known as Q9H9S0. Secondary accessions number for NANOG is D3DUU4, Q2TTG0, or Q6JZS5.
  • hNanog_wild nt sequence Homo sapiens Nanog homeobox (NANOG), transcript variant 1, mRNA NCBI Reference Sequence: NM_024865.4. From residues 214 to 1131 (SEQ ID NO: 3) ATGAGTGTGGATCCAGCTTGTCCCCAAAGCTTGCCTTGCTTTGAAGCATCCGACTGTAAAGAATCTTCACCTATGCCT GTGATTTGTGGGCCTGAAGAAAACTATCCATCCTTGCAAATGTCTTCTGCTGAGATGCCTCACACGGAGACTGTCTCT CCTCCATGGATCTGCTTATTCAGGACAGCCCTGATTCTTCCACCAGTCCCAAAGGCAAACAACCCACT TCTGCAGAGAAGAGTGTCGCAAAAAAGGAAGACAAGGTCCCGGTCAAGAAACAGAAGACCAGAACTGTGTTCTCTTCC ACCCAGCTGTGTGTACTCAATGATAGATTTCAGAGACAGAAATACCTCAGCCTCCAGCAGATGCAAGAACTCTCT
  • the NANOG useful for the present method comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 4.
  • the nucleotide sequence encoding a NANOG protein useful for the present method comprises a nucleotide sequence having at least about 75%, at least about 76%, at least about 77%, at least 78%, at least about 79%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 3.
  • SV40 Large T antigen is known as Genbank SV40:NCBI Reference Sequence: NF 043127. 1 large T antigen [Macacamulatta polyomavirus 1] .
  • the SV40 large T antigen useful for the present method comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 13.
  • the reprogramming factor comprises a molecule that increases expression of an OCT4 protein, a SOX2 protein, a KLF4 protein, a c-MYC protein, a LIN28 protein, a NANOG protein, or any combination thereof.
  • the reprogramming factor comprises (i) a molecule that increases expression of an OCT4 protein (e.g., a vector or mRNA encoding OCT4 protein), a molecule that increases expression of a SOX2 protein(e.g., a vector or mRNA encoding SOX2 protein), a molecule that increases expression of a KLF4 protein(e.g., a vector or mRNA encoding KLF4 protein), and a molecule that increases expression of a c-MYC protein(e.g., a vector or mRNA encoding c-MYC protein).
  • OCT4 protein e.g., a vector or mRNA encoding OCT4 protein
  • SOX2 protein e.g., a vector or mRNA encoding SOX2 protein
  • KLF4 protein e.g., a vector or mRNA encoding KLF4 protein
  • c-MYC protein e.g., a vector or
  • the reprogramming factor comprises a molecule that increases expression of an OCT4 protein (e.g., a vector or mRNA encoding OCT4 protein), a molecule that increases expression of a SOX2 protein(e.g., a vector or mRNA encoding SOX2 protein), and a molecule that increases expression of a KLF4 protein(e.g., a vector or mRNA encoding KLF4 protein).
  • an OCT4 protein e.g., a vector or mRNA encoding OCT4 protein
  • SOX2 protein e.g., a vector or mRNA encoding SOX2 protein
  • KLF4 protein e.g., a vector or mRNA encoding KLF4 protein
  • the reprogramming factor comprises a molecule that increases expression of an OCT4 protein (e.g., a vector or mRNA encoding OCT4 protein), a molecule that increases expression of a SOX2 protein (e.g., a vector or mRNA encoding SOX2 protein), a molecule that increases expression of a KLF4 protein (e.g., a vector or mRNA encoding KLF4 protein), a molecule that increases expression of a c-MYC protein (e.g., a vector or mRNA encoding c-MYC protein), a molecule that increases expression of a LIN28 protein (e.g., a vector or mRNA encoding LIN28 protein), and a molecule that increases expression of a NANOG protein (e.g., a vector or mRNA encoding NANOG protein).
  • OCT4 protein e.g., a vector or mRNA encoding OCT4 protein
  • the reprogramming factors are expressed in one vector. In some aspects, the reprogramming factors are expressed from a polycistronic vector. In some aspects, the reprogramming factors are expressed in multiple vectors. In some aspects, the recombinant expression vector is a viral vector. Suitable viral vectors include, without limitation, Sendai virus vectors, retroviral vectors, lentiviral vectors, alphaviral, vaccinia, adenoviral, adeno-associated viral, herpes viral, and fowl pox viral vectors, and preferably have a native or engineered capacity to transform immune cells (e.g., T cells). In some aspects, the viral vectors are pseudotyped with a heterologous viral envelope protein.
  • the viral vector is a lentivirus or Sendai virus vector pseudotyped with a suitable envelope, e.g., a virus envelope (see, e.g., Blood Adv 2017 Oct 24;l(23):2088-2104; Mol Ther 2012 Sep;20(9): 1699-712).
  • the viral vector is an integration deficient lentiviral vector. Suitable viral vectors are known in the art and described, for example, in J. Biol. Chem. (2011) 286:4760-4771; Stem Cell Research & Therapy (2019) 10:185.
  • the recombinant expression vector is delivered in a nanoparticle.
  • the reprogramming factors are delivered using a nanoparticle.
  • Such nanoparticles can be designed to deliver specific mRNA or other macromolecules to lymphocytes in a transient and dose-controlled manner. These nanoparticles can be designed to target specific cell subtypes and, upon binding to them, stimulate receptor mediated endocytosis, thereby introducing the synthetic mRNA they carry which the cells can now express. Because nuclear transport and transcription of the transgene are not required, this process is fast and efficient.
  • the partial reprogramming comprises (a) contacting immune cells with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT4, SOX2 and C-MYC, and (ii) optionally SV40 Large T cell antigen, for a period of time sufficient for reprogrammed cells to form.
  • one or more reprogramming vectors are delivered to the T cells in separate vectors.
  • KLF4, OCT4, and SOX2 (KOS) are delivered in a single vector.
  • the vector expressing KOS is a viral vector that is delivered at a titer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 MOIs (Multiplicity of Infection).
  • KLF4 is delivered in a viral vector at a titer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 MOIs.
  • cMyc is delivered in a viral vector at a titer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 MOIs.
  • a vector expressing SV40 Large T cell antigen is further delivered to the cell and is a viral vector delivered at a titer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 MOIs.
  • the T cells are contacted with a Sendai vector expressing KOS at 10 MOI, a Sendai vector expressing KLF4 at 10 MOI, a Sendai vector expressing C-MYC at 3 MOI, and a Sendai vector expressing SV40 Large T cell antigen at 5 MOI.
  • the T cell is contacted with a multi ci stronic vector, such as a lentiviral vector, a Sendai virus vector or a non-viral vector, expressing KOSM.
  • endogenous reprogramming factor expression is induced using one or more chemical compounds (see e.g., Guan et al., 2022 Nature DOI 10.1038/s41586-022-04593- 5).
  • Some aspects of the present disclosure are directed to a method of producing cells with at least one T cell marker, cells with a T cell phenotype, and/or a T cell lineage (“redirected T cells”), comprising (a) contacting immune cells obtained from a subject with (i) at least one reprogramming factor selected from the group consisting of a molecule that increases expression of a KLF4 protein (e.g., a KLF4 protein encoding vector or mRNA), a molecule that increases expression of an OCT4 protein (e.g., a OCT4 protein encoding vector or mRNA), a molecule that increases expression of a SOX2 protein (e.g., a SOX2 protein encoding vector or mRNA) and a molecule that increases expression of a C-MYC protein (e.g., a C-MYC protein encoding vector or mRNA), and (ii) optionally a molecule that increases expression of a SV40 Large T Antigen
  • expression of the at least one reprogramming factors in the reprogrammed cells is inhibited by the addition of an agent that inhibits expression of the at least one reprogramming factor.
  • the agent that inhibits expression of the at least one reprogramming factor is a small molecule inhibitor that specifically inhibits the expression of one or more of KLF4, OCT4, SOX2 and C-MYC expression.
  • Some aspects of the present disclosure relate in part to methods for reprogramming cells. Some aspects of the present disclosure are directed to a method of inducing partially reprogrammed cells to become redirected cells, comprising culturing the partially reprogrammed cells in a medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof. In some aspects, the medium further comprises a concentration of potassium higher than 5 mM. In some aspects, the partially reprogrammed cells are induced to express at least one T cell marker. In some aspects, the partially reprogrammed cells are induced to increase expression of at least one T cell marker.
  • the partially reprogrammed cells are induced to gain at least one T cell function. In some aspects, the partially reprogrammed cells are induced to gain a T cell phenotype. In some aspects, the partially reprogrammed cells are induced to become a T cell lineage cell.
  • redirected cells after the redirection phase, redirected cells re-express T cell associated genes.
  • the redirected cells prepared by the present methods do not show aberrant expression of unconventional, T or B cell markers like those observed on human iPSC- derived T cell products, such as but not limited to NCAM1, NCR2, FCGR3A, KIR2DL4, or KIR2DS4.
  • T-cell lineage or “T cell lineage” cell refers to a cell which expresses one or more cell markers within the T cell lineage differentiation pathway.
  • T cell markers include but are not limited to TCRa, TCRb, TCRg, TCRd, CD3, CD3gamma, LCK, CD3zeta, IL2 receptor alpha (IL2RA), IL2RB, IL2Rgamma, IL7RA, CD45RO, CCR7, CD45RA, CD62L, CD27, CD28, TCF7, CD4, CD5, CD7, CD8, CD45, CD69, LEF1, CXCR4, GZMB, or any combination thereof.
  • a T cell lineage cell may have any of the following T cell marker expression profiles: CD7 and CD5 (e.g., immature T cells); CD45+CD7+CD5-; CD45+CD7+CD5+CD3-CD4-CD8- (e.g., CD4CD8 double negative (DN) immature T cells); CD45+CD7+CD5+CD3-CD4+CD8+ (e.g., CD4CD8 double positive (DP) immature T cells); CD45+CD7+CD5+CD3+CD4+CD8+ (e.g., CD4CD8 DP immature T cells, late stage); CD45+CD7+CD5+CD3+CD4+ (normal CD4 SP T cells); CD45+CD7+CD5+CD3+CD8ap+ (normal CD8 SP T cells); TCRa+CD3+; TCRb+CD3+; TCRg+CD3+; TCRd+CD3+; NK1.1+ CD3+ CD3+ CD
  • T cell lineage cells include but are not limited to a T cell progenitor, immature T cells, conventional T cells, and/or a T cell of a particular class.
  • Non-limiting classes of T cells include CD4+CD8+ double positive T cells, intrathymic CD4 or CD8 single positive cells, recent thymic emigrant cells, unconventional T cells (such as CD8aa, gamma delta T cells), effector T cells (such as CD8 + T cells) and Th cells (such as CD4 + T cells).
  • a T cell lineage cell may be a Thl cell, a Th2 cell, a Tcl7 cell, a Thl7 cell, a T reg cell, or an NKT cell.
  • a T cell phenotype may be a molecular phenotype (e.g., gene/protein expression pattern) or a functional phenotype (e.g., cytotoxicity, antigen specific killing/activation, cytokine secretion, granzyme and/or perforin release etc.).
  • T cell phenotypes include Tnaive, T effector cells, central memory T cells (TCM cells), effector memory T cells (TEM cells), tissue resident memory T cells (TRM cells), stem cell-like memory T cells (TSCM cells), TH1, TH2, and regulatory T cells.
  • Some aspects of the present disclosure are directed to a method of redirecting partially reprogrammed cells, comprising culturing the partially reprogrammed cells in a medium comprising a concentration of potassium higher than 5 mM.
  • the redirected cells have at least one T cell marker.
  • the redirected cells have an increased expression of at least one T cell marker.
  • the redirected cells have a T cell phenotype.
  • the redirected T cells have one or more T cell functions.
  • the redirected cells are a T cell lineage cell.
  • Some aspects of the present disclosure are directed to a method of producing redirected cells from partially reprogrammed cells comprising culturing the partially reprogrammed cells in a medium comprising a concentration of potassium higher than 5 mM (HKM).
  • Some aspects of the present disclosure are directed to a method of producing redirected cells from partially reprogrammed cells comprising culturing the partially reprogrammed cells in a basal media suitable for culturing immune cells, optionally comprising one or more cytokines, growth factors, with or without serum or serum replacement, and further comprising 2DG, metformin, an mTOR inhibitor (e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof), a phosphoAKT inhibitor (e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 751, ipatasertib, and any combination thereof), or a combination of any one or more of the foregoing compounds.
  • the redirected cells exhibit an increased expression of at least one T cell marker compared to the partially reprogrammed cells
  • the partially reprogrammed cells do not express the at least one T cell marker expressed by the redirected cells.
  • the expression of the at least one T cell marker can be reduced to the point that the marker is no longer detectable on the surface of the cell.
  • the at least one T cell marker is no longer detected on the cell surface, i.e., the T cell marker is no longer detectable on the cell surface.
  • the partially reprogrammed cells do not exhibit the T cell phenotype exhibited by the redirected cells.
  • the partially reprogrammed cells do not exhibit T cell identity exhibited by the redirected cells.
  • the partially reprogrammed cells are not T cell lineage cells.
  • the redirected T cells further exhibit an increased expression of one or more T cell markers compared to the partially reprogrammed cells.
  • the redirected T cells further exhibit a decreased expression of one or more stem cell markers or non-T cell lineage markers compared to the partially reprogrammed cells.
  • the expression of the T cell marker is measured by Flow cytometry, RNA Seq, CITE-seq, PCR, Reverse Transcription PCR (RT-PCR), quantitative PCR (qPCR) or other PCR methods, Western blot, ELISA, Meso Scale Discovery (MSD), Cytometry by time-of flight (CYTOF®), immunofluorescent tissue staining, or any combination thereof. These methods are well known in the art.
  • Assays useful for measuring cell function, phenotype and gene and protein expression profiles are known in the art and include, for example flow cytometry, intracellular staining (ICS), INCUCYTE immune cell killing analysis, Meso Scale Discovery (MSD) or similar assay, persistent antigen stimulation assays, bulk and single cell RNAseq (see e.g., Fron Genet. 2020; 11 :220; 2019 Bioinformatics 35:i436-445; 2019 Annual Review of Biomed. Data Sci.
  • CITE-seq uses DNA-barcoded antibodies to convert detection of proteins into a quantitative, sequenceable readout.
  • Antibody-bound oligos act as synthetic transcripts that are captured during most large-scale oligodT-based scRNA-seq library preparation protocols (e.g. lOx Genomics, Drop-seq, ddSeq).
  • MSD Meso Scale Discovery
  • MSD provides quantitation of protein markers by using multiplex immunoassays based on electrochemiluminescence and multiarray technology for detection of multiple proteins in a single sample.
  • Mass cytometry or CYTOF® (Fluidigm) is a variation of flow cytometry in which antibodies are labeled with heavy metal ion tags rather than fluorochromes.
  • CYTOF® is used to quantify labeled targets on the surface and interior of single cells. Readout is by time-of-flight mass spectrometry.
  • the redirected T cells express at least two more, at least three more, at least four more, at least five more, at least six more, at least seven more, at least eight more, at least nine more, or at least ten more T cell markers compared to the partially reprogrammed cells.
  • the redirected T cells express at least two less, at least three less, at least four less, at least five less, at least six less, at least seven less, at least eight less, at least nine less, or at least ten less stem cell markers compared to the partially reprogrammed cells.
  • the T cell marker comprises one or more of TCRa, TCRb, TCRg, TCRd, CD3, CD3gamma, LCK, CD3zeta, IL2 receptor alpha (IL2RA), IL2RB, IL2Rgamma, IL7RA, CD45RO, CCR7, CD45RA, CD62L, CD27, CD28, TCF7, CD4, CD5, CD7, CD8, CD45, CD69, LEF1, CXCR4, and GZMB.
  • IL2 receptor alpha IL2RA
  • IL2RB IL2 receptor alpha
  • IL7RA IL7RA
  • CD45RO CCR7, CD45RA, CD62L
  • redirected T cell refers to a partially reprogrammed cell that has become a T cell lineage cell after partial reprogramming.
  • a redirected T cell has been induced to become a T cell lineage cell after partial reprogramming.
  • a redirected T cell is a T cell that has regained T cell phenotype and/or function after partial reprogramming.
  • a redirected T cell is a T cell that has been partially reprogrammed and has returned to the T cell lineage following contact with a T cell activating and/or stimulating agent.
  • a redirected T cell is a T cell that has been partially reprogrammed and has regained expression of one or more T cell markers and/or regained one or more T cell functions following contact with a T cell activating or stimulating agent.
  • the redirected T cells can express at least one T cell marker that is not expressed in the partially reprogrammed cell or have increased expression of the T cell marker compared to the expression of the marker in the partially reprogrammed cell.
  • a redirected T cell loses expression of one or more non T cell lineage-specific markers and/or one or more stem cell markers that was expressed in the partially reprogrammed cell.
  • the redirected T cells show characteristics of cellular rejuvenation while retaining lineage stability and retaining antigen-specificity, as described in further detail herein.
  • a redirected T cell has regained one or more T cell functions.
  • T cell functions include but are not limited to, cytotoxicity, antigen specific killing/activation, cytokine expression/secretion (e.g., IFN-y, TNF-a, and/or IL-2) granzyme release and/or perforin release.
  • the methods described herein produce redirected T cells that provide any one or more of increased proliferation, survival, persistence, and/or cytotoxicity; reduced epigenetic age, increased telomere length, improved metabolism, decreased apoptosis and other markers associated with cell senescence and improved anti-tumor activity as compared to a population of reference T cells, e.g., terminally differentiated T cells or T cells that have not been partially reprogrammed and redirected.
  • redirected T cells exhibit a polyclonal TCR repertoire and the capacity for long term in vivo engraftment.
  • Redirected T cells may exhibit biological and phenotypic characteristics of younger T cells in terms of epigenetic signature, telomere length, metabolic activity and functionality.
  • the partial reprogramming process as described herein may cause the T cell to lose some aspects of the T cell phenotype, which are regained through T cell activation through TCR signaling and/or culturing in an appropriate media.
  • the redirected T cells can be used to target cancers, viruses, or autoimmune diseases.
  • the partially reprogrammed cells can be used to reconstitute the whole adaptive immune system or alleviate immunocompromised patients.
  • the redirected T cells are reprogrammed and redirected through transient expression of reprogramming factors.
  • the morphology and cell surface expression profile of the T cells begins to change.
  • the cell begins to express certain cell surface markers and the expression of T cell markers decreases.
  • the cell begins to express certain cell surface markers and the expression of T cell markers decreases around day 5 of partial reprogramming, indicating that the dedifferentiation process has initiated.
  • the partially reprogrammed cells are stimulated with one or more T cell activating molecules, such as anti-CD3 antibodies or other CD3 agonist, and cultured in HKM.
  • this T cell stimulation also referred to herein as “reactivation”
  • reactivation can drive the reprogrammed cells to return to the T cell lineage.
  • the redirected T cells have characteristics of much younger cells based on the epigenetic clock analysis, have a more stemlike phenotype and have acquired high proliferating potential.
  • an mTOR inhibitor e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof
  • a phosphoAKT inhibitor e.g.,
  • an mTOR inhibitor e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof
  • a phosphoAKT inhibitor e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 75
  • an mTOR inhibitor e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof
  • a phosphoAKT inhibitor e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 75
  • an mTOR inhibitor e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof
  • a phosphoAKT inhibitor e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 75
  • an mTOR inhibitor e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof
  • a phosphoAKT inhibitor e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 75
  • an mTOR inhibitor e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof
  • a phosphoAKT inhibitor e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 75
  • an mTOR inhibitor e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof
  • a phosphoAKT inhibitor e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 75
  • an mTOR inhibitor e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof
  • a phosphoAKT inhibitor e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 75
  • an mTOR inhibitor e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof
  • a phosphoAKT inhibitor e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 75
  • an mTOR inhibitor e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof
  • a phosphoAKT inhibitor e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 75
  • an mTOR inhibitor e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof
  • a phosphoAKT inhibitor e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 75
  • an mTOR inhibitor e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof
  • a phosphoAKT inhibitor e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 75
  • an mTOR inhibitor e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof
  • a phosphoAKT inhibitor e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 75
  • the culturing in culture medium comprising one or more of the foregoing compounds is for between 1 day and 7 days. In some aspects, the culturing in culture medium comprising one or more of the foregoing compounds is for between 2 days and 7 days. In some aspects, the culturing in culture medium comprising one or more of the foregoing compounds is for between 3 days and 7 days. In some aspects, the culturing in culture medium comprising one or more of the foregoing compounds is for between 4 days and 7 days. In some aspects, the culturing in culture medium comprising one or more of the foregoing compounds is for between 5 days and 7 days. In some aspects, the culturing in culture medium comprising one or more of the foregoing compounds is for between 6 days and 7 days.
  • the culturing is in culture medium comprising at least about 5 mM potassium ion.
  • the culturing in culture medium comprising at least about 5 mM potassium ion is for between one day and seven days.
  • the culturing in culture medium comprising at least about 5 mM potassium ion is for about one day, about two days, about three days, about four days, about five days, about six days, or about seven days.
  • the medium further comprises calcium ion, glucose, or any combination thereof.
  • the medium is capable of increasing the speed of or otherwise decreasing the time required to redirect partially reprogrammed cells. In some aspects, the medium is capable of increasing the number of redirected cells and/or maintaining a reduced epigenetic age of the redirected cells in the final cell product as compared to the starting cell population or the cells that are redirected in a control media. In some aspects, the redirected T cells have one or more sternness properties including high epigenetic plasticity. Other advantageous phenotypic markers that the redirected cells can acquire include expression of L-selectin (CD62L), IL-7Ra, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, CXCR3, TCF7 and LFA-1.
  • the redirected cells acquire T memory stem cell expression markers, e.g., are memory T cells that express CD95, CD45RA, CCR7, and CD62L and are endowed with the stem cell-like ability to self-renew and the multipotent capacity to reconstitute the entire spectrum of memory and effector T cell subsets.
  • the redirected cells are "central memory T cells” or "TCM cells” which are memory T cells that express CD45RO, CCR7, and CD62L.
  • redirected cells have effector memory T cell markers such as expression of CD45RO but lack of expression of CCR7 and CD62L.
  • the redirected cells herein have a stem-like phenotype and have an improved capacity for proliferation.
  • the redirected cells have characteristics of a naive T cell, "TN cells,” which are T cells that express CD45RA, CCR7, and CD62L, but which do not express CD95.
  • the redirected cells can exhibit biological and phenotypic characteristics of younger T cells in terms of epigenetic signature, telomere length and functionality.
  • the “Horvath Clock” as described in Horvath and Raj is used to determine the epigenetic age of the cells described herein including the redirected immune cell (e.g., a T cell).
  • the Horvath Clock is used to determine whether partial reprogramming has occurred.
  • the Horvath Clock is used to predict the counts of exhausted CD8 T cells.
  • the Horvath clock is used to determine whether a population of immune cell (e.g., a T cell) is suitable for administration of patient.
  • other epigenetic clocks or algorithms for example, the Hannum Clock or the Levine Clock are used. See, e.g., Hannum et al., 2013, Mol. Cell., 49:359-369; Levine et al., 2018, Aging, 10(4):573-591.
  • redirected cells exhibit a reduced epigenetic age (eAge) as compared to control T cells (e.g., T cells that have been cultured without contact with one or more reprogramming factors).
  • eAge can be determined using a known epigenetic clock determination method, such as the Horvath clock as described elsewhere herein.
  • the redirected cells have increased telomere length as compared to control T cells. Telomere length can be measured using methods known in the art (see e.g., Rosenberg et al., 2011, Clin Cancer Res 17, 4550-4557) and commercially available kits (e.g., TELOTAGGG, Sigma-Aldrich).
  • the telomere length is elongated by between about 0.1-3kb. In some aspects, the telomere length is elongated by about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or about 1 kb. In some aspects, the telomere is elongated by about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 kb.
  • the epigenetic age of the redirected cells is at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% younger than the starting T cell population.
  • the epigenetic age of the redirected cells is about 5% to about 90%, about 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 90%, about 15% to about 75%, about 15% to about 50%, about 20% to about 75%, or about 20% to about 50% younger than the starting T cell population.
  • the epigenetic age of the redirected cells is about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% younger than the starting T cell population.
  • the present disclosure provides a population of redicted cells whose epigenetic age is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% younger than the starting cell population (e.g., the chronological age) or an appropriate control cell and wherein the redirected cells do not express NCAM1, NCR2, FCGR3A, KIR2DL4, or KIR2DS4.
  • the present disclosure provides a redirected T cell whose epigenetic age is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% younger than the starting T cell population (e.g., the chronological age) (or an appropriate control T cell) and wherein the T cell is enriched for oxidative phosphorylation, fatty acid metabolism, glycolysis and hypoxia gene sets as determined, for example, by transcriptome analysis.
  • the present disclosure provides a redirected T cell or a population of such redirected T cells, whose epigenetic age is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% younger than its chronological age (or an appropriate control T cell) and wherein the redirected T cells is enriched for both oxidative phosphorylation and glycolysis gene sets as compared to control T cells as determined, for example, by transcriptome analysis.
  • Some aspects of the present disclosure are directed to a method of producing redirected T cells, comprising (a) contacting immune cells obtained from a subject with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT4, SOX2 and C-MYC, and (ii) optionally SV40 Large T cell antigen, LIN28 and/or NANOG, for a period of time sufficient to partially reprogram cells; and (b) culturing the reprogrammed cells in a medium comprising 2DG, metformin, an mTOR inhibitor (e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof), a phosphoAKT inhibitor (e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 751, ipatasertib, and any combination thereof),
  • Some aspects of the present disclosure are directed to a method of producing redirected T cells, comprising (a) contacting immune cells obtained from a subject with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT4, SOX2 and C-MYC, and (ii) optionally SV40 Large T cell antigen, LIN28 and/or NANOG, for a period of time sufficient for reprogrammed cells to form; and (b) culturing the reprogrammed cells in a medium comprising 2DG, metformin, an mTOR inhibitor (e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof), a phosphoAKT inhibitor (e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 751, ipatasertib, and
  • the partially reprogrammed cells are contacted with one or more T cell activating and/or costimulatory and/or immunomodulatory agents as described elsewhere herein in a medium comprising 2DG, metformin, an mTOR inhibitor (e.g., rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof), a phosphoAKT inhibitor (e.g., phosphoAKT inhibitor VIII, AZD5363, perifosine, MK-2206, GSK-2141795, afuresertib, ARQ 092, ARQ 751, ipatasertib, and any combination thereof), or a combination of any one or more of the foregoing compounds, with the addition of one or more supplements such as OpTmizer Cell Supplement, in some aspects with the addition of Immune Cell Serum Replacement (e.g., T cell serum replacement) and other supplements such as L-Glutamine; GlutaMAX; cytokines such as IL
  • the cells are activated (e.g., prior to reprogramming or for the redirection phase of the methods herein) using commercially available reagents such as TRANSACT (Miltenyi Biotec).
  • TRANSACT Microwave Access Control Protocol
  • the partially reprogrammed cells are activated using TRANSACT at a ratio that allows the partially reprogrammed cells to regain its T cell phenotype.
  • the partially reprogrammed cells are activated with TRANSACT at 1 : 10, 1 :50 1 : 100, 1 : 150, 1 :200, 1 :250, 1 :300, 1 :350, 1 :400, 1 :450, 1 :500, 1 :550, 1 :600, 1 :650, 1 :700, 1 :750, 1 :800, 1 :850, 1 :900, 1 : 1,000, 1 :2000, 1 :3000, 1 :4000 or 1 : 10,000.
  • the partially reprogrammed cells are activated with an antigen of interest, target cells expressing antigen, or tumor cells. Antigens of interest are disclosed elsewhere herein, e.g., as CAR or TCR targets. Such antigens can be used here for activating partially reprogrammed cells.
  • the redirected T cells express at least one T cell marker compared to the partially reprogrammed cells. In some aspects, the redirected T cells gain one or more T cell functions compared to the partially reprogrammed cells. In some aspects, the redirected T cells gain a T cell phenotype compared to the partially reprogrammed cells. In some aspects, the redirected T cells become a T cell lineage cell. In some aspects, the redirected T cells exhibit an increased expression of at least one T cell marker.
  • the redirected T cells further exhibit an increased expression of one or more T cell markers (e.g., TCRa, TCRb, TCRg, TCRd, CD3, CD3gamma, LCK, CD3zeta, IL2 receptor alpha (IL2RA), IL2RB, IL2Rgamma, IL7RA, CD45RO, CCR7, CD45RA, CD62L, CD27, CD28, TCF7, CD4, CD5, CD7, CD8, CD45, CD69, LEF1, CXCR4, or GZMB) compared to the partially reprogrammed cells.
  • T cell markers e.g., TCRa, TCRb, TCRg, TCRd, CD3, CD3gamma, LCK, CD3zeta, IL2 receptor alpha (IL2RA), IL2RB, IL2Rgamma, IL7RA, CD45RO, CCR7, CD45RA, CD62L, CD27, CD28, TCF7, CD4, CD5,
  • expression is increased by about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% or more compared to the partially reprogrammed cells.
  • the redirected T cells exhibit a decreased expression of one or more non-T cell lineage markers (e.g., SSEA4, CD9, CD90, CD164, CD63, CD71, CD326, TRA-1-81 or TRA-1-60-R) compared to the partially reprogrammed cells.
  • expression is decreased by about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% or more compared to the partially reprogrammed cells.
  • the expression of one or more non-T cell lineage markers is non-detectable.
  • the redirected T cells further exhibit a decreased expression of one or more stem cell markers and/or other non-T cell lineage markers compared to the partially reprogrammed cells.
  • expression is decreased by about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% compared to the partially reprogrammed cells.
  • the redirected T cells further exhibit a decreased expression of one or more stem cell markers and/or other non-T cell lineage markers (e.g., SSEA4, CD9, CD90, CD164, CD63, CD71, CD326, TRA-1-81 or TRA-1-60-R) and exhibit an increased expression of one or more T cell lineage markers (e.g., TCRa, TCRb, TCRg, TCRd, CD3, CD3gamma, LCK, CD3zeta, IL2 receptor alpha (IL2RA), IL2RB, IL2Rgamma, IL7RA, CD45RO, CCR7, CD45RA, CD62L, CD27, CD28, TCF7, CD4, CD5, CD7, CD8, CD45, CD69, LEF1, CXCR4, or GZMB) compared to the partially reprogrammed cells.
  • expression of non-T cell lineage markers is decreased by about 5% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased by about 10% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased by about 15% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased by about 20% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased by about 25% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased by about 30% compared to the partially reprogrammed cells.
  • expression of non-T cell lineage markers is decreased by about 35% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased by about 40% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased by about 45% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased by about 50% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased by about 55% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased by about 60% compared to the partially reprogrammed cells.
  • expression of non-T cell lineage markers is decreased by about 65% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased by about 70% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased by about 75% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased by about 80% compared to the partially reprogrammed cells.
  • expression of non-T cell lineage markers is decreased by about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or at about 80% compared to the partially reprogrammed cells and expression of T-cell lineage markers is increased by about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% or more compared to the partially reprogrammed cells.
  • expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 5% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 10% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 15% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 20% compared to the partially reprogrammed cells.
  • expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 25% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 30% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 35% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 40% compared to the partially reprogrammed cells.
  • expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 45% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 50% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 55% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 60% compared to the partially reprogrammed cells.
  • expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 65% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 70% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 75% compared to the partially reprogrammed cells. In some aspects, expression of non-T cell lineage markers is decreased and expression of T-cell lineage markers is increased by at least about 80% compared to the partially reprogrammed cells.
  • the expression of one or more non-T cell lineage markers is non- detectable.
  • the expression of the T cell marker is measured by Flow cytometry, RNA Seq, CITE-seq, PCR, Reverse Transcription PCR (RT-PCR), quantitative PCR (qPCR) or other PCR methods, Western blot, ELISA, Meso Scale Discovery (MSD), Cytometry by time-of flight (CYTOF®), immunofluorescent tissue staining, or any combination thereof. These methods are well known in the art.
  • Assays useful for measuring cell function, phenotype and gene and protein expression profiles are known in the art and include, for example flow cytometry, intracellular staining (ICS), INCUCYTE immune cell killing analysis, Meso Scale Discovery (MSD) or similar assay, persistent antigen stimulation assays, bulk and single cell RNAseq (see e.g., Fron Genet. 2020; 11 :220; 2019 Bioinformatics 35:i436-445; 2019 Annual Review of Biomed. Data Sci.
  • CITE-seq uses DNA-barcoded antibodies to convert detection of proteins into a quantitative, sequenceable readout.
  • Antibody-bound oligos act as synthetic transcripts that are captured during most large-scale oligodT-based scRNA-seq library preparation protocols (e.g. lOx Genomics, Drop-seq, ddSeq).
  • MSD Meso Scale Discovery
  • MSD provides quantitation of protein markers by using multiplex immunoassays based on electrochemiluminescence and multiarray technology for detection of multiple proteins in a single sample.
  • Mass cytometry or CyTOF® (Fluidigm) is a variation of flow cytometry in which antibodies are labeled with heavy metal ion tags rather than fluorochromes. CyTOF® is used to quantify labeled targets on the surface and interior of single cells. Readout is by time-of-flight mass spectrometry.
  • the redirected cells express at least two more, at least three more, at least four more, at least five more, at least six more, at least seven more, at least eight more, at least nine more, or at least ten more T cell markers compared to the partially reprogrammed cells.
  • the redirected T cells express at least two less, at least three less, at least four less, at least five less, at least six less, at least seven less, at least eight less, at least nine less, or at least ten less stem cell markers and/or non-T cell lineage markers compared to the partially reprogrammed cells.
  • the T cell marker comprises one or more of TCRa, TCRb, TCRg, TCRd, CD3, CD3gamma, LCK, CD3zeta, IL2Rgamma, IL2RA, IL2RB, IL7RA, CCR7, CD62L, CD27, CD28, TCF7, CD4, CD5, CD7, CD8, CD45, CD69, LEF1, CXCR4, and GZMB.
  • the partially reprogrammed cells are capable of adhering to a culture vessel surface. In some aspects, the partially reprogrammed cells are loosely attached to the culture vessel surface. In some aspects, the partially reprogrammed cells are semi -adherent, loosely adherent, adherent, or strongly adherent (strongly attached).
  • the cells disclosed in the present disclosure can be expanded in a variety of different culture systems.
  • immune cells are expanded in flasks.
  • standard laboratory culture flasks such as T-flasks
  • the cells can be expanded in a closed system.
  • the simplest closed culture systems are cell culture bags. Bags that are specifically designed for cell culture are available in a variety of sizes, which allows bag culture systems to easily accommodate a wide variety of culture volumes. Culturing cells in bags is limited by the relatively low cell concentration that must be maintained.
  • cells collected in bags can be sterilely connected to cell culture bags or closed-system culture flasks and bioreactors.
  • culture media and other reagents can be manufactured in bags, which can be sterilely connected to maintain the closed nature of these culture systems.
  • Cells and culture media can also be removed sterilely from these closed- system bags, flasks, and bioreactors.
  • cells may be cultured in 3-dimensional (3D) cell culture systems.
  • Such 3D systems include 3D microfluidic systems, encapsulating cells in hydrogel (hydrogel microspheres), or culture using emulsion-based encapsulation (see e.g., WO22058615; US11154506; US2022112456; EP3235899).
  • the culturing in cell culture medium is carried out for is for between about one day and about 20 days, about 19 days, about 18 days, about 17 days, about 16 days, about 15 days, about 14 days, about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, or about 2 days.
  • the culturing in cell culture medium is carried out for about one day, about two days, about three days, about four days, about five days, about six days, or about seven days.
  • the culturing is about one day.
  • the culturing in cell culture medium is for about two days.
  • the culturing in cell culture medium is for about three days. In some aspects, the culturing in cell culture medium is for about four days. In some aspects, the culturing in cell culture medium is for about five days. In some aspects, the culturing in cell culture medium is for about six days. In some aspects, the culturing in cell culture medium is for about seven days. In some aspects, the culturing in cell culture medium is for about eight days. In some aspects, the culturing in cell culture medium is for about nine days. In some aspects, the culturing in cell culture medium is for about ten days.
  • the culturing is carried out in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof and is for between about one day and about 20 days, about 19 days, about 18 days, about 17 days, about 16 days, about 15 days, about 14 days, about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, or about 2 days.
  • the culturing in medium comprising comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is between about one day and about 20 days. In some aspects, the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is between about one day and about 19 days.
  • the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is between about one day and about 18 days. In some aspects, the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is between about one day and about 17 days.
  • the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is between about one day and about 16 days. In some aspects, the culturing in medium comprising (i) a phosphoAKT inhibitor,
  • the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is between about one day and about 15 days.
  • the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is between about one day and about 14 days.
  • the culturing in medium comprising
  • a phosphoAKT inhibitor (i) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is between about one day and about 13 days.
  • the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is between about one day and about 12 days.
  • the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is between about one day and about 11 days.
  • the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin,
  • an mTOR inhibitor (iv) 2DG, or (v) any combination thereof is between about one day and about 10 days.
  • the culturing in medium comprising (i) a phosphoAKT inhibitor,
  • the culturing in medium (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is for between about one day and about 9 days.
  • the culturing in medium (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is for between about one day and about 8 days.
  • the culturing in medium comprising a concentration of potassium higher than 5 mM is for between about one day and about 7 days.
  • the culturing in medium comprising a concentration of potassium higher than 5 mM is for between about one day and about 6 days.
  • the culturing in medium comprising a concentration of potassium higher than 5 mM is for between about one day and about 5 days. In some aspects, the culturing in medium comprising a concentration of potassium higher than 5 mM is for between about one day and about 4 days. In some aspects, the culturing in medium comprising a concentration of potassium higher than 5 mM is for between about one day and about 3 days. In some aspects, the culturing in medium comprising a concentration of potassium higher than 5 mM is for between about one day and about 2 days.
  • the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is for about one day, about two days, about three days, about four days, about five days, about six days, about seven days, about eight days, about nine days, about ten days, about eleven days, about twelve days, about thirteen days, about fourteen days, or about fifteen days. In some aspects, the culturing is about one day.
  • the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is for about two days. In some aspects, the culturing in medium (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is for about three days. In some aspects, the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is for about four days.
  • the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is for about five days. In some aspects, the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is for about six days. In some aspects, the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is for about seven days.
  • the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is for about eight days. In some aspects, the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is for about nine days. In some aspects, the culturing in medium comprising (i) a phosphoAKT inhibitor, (ii) metformin, (iii) an mTOR inhibitor, (iv) 2DG, or (v) any combination thereof is for about ten days.
  • the culturing in medium is carried out in media comprising a concentration of potassium higher than 5 mM and is for about one day, about two days, about three days, about four days, about five days, about six days, or about seven days. In some aspects, the culturing is about one day. In some aspects, the culturing in medium comprising a concentration of potassium higher than 5 mM is for about two days. In some aspects, the culturing in medium comprising a concentration of potassium higher than 5 mM is for about three days. In some aspects, the culturing in medium comprising a concentration of potassium higher than 5 mM is for about four days.
  • the culturing in medium comprising a concentration of potassium higher than 5 mM is for about five days. In some aspects, the culturing in medium comprising a concentration of potassium higher than 5 mM is for about six days. In some aspects, the culturing in medium comprising a concentration of potassium higher than 5 mM is for about seven days. In some aspects, the culturing in medium comprising a concentration of potassium higher than 5 mM is for about eight days. In some aspects, the culturing in medium comprising a concentration of potassium higher than 5 mM is for about nine days. In some aspects, the culturing in medium comprising a concentration of potassium higher than 5 mM is for about ten days.
  • the redirected T cells express a chimeric antigen receptor (CAR). In some aspects, the redirected T cells are engineered to express a CAR. In some aspects, the cells are engineered to express a CAR prior to reprogramming. In some aspects, the cells are engineered to express a CAR prior to redirection. In some aspects, the cells are engineered to express a CAR after redirection.
  • CAR chimeric antigen receptor
  • the redirected T cells express an engineered T cell receptor (TCR). In some aspects, the redirected T cells are engineered to express a TCR. In some aspects, the cells are engineered to express a TCR prior to reprogramming. In some aspects, the cells are engineered to express a TCR prior to redirection. In some aspects, the cells are engineered to express a TCR after redirection.
  • TCR T cell receptor
  • the cell comprises a construct expressing an antigen receptor and/or another additional polypeptide.
  • the antigen receptor comprises an antibody, an engineered antibody such as scFv, a CAR, an engineered TCR, a TCR mimic (e.g., an antibody - T cell receptor (abTCR) or a chimeric antibody-T cell receptor (caTCR)), or a chimeric signaling receptor (CSR).
  • a TCR can comprise an engineered TCR in which the antigenbinding domain of a TCR (e.g., an alpha/beta TCR or a gamma/delta TCR) has been replaced by that of an antibody (with or without the antibody’s constant domains); the engineered TCR then becomes specific for the antibody’s antigen while retaining the TCR’s signaling functions.
  • a TCR e.g., an alpha/beta TCR or a gamma/delta TCR
  • a chimeric signaling receptor can comprise (1) an extracellular binding domain (e.g., natural/modified receptor extracellular domain, natural/modified ligand extracellular domain, scFv, nanobody, Fab, DARPin, and affibody), (2) a transmembrane domain, and (3) an intracellular signaling domain (e.g., a domain that activates transcription factors, or recruits and/or activates JAK/STAT, kinases, phosphatases, and ubiquitin; SH3; SH2; and PDZ).
  • an extracellular binding domain e.g., natural/modified receptor extracellular domain, natural/modified ligand extracellular domain, scFv, nanobody, Fab, DARPin, and affibody
  • an intracellular signaling domain e.g., a domain that activates transcription factors, or recruits and/or activates JAK/STAT, kinases, phosphatases, and ubiquitin
  • an extracellular binding domain e
  • the antigen receptor targets an antigen of interest e.g., a tumor antigen or an antigen of a pathogen).
  • the antigens can include, without limitation, AFP (alphafetoprotein), avP6 or another integrin, BCMA, B7-H3, B7-H6, Braf, CA9 (carbonic anhydrase 9), CCL-1 (C-C motif chemokine ligand 1), CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7/8, CD45, CD47, CD56, CD66e, CD70, CD74, CD79a, CD79b, CD98, CD123, CD138, CD171, CD352, CEA (carcinoembryonic antigen), Claudin 18.2, Claudin 6, c-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (ectonucleotide
  • the antigen receptor targets hTERT. In some aspects, the antigen receptor targets KRAS. In some aspects, the antigen receptor targets Braf. In some aspects, the antigen receptor targets mesothelin. In some aspects, the antigen receptor targets ROR1. In some aspects, the antigen receptor targets HPV. In some aspects, the antigen receptor targets TGFpRII. In some aspects, the antigen receptor targets MAGE A10/A4. In some aspects, the antigen receptor targets AFP. In some aspects, the antigen receptor targets PRAME. In some aspects, the antigen receptor targets MAGE Al. In some aspects, the antigen receptor targets WT-1. In some aspects, the antigen receptor targets NY-ESO. In some aspects, the antigen receptor targets PRAME. In some aspects, the antigen receptor targets CD 19.
  • the antigen receptor targets BCMA. In some aspects, the antigen receptor targets CD 147. In some aspects, the antigen receptor targets CD 19. In some aspects, the antigen receptor targets CD 19 and CD22. In some aspects, the antigen receptor targets CD 19 and CD28. In some aspects, the antigen receptor targets CD20. In some aspects, the antigen receptor targets CD20 and CD 19. In some aspects, the antigen receptor targets CD22. In some aspects, the antigen receptor targets CD30. In some aspects, the antigen receptor targets CEA. In some aspects, the antigen receptor targets DLL3. In some aspects, the antigen receptor targets EGFRvIII. In some aspects, the antigen receptor targets GD2. In some aspects, the antigen receptor targets HER2.
  • the antigen receptor targets IL-1RAP. In some aspects, the antigen receptor targets mesothelin. In some aspects, the antigen receptor targets methothelin. In some aspects, the antigen receptor targets NKG2D. In some aspects, the antigen receptor targets PSMA. In some aspects, the antigen receptor targets TNMUC1.
  • one or more of the polynucleotides comprise DNA, RNA, or any combination thereof. In some aspects, one or more of the polynucleotides comprise mRNA. In some aspects, one or more of the polynucleotides comprise plasmid DNA.In some aspects, one or more of the polynucleotides comprise an expression vector. In some aspects, two or more of the polynucleotides are on the same DNA or RNA sequence. In some aspects, the two or more of the polynucleotides are on two different vectors.
  • the redirected T cells comprise a chimeric antigen receptor (CAR), an engineered T cell receptor, or any combination thereof.
  • CAR chimeric antigen receptor
  • the partially reprogrammed cells are genetically modified before the culturing with media comprising a phosphoAKT inhibitor, metformin, an mTOR inhibitor, 2DG, or any combination thereof.
  • the redirected cells are genetically modified during the culturing with media comprising a phosphoAKT inhibitor, metformin, an mTOR inhibitor, 2DG, or any combination thereof.
  • the redirected cells are genetically modified after the culturing with media comprising a phosphoAKT inhibitor, metformin, an mTOR inhibitor, 2DG, or any combination thereof..
  • the cells are genetically modified with a transgene encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a TCR mimic, prior to reprogramming, during reprogramming, or after reprogramming.
  • the cells are genetically modified with a transgene encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a TCR mimic, prior to redirection, during redirection, or after redirection.
  • the CAR, TCR or TCR mimic targets a tumor antigen.
  • Non-limiting examples of tumor-associted antigens include: AFP (alphafetoprotein), avP6 or another integrin, BCMA, BRAF, B7-H3, B7-H6, CA9 (carbonic anhydrase 9), (carcinoembryonic antigen), Claudin 18.2, Claudin 6, c-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase family member 3), EpCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, ephrinB2, EPHa2 (ephrine receptor A2), ERBB dimers, estrogen receptor, ETBR (endothelin B receptor), FAP-a (fibroblast activation protein a), fetal AchR (fetal acetylcholine receptor), FBP (a folate binding protein), FCRL5, FR-a (folate receptor alpha), GCC (guany
  • the CAR targets CD19, TRAC, TCRP, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL- 13Ra2, mesothelin, IL-1 IRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA- 4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF -I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase,
  • the TCR targets AFP, CD19, TRAC, TCRP, BCMA, CLL-1, CS1,
  • CD38 CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL- 13Ra2, mesothelin, IL-1 IRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF -I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2,
  • a transgene such as an expression vector encoding a chimeric antigen receptor, is delivered using RNA, a DNA vector, a retroviral vector, a lentiviral vector, an adeno-associated virus (AAV), an adenovirus, an AAV hybrid virus, a baculovirus, or any combination thereof.
  • RNA Ribonucleic acid
  • AAV adeno-associated virus
  • AAV hybrid virus a baculovirus
  • the partially reprogrammed T cells are activated or stimulated using a T cell activating or stimulating agent and/or costimulatory agent as described elsewhere herein, while being cultured in cell culture medium.
  • the medium comprises a phosphoAKT inhibitor, metformin, an mTOR inhibitor, 2DG, or any combination thereof and further comprises a T cell activating or stimulating agent as described elsewhere herein.
  • the T cell activating or stimulating agent and/or the costimulatory agent may be coated or adsorbed onto one or more surfaces.
  • the T cell activating or stimulating agent comprises an antigen, such as a tumor associated antigen.
  • the T cell activating or stimulating agent is capable of inhibiting the expression of the at least one reprogramming factor. In some aspects, activating and stimulating the partially reprogrammed cells inhibits the expression of the at least one reprogramming factor.
  • the redirected T cells comprise an incomplete set of V, D, and I segments of T cell receptor genes.
  • T cell receptor V, D, and I gene segments rearrange during T cell development to form complete variable domain exons. These gene rearrangements take place in the thymus.
  • the redirected T cells produced herein are produced from isolated T cells that have a rearranged TCR locus and express a TCR.
  • the redirected T cells produced herein are epigenetically younger than the source isolated T cells yet have a functional TCR (i.e., an incomplete set of V, D, and I segment of T cell receptor genes), which distinguishes them from the isolated T cells.
  • the method further comprises measuring the epigenetic age of the redirected T cells.
  • the redirected T cells are capable of expanding at least about 5- fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21 -fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, or at least about 25-fold, at least about 50-fold, at least about 100-fold, at least about 150-fold at least about 200-fold, at least about 300-fold, at least about 400- fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, at least about 1100-fold, at least about
  • the redirected T cells are capable of expanding between about 5- fold to about 2000-fold, about 5-fold to about 1500-fold, about 5-fold to about 1000-fold, about 5- fold to about 900-fold, about 5-fold to about 800-fold, about 5-fold to about 700-fold, about 5-fold to about 600-fold, about 5-fold to about 500-fold, about 5-fold to about 400-fold, about 5-fold to about 300-fold, about 5-fold to about 200-fold, about 5-fold to about 150-fold, about 5-fold to about 100-fold, about 5-fold to about 50-fold, about 5-fold to about 25-fold, about 5-fold to about 20-fold, about 5-fold to about 15-fold, about 5-fold to about 10-fold, about 5-fold to about 9-fold, about 5-fold to about 8-fold, about 5-fold to about 7-fold, about 5-fold to about 6-fold, about 10- fold to about 25-fold, about 10-fold to about 20-fold, about 10-fold to about 15-fold, about 15- fold to about 25-fold, about 15-fold, about 15-fold,
  • the method further comprises expanding the redirected T cells.
  • the partially reprogrammed and redirected T cells herein may be further stimulated and expanded using the same activation methods as described elsewhere herein. Such methods for activating and expanding T cells are known in the art and in some aspects may be carried out in any media for culturing immune cells, e.g., T cells, NK cells, and/or TILs.
  • the methods herein comprising the partial reprogramming followed by redirection and optionally, further expansion, will be sufficient for the redirected T cells to acquire any one or more of the characteristics disclosed herein.
  • a population of T cells produced by a method disclosed herein acquire one or more of the characteristics disclosed herein, wherein the method comprises: a. culturing T cells in a first medium comprising IL-2 and activating said T cells with at least one antibody and antigen-binding fragment thereof, specific for CD3 and/or for CD28; b.
  • T cells contacting said activated T cells with KLF4, OCT3/4, SOX2 and C-MYC, and optionally SV40, LIN28 and/or NANOG, for a period of about five days to about 10 days in a second culture medium that does not comprise IL-2, or antibody specific for CD3 or CD28; wherein said T cells are not completely reprogrammed into iPS or totipotent cells; and c.
  • a T cell activating or stimulating agent e.g., an antibody or antigen-binding fragment thereof, specific for CD3 and/or CD28
  • a population of T cells produced by a method disclosed herein acquire one or more of the characteristics disclosed herein, wherein the method comprises: a. culturing T cells and activating said T cells with at least one antibody and antigen-binding fragment thereof, specific for CD3 and/or for CD28; b. contacting said activated T cells with KLF4, OCT3/4, SOX2 and C-MYC, and optionally SV40, LIN28 and/or NANOG, for a period of about five days to about 10 days; wherein said T cells are not completely reprogrammed into iPS or totipotent cells; and c.
  • step (b) culturing the partially reprogrammed cells of step (b) in a media comprising at least one T cell activating or stimulating agent (e.g., an antibody or antigen-binding fragment thereof, specific for CD3 and/or CD28; wherein the contacted T cells are further cultured in an appropriate culture mediacomprising a phosphoAKT inhibitor, metformin, an mTOR inhibitor, 2DG, or any combination thereof for at least about 5 days.
  • T cell activating or stimulating agent e.g., an antibody or antigen-binding fragment thereof, specific for CD3 and/or CD28
  • an appropriate culture media comprising a phosphoAKT inhibitor, metformin, an mTOR inhibitor, 2DG, or any combination thereof for at least about 5 days.
  • Non-limiting examples of the characteristics of the redirected T cells are included in the following:
  • the redirected T cells have one or more sternness properties including high epigenetic plasticity.
  • Other advantageous phenotypic markers that the redirected cells may acquire include expression of L-selectin (CD62L), IL-7Ra, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, CXCR3, TCF7 and LFA-1.
  • the redirected T cells acquire T memory stem cell expression markers, e.g., are memory T cells that express CD95, CD45RA, CCR7, and CD62L and are endowed with the stem cell-like ability to self-renew and the multipotent capacity to reconstitute the entire spectrum of memory and effector T cell subsets.
  • the redirected T cells are "central memory T cells” or "TCM cells” which are memory T cells that express CD45RO, CCR7, and CD62L.
  • the redirected T cells have effector memory T cell markers such as expression of CD45RO but lack of expression of CCR7 and CD62L.
  • the redirected T cells herein have a stem-like phenotype and have an improved capacity for proliferation.
  • the redirected T cells have characteristics of a naive T cell, "TN cells,” which are T cells that express CD45RA, CCR7, and CD62L, but which do not express CD95.
  • the metabolic state of redirected T cells is improved (see e.g., Cell Metabolism 14, 264-271).
  • metabolic gene sets corresponding to oxidative phosphorylation, fatty acid metabolism, glycolysis and hypoxia are enriched in the redirected T cells described herein as compared to appropriate controls (see e.g., Nishimura 2019 Int. J. Mol. Sci. 20:2254).
  • the redirected T cells described herein have upregulated glycolytic enzymes and downregulated electron transport chain subunits.
  • the redirected T cells herein demonstrate one or more of the following characteristics: a metabolic switch converting somatic oxidative metabolism into a glycolytic flux-dependent, mitochondria independent state; downregulated expression of age-related stress response genes in the p53 tumor suppressor pathway, including pl6INK4a, p21CIPl, Atf3, and Gadd45B; downregulation of the senescence-associated metalloprotease MMP13 and interleukin-6; reduced senescence-associated P -galactosidase activity; reduced production of mitochondrial reactive oxygen species (ROS); restored levels of H3K9me3 and H4K20me3 (epigenetic modifications involved in the maintenance of heterochromatin) (see e.g., Ocampo, Cell.
  • ROS mitochondrial reactive oxygen species
  • the redirected T cells described herein are enriched for sternness gene signatures as compared to non-redirected control T cells (see, e.g., Gattinoni et al., 2011 Nature Medicine 17(10): 1290-1297). 5. Cell Culture Medium
  • the present disclosure provides methods of culturing redirected cells in a cell culture medium comprising one or more agents that increases the sternness of the redirected cells, increases the cell killing ability of the redirected cells, increases cytokine expression by the redirected cells, decreases exhaustion by the redirected cells, or any combination thereof.
  • the one or more agents comprises a phosphoAKT inhibitor.
  • the phosphoAKT inhibitor is selected from phosphoAKT inhibitor VIII, AZD5363, perifosine, MK- 2206, GSK-2141795, afuresertib, ARQ 092, ARQ 751, ipatasertib, and any combination thereof.
  • the phosphoAKT inhibitor is selected from MK-2206 2HC1, Perifosine (KRX- 0401), GSK690693, Ipatasertib (GDC-0068), Capivasertib (AZD5363), PF-04691502, AT7867, Triciribine (NSC 154020), CCT128930, A-674563, PHT-427, A-443654, Miransertib (ARQ-092), BAY1 125976, Borussertib, Miransertib (ARQ 092) HC1, Akti-1/2, Uprosertib (GSK2141795), Afuresertib (GSK2110183), AT13148, Honokiol (NSC 293100), TIC10 Analogue, BIA, a- Linolenic acid, SPP-86, Hematein, RPI-1, Urolithin B, ML-9 HC1, ABTL-0812, Alobre
  • the medium comprises at least about 0.1 pM to at least about 100 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 0.1 pM to at least about 100 pM, at least about 1 pM to at least about 100 pM, at least about 10 pM to at least about 100 pM, at least about 20 pM to at least about 100 pM, at least about 30 pM to at least about 100 pM, at least about 1 pM to at least about 75 pM, at least about 1 pM to at least about 50 pM, at least about 1 pM to at least about 45 pM, at least about 1 pM to at least about 40 pM, at least about 1 pM to at least about 35 pM, at least about 1 pM to at least about 30 pM, at least about 1 pM to at least about 25 pM, at least about 5 pM to at least about 25 pM, at least about 10
  • the medium comprises at least about 10 pM to at least about 15 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 15 pM to at least about 20 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 20 pM to at least about 25 pM of the phosphoAKT inhibitor.
  • the medium comprises at least about 0.1 pM, at least about 0.5 pM, at least about 1.0 pM, at least about 1.5 pM, at least about 2.0 pM, at least about 2.5 pM, at least about 3.0 pM, at least about 3.5 pM, at least about 4.0 pM, at least about 4.5 pM, at least about 5.0 pM, at least about 5.5 pM, at least about 6.0 pM, at least about 6.5 pM, at least about 7.0 pM, at least about 7.5 pM, at least about 8.0 pM, at least about 8.5 pM, at least about 9.0 pM, at least about 9.5 pM, at least about 10 pM, at least about 11 pM, at least about 12 pM, at least about 13 pM, at least about 14 pM, at least about 15 pM, at least about 16 pM, at least about 17 pM, at least about
  • the medium comprises at least about 10 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 11 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 12 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 13 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 14 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 15 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 16 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 17 pM of the phosphoAKT inhibitor.
  • the medium comprises at least about 18 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 19 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 20 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 21 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 22 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 23 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 24 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 25 pM of the phosphoAKT inhibitor.
  • the medium comprises at least about 0.1 pM to at least about 100 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 0.1 pM to at least about 100 pM, at least about 1 pM to at least about 100 pM, at least about 10 pM to at least about 100 pM, at least about 20 pM to at least about 100 pM, at least about 30 pM to at least about 100 pM, at least about 1 pM to at least about 75 pM, at least about 1 pM to at least about 50 pM, at least about 1 pM to at least about 45 pM, at least about 1 pM to at least about 40 pM, at least about 1 pM to at least about 35 pM, at least about 1 pM to at least about 30 pM, at least about 1 pM to at least about 25 pM, at least about 5 pM to at least about 25 pM, at least about 10
  • the medium comprises at least about 10 pM to at least about 15 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 15 pM to at least about 20 pM of the phosphoAKT inhibitor. In some aspects, the medium comprises at least about 20 pM to at least about 25 pM of the phosphoAKT inhibitor.
  • the medium comprises at least about 0.1 pM, at least about 0.5 pM, at least about 1.0 pM, at least about 1.5 pM, at least about 2.0 pM, at least about 2.5 pM, at least about 3.0 pM, at least about 3.5 pM, at least about 4.0 pM, at least about 4.5 pM, at least about 5.0 pM, at least about 5.5 pM, at least about 6.0 pM, at least about 6.5 pM, at least about 7.0 pM, at least about 7.5 pM, at least about 8.0 pM, at least about 8.5 pM, at least about 9.0 pM, at least about 9.5 pM, at least about 10 pM, at least about 11 pM, at least about 12 pM, at least about 13 pM, at least about 14 pM, at least about 15 pM, at least about 16 pM, at least about 17 pM, at least about
  • the medium comprises at least about 10 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises at least about 11 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises at least about 12 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises at least about 13 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises at least about 14 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises at least about 15 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises at least about 16 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises at least about 17 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises at least about 18 pM phosphoAKT inhibitor VIII.
  • the medium comprises at least about 19 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises at least about 20 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises at least about 21 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises at least about 22 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises at least about 23 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises at least about 24 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises at least about 25 pM phosphoAKT inhibitor VIII.
  • the one or more agents comprises metformin.
  • the medium comprises at least about 1 pM to at least about 1000 pM metformin.
  • the medium comprises at least about 1 pM to at least about 900 pM, at least about 1 pM to at least about 800 pM, at least about 1 pM to at least about 700 pM, at least about 1 pM to at least about 600 pM, at least about 1 pM to at least about 500 pM, at least about 1 pM to at least about 450 pM, at least about 1 pM to at least about 400 pM, at least about 1 pM to at least about 350 pM, at least about 1 pM to at least about 300 pM, at least about 1 pM to at least about 275 pM, at least about 1 pM to at least about 250 pM, at least about 1 pM to at least about 225 pM, at least about 1 pM to
  • the medium comprises at least about 150 pM to at least about 500 pM metfromin. In some aspects, the medium comprises at least about 150 pM to at least about 400 pM metfromin. In some aspects, the medium comprises at least about 150 pM to at least about 300 pM metfromin. In some aspects, the medium comprises at least about 150 pM to at least about 250 pM metfromin. In some aspects, the medium comprises at least about 150 pM to at least about 200 pM metfromin. In some aspects, the medium comprises at least about 100 pM to at least about 500 pM metfromin. In some aspects, the medium comprises at least about 100 pM to at least about 400 pM metfromin.
  • the medium comprises at least about 100 pM to at least about 300 pM metfromin. In some aspects, the medium comprises at least about 100 pM to at least about 250 pM metfromin. In some aspects, the medium comprises at least about 100 pM to at least about 200 pM metfromin. In some aspects, the medium comprises at least about 50 pM to at least about
  • the medium comprises at least about 50 pM to at least about
  • the medium comprises at least about 50 pM to at least about
  • the medium comprises at least about 50 pM to at least about
  • the medium comprises at least about 50 pM to at least about
  • the medium comprises at least about 50 pM to at least about
  • the medium comprises at least about 50 pM to at least about
  • the medium comprises at least about 1 pM, at least about 10 pM, at least about 20 pM, at least about 30 pM, at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 110 pM, at least about 120 pM, at least about 130 pM, at least about 140 pM, at least about 150 pM, at least about 160 pM, at least about 170 pM, at least about 180 pM, at least about 190 pM, at least about 200 pM, at least about 210 pM, at least about 220 pM, at least about 230 pM, at least about 240 pM, at least about 250 pM, at least about 260 pM, at least about 270 pM, at least about 280 pM, at least about
  • the medium comprises at least about 50 pM metformin. In some aspects, the medium comprises at least about 60 pM metformin. In some aspects, the medium comprises at least about 70 pM metformin. In some aspects, the medium comprises at least about 80 pM metformin. In some aspects, the medium comprises at least about 90 pM metformin. In some aspects, the medium comprises at least about 100 pM metformin. In some aspects, the medium comprises at least about 110 pM metformin. In some aspects, the medium comprises at least about 120 pM metformin. In some aspects, the medium comprises at least about 130 pM metformin. In some aspects, the medium comprises at least about 140 pM metformin.
  • the medium comprises at least about 150 pM metformin. In some aspects, the medium comprises at least about 160 pM metformin. In some aspects, the medium comprises at least about 170 pM metformin. In some aspects, the medium comprises at least about 180 pM metformin. In some aspects, the medium comprises at least about 190 pM metformin. In some aspects, the medium comprises at least about 200 pM metformin. In some aspects, the medium comprises at least about 210 pM metformin. In some aspects, the medium comprises at least about 220 pM metformin. In some aspects, the medium comprises at least about 230 pM metformin. In some aspects, the medium comprises at least about 240 pM metformin.
  • the medium comprises at least about 250 pM metformin. In some aspects, the medium comprises at least about 260 pM metformin. In some aspects, the medium comprises at least about 270 pM metformin. In some aspects, the medium comprises at least about 280 pM metformin. In some aspects, the medium comprises at least about 290 pM metformin. In some aspects, the medium comprises at least about 300 pM metformin.
  • the one or more agents comprises an mTOR inhibitor.
  • the mTOR inhibitor is selected from rapamycin, temsirolimus, everolimus, ridaforolimus, and any combination thereof.
  • the mTOR inhibitor is selected from Dactolisib (BEZ235), AZD8055, PI-103, NU7441 (KU-57788), KU-0063794, Torkinib (PP242), Ridaforolimus (Deforolimus, MK-8669), Sapanisertib (MLN0128), Voxtalisib (XL765) Analogue, Torin 1, Omipalisib (GSK2126458), OSI-027, PF-04691502, Apitolisib (GDC-0980), GSK1059615, Gedatolisib (PKI-587), WYE-354, Vistusertib (AZD2014), Torin 2, WYE-125132 (WYE-
  • the medium comprises at least about 0.1 pM to at least about 100 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 0.1 pM to at least about 100 pM, at least about 1 pM to at least about 100 pM, at least about 10 pM to at least about 100 pM, at least about 20 pM to at least about 100 pM, at least about 30 pM to at least about 100 pM, at least about 1 pM to at least about 75 pM, at least about 1 pM to at least about 50 pM, at least about 1 pM to at least about 45 pM, at least about 1 pM to at least about 40 pM, at least about 1 pM to at least about 35 pM, at least about 1 pM to at least about 30 pM, at least about 1 pM to at least about 25 pM, at least about 5 pM to at least about 25 pM, at least about 10
  • the medium comprises at least about 0.5 pM to at least about 15 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 1 pM to at least about 15 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 1 pM to at least about 10 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 5 pM to at least about 10 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 5 pM to at least about 15 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 10 pM to at least about 15 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 10 pM to at least about 20 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 15 pM to at least about 20 pM of the mTOR inhibitor.
  • the medium comprises at least about 0.1 pM, at least about 0.5 pM, at least about 1.0 pM, at least about 1.5 pM, at least about 2.0 pM, at least about 2.5 pM, at least about 3.0 pM, at least about 3.5 pM, at least about 4.0 pM, at least about 4.5 pM, at least about 5.0 pM, at least about 5.5 pM, at least about 6.0 pM, at least about 6.5 pM, at least about 7.0 pM, at least about 7.5 pM, at least about 8.0 pM, at least about 8.5 pM, at least about 9.0 pM, at least about 9.5 pM, at least about 10 pM, at least about 11 pM, at least about 12 pM, at least about 13 pM, at least about 14 pM, at least about 15 pM, at least about 16 pM, at least about 17 pM, at least about
  • the medium comprises at least about 10 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 11 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 12 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 13 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 14 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 15 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 16 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 17 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 18 pM of the mTOR inhibitor.
  • the medium comprises at least about 19 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 20 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 21 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 22 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 23 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 24 pM of the mTOR inhibitor. In some aspects, the medium comprises at least about 25 pM of the mTOR inhibitor.
  • the medium comprises at least about 0.1 pM to at least about 100 pM of rapamycin. In some aspects, the medium comprises at least about 0.1 pM to at least about 100 pM, at least about 1 pM to at least about 100 pM, at least about 10 pM to at least about 100 pM, at least about 20 pM to at least about 100 pM, at least about 30 pM to at least about 100 pM, at least about 1 pM to at least about 75 pM, at least about 1 pM to at least about 50 pM, at least about 1 pM to at least about 45 pM, at least about 1 pM to at least about 40 pM, at least about 1 pM to at least about 35 pM, at least about 1 pM to at least about 30 pM, at least about 1 pM to at least about 25 pM, at least about 5 pM to at least about 25 pM, at least about 10 pM
  • the medium comprises at least about 0.5 pM to at least about 15 pM of rapamycin. In some aspects, the medium comprises at least about 1 pM to at least about 15 pM of rapamycin. In some aspects, the medium comprises at least about 1 pM to at least about 10 pM of rapamycin. In some aspects, the medium comprises at least about 5 pM to at least about 10 pM of rapamycin. In some aspects, the medium comprises at least about 5 pM to at least about 15 pM of rapamycin. In some aspects, the medium comprises at least about 10 pM to at least about 15 pM of rapamycin. In some aspects, the medium comprises at least about 10 pM to at least about 20 pM of rapamycin. In some aspects, the medium comprises at least about 15 pM to at least about 20 pM of rapamycin.
  • the medium comprises at least about 0.1 pM, at least about 0.5 pM, at least about 1.0 pM, at least about 1.5 pM, at least about 2.0 pM, at least about 2.5 pM, at least about 3.0 pM, at least about 3.5 pM, at least about 4.0 pM, at least about 4.5 pM, at least about 5.0 pM, at least about 5.5 pM, at least about 6.0 pM, at least about 6.5 pM, at least about 7.0 pM, at least about 7.5 pM, at least about 8.0 pM, at least about 8.5 pM, at least about 9.0 pM, at least about 9.5 pM, at least about 10 pM, at least about 11 pM, at least about 12 pM, at least about 13 pM, at least about 14 pM, at least about 15 pM, at least about 16 pM, at least about 17 pM, at least about
  • the medium comprises at least about 10 pM of rapamycin. In some aspects, the medium comprises at least about 11 pM of rapamycin. In some aspects, the medium comprises at least about 12 pM of rapamycin. In some aspects, the medium comprises at least about 13 pM of rapamycin. In some aspects, the medium comprises at least about 14 pM of rapamycin. In some aspects, the medium comprises at least about 15 pM of rapamycin. In some aspects, the medium comprises at least about 16 pM of rapamycin. In some aspects, the medium comprises at least about 17 pM of rapamycin. In some aspects, the medium comprises at least about 18 pM of rapamycin.
  • the medium comprises at least about 19 pM of rapamycin. In some aspects, the medium comprises at least about 20 pM of rapamycin. In some aspects, the medium comprises at least about 21 pM of rapamycin. In some aspects, the medium comprises at least about 22 pM of rapamycin. In some aspects, the medium comprises at least about 23 pM of rapamycin. In some aspects, the medium comprises at least about 24 pM of rapamycin. In some aspects, the medium comprises at least about 25 pM of rapamycin.
  • the one or more agents comprises 2DG.
  • the medium comprises at least about 0.1 nM to at least about 100 mM of 2DG. In some aspects, the medium comprises at least about 1 nM to at least about 100 mM 2DG. In some aspects, the medium comprises at least about 1 nM to at least about 10 mM 2DG. In some aspects, the medium comprises at least about 1 nM to at least about 5 mM 2DG. In some aspects, the medium comprises at least about 1 nM to at least about 4 mM 2DG.
  • the medium comprises at least about 10 nM to at least about 100 mM, at least about 20 nM to at least about 100 mM, at least about 30 nM to at least about 100 mM, at least about 1 nM to at least about 75 mM, at least about 1 nM to at least about 50 mM, at least about 1 nM to at least about 45 mM, at least about 1 nM to at least about 40 mM, at least about 1 nM to at least about 30 mM, at least about 1 nM to at least about 20 mM, at least about 1 nM to at least about 10 mM, at least about 25 nM to at least about 75 mM, at least about 30 nM to at least about 60 mM, at least about 40 nM to at least about 50 mM, at least about 40 nM to at least about 45 mM, at least about 45 nM to at least about 50 mM, at least about 45 nM to
  • the one or more agents comprises 2DG.
  • the medium comprises at least about 0.1 nM to at least about 100 nM of 2DG.
  • the medium comprises at least about 0.1 nM to at least about 100 nM, at least about 1 nM to at least about 100 nM, at least about 10 nM to at least about 100 nM, at least about 20 nM to at least about 100 nM, at least about 30 nM to at least about 100 nM, at least about 1 nM to at least about 75 nM, at least about 1 nM to at least about 50 nM, at least about 1 nM to at least about 45 nM, at least about 25 nM to at least about 75 nM, at least about 30 nM to at least about 60 nM, at least about 40 nM to at least about 50 nM, at least about 40 nM to at least about 45 nM, or at least about 45 nM to at least
  • the medium comprises at least about 40 nM to at least about 50 nM of 2DG. In some aspects, the medium comprises at least about 40 nM to at least about 45 nM of 2DG. In some aspects, the medium comprises at least about 45 nM to at least about 50 nM of 2DG.
  • the one or more agents comprises 2DG.
  • the medium comprises at least about 0.1 pM to at least about 100 pM of 2DG.
  • the medium comprises at least about 0.1 pM to at least about 100 pM, at least about 1 pM to at least about 100 pM, at least about 10 pM to at least about 100 pM, at least about 20 pM to at least about 100 pM, at least about 30 pM to at least about 100 pM, at least about 1 pM to at least about 75 pM, at least about 1 pM to at least about 50 pM, at least about 1 pM to at least about 45 pM, at least about 25 pM to at least about 75 pM, at least about 30 pM to at least about 60 pM, at least about 40 pM to at least about 50 pM, at least about 40 pM to at least about 45 pM, or at least about 45 pM to at least
  • the medium comprises at least about 40 pM to at least about 50 pM of 2DG. In some aspects, the medium comprises at least about 40 pM to at least about 45 pM of 2DG. In some aspects, the medium comprises at least about 45 pM to at least about 50 pM of 2DG.
  • the medium comprises at least about 20 pM, at least about 25 pM, at least about 30 pM, at least about 35 pM, at least about 40 pM, at least about 41 pM, at least about 42 pM, at least about 43 pM, at least about 44 pM, at least about 45 pM, at least about 46 pM, at least about 47 pM, at least about 48 pM, at least about 49 pM, at least about 50 pM, at least about 55 pM, at least about 60 pM, at least about 65 pM, at least about 70 pM, at least about 75 pM, at least about 80 pM, at least about 85 pM, at least about 90 pM, at least about 95 pM, or at least about 100 pM of 2DG.
  • the medium comprises at least about 40 nM of 2DG. In some aspects, the medium comprises at least about 45 nM of 2DG. In some aspects, the medium comprises at least about 50 nM of 2DG. In some aspects, the medium comprises at least about 13 nM of 2DG. In some aspects, the medium comprises at least about 10 nM of 2DG. In some aspects, the medium comprises at least about 9 nM of 2DG. In some aspects, the medium comprises at least about 8 nM of 2DG. In some aspects, the medium comprises at least about 7 nM of 2DG. In some aspects, the medium comprises at least about 6 nM of 2DG. In some aspects, the medium comprises at least about 5 nM of 2DG.
  • the medium comprises at least about 4 nM of 2DG. In some aspects, the medium comprises at least about 3 nM of 2DG. In some aspects, the medium comprises at least about 2 nM of 2DG. In some aspects, the medium comprises at least about 1 nM of 2DG. In some aspects, the medium comprises at least about 0.5 nM of 2DG. In some aspects, the medium comprises at least about 0.1 nM of 2DG.
  • the medium comprises about 0.1 nM to about 100 mM of 2DG. In some aspects, the medium comprises about 1 nM to about 100 mM 2DG. In some aspects, the medium comprises about 1 nM to about 10 mM 2DG. In some aspects, the medium comprises about 1 nM to about 5 mM 2DG. In some aspects, the medium comprises about 1 nM to about 4 mM 2DG.
  • the medium comprises about 10 nM to about 100 mM, about 20 nM to about 100 mM, about 30 nM to about 100 mM, about 1 nM to about 75 mM, about 1 nM to about 50 mM, about 1 nM to about 45 mM, about 1 nM to about 40 mM, about 1 nM to about 30 mM, about 1 nM to about 20 mM, about 1 nM to about 10 mM, about 25 nM to about 75 mM, about 30 nM to about 60 mM, about 40 nM to about 50 mM, about 40 nM to about 45 mM, about 45 nM to about 50 mM, about 45 nM to about 40 mM, about 45 nM to about 30 mM, about 45 nM to about 20 mM, about 45 nM to about 10 mM 2DG.
  • the medium comprises at least about 0.1 mM to at least about 100 mM of 2DG. In some aspects, the medium comprises at least about 1 mM to at least about 100 mM 2DG. In some aspects, the medium comprises at least about 1 mM to at least about 10 mM 2DG. In some aspects, the medium comprises at least about 1 mM to at least about 5 mM 2DG. In some aspects, the medium comprises at least about 1 mM to at least about 4 mM 2DG.
  • the medium comprises at least about 10 mM to at least about 100 mM, at least about 20 mM to at least about 100 mM, at least about 30 mM to at least about 100 mM, at least about 1 mM to at least about 75 mM, at least about 1 mM to at least about 50 mM, at least about 1 mM to at least about 45 mM, at least about 1 mM to at least about 40 mM, at least about 1 mM to at least about 30 mM, at least about 1 mM to at least about 20 mM, at least about 1 mM to at least about 10 mM, at least about 25 mM to at least about 75 mM, at least about 30 mM to at least about 60 mM, at least about 40 mM to at least about 50 mM, at least about 40 mM to at least about 45 mM, at least about 45 mM to at least about 50 mM, at least about 45 mM to
  • the one or more agents comprises 2DG.
  • the medium comprises at least about 0.1 mM to at least about 100 mM of 2DG.
  • the medium comprises at least about 0.1 mM to at least about 100 mM, at least about 1 mM to at least about 100 mM, at least about 10 mM to at least about 100 mM, at least about 20 mM to at least about 100 mM, at least about 30 mM to at least about 100 mM, at least about 1 mM to at least about 75 mM, at least about 1 mM to at least about 50 mM, at least about 1 mM to at least about 45 mM, at least about 25 mM to at least about 75 mM, at least about 30 mM to at least about 60 mM, at least about 40 mM to at least about 50 mM, at least about 40 mM to at least about 45 mM, or at least about 45 mM to at least
  • the medium comprises at least about 40 mM to at least about 50 mM of 2DG. In some aspects, the medium comprises at least about 40 mM to at least about 45 mM of 2DG. In some aspects, the medium comprises at least about 45 mM to at least about 50 mM of 2DG.
  • the medium comprises at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 41 mM, at least about 42 mM, at least about 43 mM, at least about 44 mM, at least about 45 mM, at least about 46 mM, at least about 47 mM, at least about 48 mM, at least about 49 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, or at least about 100 mM of 2DG.
  • the medium comprises at least about 40 mM of 2DG. In some aspects, the medium comprises at least about 45 mM of 2DG. In some aspects, the medium comprises at least about 50 mM of 2DG. In some aspects, the medium comprises at least about 13 mM of 2DG. In some aspects, the medium comprises at least about 10 mM of 2DG. In some aspects, the medium comprises at least about 9 mM of 2DG. In some aspects, the medium comprises at least about 8 mM of 2DG. In some aspects, the medium comprises at least about 7 mM of 2DG. In some aspects, the medium comprises at least about 6 mM of 2DG. In some aspects, the medium comprises at least about 5 mM of 2DG.
  • the medium comprises at least about 4 mM of 2DG. In some aspects, the medium comprises at least about 3 mM of 2DG. In some aspects, the medium comprises at least about 2 mM of 2DG. In some aspects, the medium comprises at least about 1 mM of 2DG. In some aspects, the medium comprises at least about 0.5 mM of 2DG. In some aspects, the medium comprises at least about 0.1 mM of 2DG. [0432] In some aspects, the medium comprises about 0.1 mM to about 100 mM of 2DG. In some aspects, the medium comprises about 1 mM to about 100 mM 2DG. In some aspects, the medium comprises about 1 mM to about 10 mM 2DG.
  • the medium comprises about 1 mM to about 5 mM 2DG. In some aspects, the medium comprises about 1 mM to about 4 mM 2DG. In some aspects, the medium comprises about 10 mM to about 100 mM, about 20 mM to about 100 mM, about 30 mM to about 100 mM, about 1 mM to about 75 mM, about 1 mM to about 50 mM, about 1 mM to about 45 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 25 mM to about 75 mM, about 30 mM to about 60 mM, about 40 mM to about 50 mM, about 40 mM to about 45 mM, about 45 mM to about 50 mM, about 45 mM to about 40 mM, about 45 mM to about 30 mM, about 45 mM
  • the redirected cells are cultured in the medium comprising 2DG for about 1 day to about 14 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 1 day to about 13 days, about 1 day to about 12 days, about 1 day to about 11 days, about 1 day to about 10 days, about 1 day to about 9 days, about 1 day to about 8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about
  • the redirected cells are cultured in the medium comprising 2DG for about 2 days to about 14 days, about 2 days to about 13 days, about 2 days to about 12 days, about 2 days to about 11 days, about
  • the redirected cells are cultured in the medium comprising 2DG for about 3 days to about 14 days, about 3 days to about 13 days, about
  • the redirected cells are cultured in the medium comprising 2DG for about 4 days to about 14 days, about 4 days to about 13 days, about 4 days to about 12 days, about 4 days to about 11 days, about 4 days to about 10 days, about 4 days to about 9 days, about 4 days to about 8 days, about 4 days to about 7 days, about 4 days to about 6 days, about 3 days to about 5 days, about 3 days to about 4 days.
  • the redirected cells are cultured in the medium comprising 2DG for about 4 days to about 14 days, about 4 days to about 13 days, about 4 days to about 12 days, about 4 days to about 11 days, about 4 days to about 10 days, about 4 days to about 9 days, about 4 days to about 8 days, about 4 days to about 7 days, about 4 days to about 6 days, about 4 days to about 5 days.
  • the redirected cells are cultured in the medium comprising 2DG for about 5 days to about 14 days, about 5 days to about 13 days, about 5 days to about 12 days, about 5 days to about 11 days, about 5 days to about 10 days, about 5 days to about 9 days, about 5 days to about 8 days, about 5 days to about 7 days, about 5 days to about 6 days.
  • the redirected cells are cultured in the medium - I l l - comprising 2DG for about 6 days to about 14 days, about 6 days to about 13 days, about 6 days to about 12 days, about 6 days to about 11 days, about 6 days to about 10 days, about 6 days to about 9 days, about 6 days to about 8 days, about 6 days to about 7 days.
  • the redirected cells are cultured in the medium comprising 2DG for about 7 days to about 14 days, about 7 days to about 13 days, about 7 days to about 12 days, about 7 days to about 11 days, about 7 days to about 10 days, about 7 days to about 9 days, or about 7 days to about 8 days.
  • the redirected cells are cultured in the medium comprising 2DG for about 7 days to about 14 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 1 day to about 7 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 2 days to about 7 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 3 days to about 7 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 4 days to about 7 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 5 days to about 7 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 6 days to about 7 days.
  • the redirected cells are cultured in the medium comprising 2DG for about 4 days to about 10 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 5 days to about 9 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 6 days to about 8 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 7 days to about 8 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 7 days to about 9 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 7 days to about 10 days.
  • the redirected cells are cultured in the medium comprising 2DG for about 2 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 3 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 4 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 5 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 6 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 7 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 8 days.
  • the redirected cells are cultured in the medium comprising 2DG for about 9 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 10 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 11 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 12 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 13 days. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 14 days.
  • the redirected cells are cultured in the medium comprising 2DG for about 2 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 3 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 4 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 5 days, wherein the redirected cells are generated from TILs.
  • the redirected cells are cultured in the medium comprising 2DG for about 6 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 7 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 8 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 9 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 10 days, wherein the redirected cells are generated from TILs.
  • the redirected cells are cultured in the medium comprising 2DG for about 11 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 12 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 13 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising 2DG for about 14 days, wherein the redirected cells are generated from TILs. [0438] In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 2 days.
  • the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 3 days. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 4 days. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 5 days. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 6 days. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 7 days. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 8 days.
  • the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 9 days. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 10 days. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 11 days. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 12 days. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 13 days. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 14 days.
  • the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 2 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 3 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 4 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 5 days, wherein the redirected cells are generated from TILs.
  • the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 6 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 7 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 8 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 9 days, wherein the redirected cells are generated from TILs.
  • the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 10 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 11 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 12 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 13 days, wherein the redirected cells are generated from TILs. In some aspects, the redirected cells are cultured in the medium comprising about 4 mM 2DG for about 14 days, wherein the redirected cells are generated from TILs.
  • the medium comprises a phosphoAKT inhibitor and metformin. In some aspects, the medium comprises a phosphoAKT inhibitor and an mTOR inhibitor. In some aspects, the medium comprises metformin and an mTOR inhibitor. In some aspects, the medium comprises a phosphoAKT inhibitor and 2DG. In some aspects, the medium comprises an mTOR inhibitor and 2DG. In some aspects, the medium comprises metformin and 2DG.
  • the medium comprises (i) about 12 pM phosphoAKT inhibitor VIII and (ii) about 100 pM metformin. In some aspects, the medium comprises (i) about 15 pM phosphoAKT inhibitor VIII and (ii) about 100 pM metformin. In some aspects, the medium comprises (i) about 12 pM phosphoAKT inhibitor VIII and (ii) about 200 pM metformin. In some aspects, the medium comprises (i) about 15 pM phosphoAKT inhibitor VIII and (ii) about 200 pM metformin. In some aspects, the medium comprises (i) about 12 pM phosphoAKT inhibitor VIII and (ii) about 45 mM 2DG. In some aspects, the medium comprises (i) about 15 pM phosphoAKT inhibitor VIII and (ii) about 45 mM 2DG.
  • the medium comprises (i) about 12 pM phosphoAKT inhibitor VIII and (ii) about 1 pM rapamycin. In some aspects, the medium comprises (i) about 15 pM phosphoAKT inhibitor VIII and (ii) about 1 pM rapamycin. In some aspects, the medium comprises (i) about 12 pM phosphoAKT inhibitor VIII and (ii) about 10 pM rapamycin. In some aspects, the medium comprises (i) about 15 pM phosphoAKT inhibitor VIII and (ii) about 10 pM rapamycin.
  • the medium comprises (i) about 100 pM metformin and (ii) about 1 pM rapamycin. In some aspects, the medium comprises (i) about 100 pM metformin and (ii) about 10 pM rapamycin. In some aspects, the medium comprises (i) about 200 pM metformin and (ii) about 1 pM rapamycin. In some aspects, the medium comprises (i) about 200 pM metformin and (ii) about 10 pM rapamycin.
  • the medium comprises (i) about 200 pM metformin and (ii) about 45 mM 2DG. In some aspects, the medium comprises (i) about 100 pM metformin and (ii) about 45 mM 2DG. In some aspects, the medium comprises (i) about 1 pM rapamycin and (ii) about 45 mM 2DG. In some aspects, the medium comprises (i) about 10 pM rapamycin and (ii) about 45 mM 2DG. [0445] The methods disclosed herein confer unique properties on the redirected cells.
  • the medium further comprises at least about 5 mM potassium ion.
  • the concentration of potassium ion for the present method is higher than any known medium for T cell culture or stem cell culture. In some aspects, the concentration of potassium ion for the present method is higher than 5 mM.
  • the concentration of potassium ion is between about 5 mM and about 10 mM, about 10 mM and about 20 mM, about 20 mM and about 30 mM, about 30 mM and about 40 mM, about 40 mM and about 50 mM, about 50 mM and about 60 mM, about 60 mM and about 70 mM, about 70 mM and about 80 mM, about 80 mM and about 90 mM, or about 90 mM and about 100 mM.
  • the concentration of potassium ion is between about 5mM and about 100 mM, about 10 mM and about 100 mM, about 20 mM and about 100 mM, about 30 mM and about 100 mM, about 40 mM and about 100 mM, about 50 mM and about 100 mM, about 60 mM and about 100 mM, about 70 mM and about 100 mM, about 80 mM and about 100 mM, about 5 mM and about 90 mM, about 10 mM and about 90 mM, about 20 mM and about 90 mM, about 30 mM and about 90 mM, about 40 mM and about 90 mM, about 50 mM and about 90 mM, about
  • the concentration of potassium ion is between about 5 mM and about 10 mM. In some aspects, the concentration of potassium ion is between about 10 mM and about 20 mM. In some aspects, the concentration of potassium ion is between about 20 mM and about 30 mM. In some aspects, the concentration of potassium ion is between about 30 mM and about 40 mM. In some aspects, the concentration of potassium ion is between about 40 mM and about 50 mM.
  • the concentration of potassium ion is between about 50 mM and about 60 mM. In some aspects, the concentration of potassium ion is between about 60 mM and about 70 mM. In some aspects, the concentration of potassium ion is between about 70 mM and about 80 mM. In some aspects, the concentration of potassium ion is between about 80 mM and about 90 mM. In some aspects, the concentration of potassium ion is between about 90 mM and about 100 mM.
  • the concentration of potassium ion is about 5 mM, 10 mM, about 20 mM, about 30 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, or about 150 mM.
  • the medium comprises (i) a concentration of potassium ion between about 5 mM and about 80 mM, and (ii) about 15 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises (i) a concentration of potassium ion between about 5 mM and about 80 mM, and (ii) about 12 pM phosphoAKT inhibitor VIII.
  • the medium comprises (i) a concentration of potassium ion between about 5 mM and about 80 mM, and (ii) about 100 pM metformin. In some aspects, the medium comprises (i) a concentration of potassium ion between about 5 mM and about 80 mM, and (ii) about 200 pM metformin.
  • the medium comprises (i) a concentration of potassium ion between about 5 mM and about 80 mM, and (ii) about 1 pM rapamycin. In some aspects, the medium comprises (i) a concentration of potassium ion between about 5 mM and about 80 mM, and (ii) about 5 pM rapamycin.
  • the medium comprises (i) a concentration of potassium ion between about 5 mM and about 80 mM, and (ii) about 45 mM 2DG.
  • the medium comprises (i) a concentration of potassium ion between about 5 mM and about 80 mM, (ii) about 12 pM phosphoAKT inhibitor VIII, and (iii) about 100 pM metformin. In some aspects, the medium comprises (i) a concentration of potassium ion between about 5 mM and about 80 mM, (ii) about 12 pM phosphoAKT inhibitor VIII, and (iii) about 100 pM metformin. In some aspects, the medium comprises (i) a concentration of potassium ion between about 5 mM and about 80 mM, (ii) about 15 pM phosphoAKT inhibitor VIII, and (iii) about 100 pM metformin.
  • the medium comprises (i) a concentration of potassium ion between about 5 mM and about 80 mM, and (ii) about 12 pM phosphoAKT inhibitor VIII. In some aspects, the medium comprises (i) a concentration of potassium ion between about 5 mM and about 80 mM, (ii) about 15 pM phosphoAKT inhibitor VIII, and (iii) about 200 pM metformin.
  • Some aspects of the present disclosure are directed to methods of culturing T cells, comprising placing the T cells in a medium comprising (i) potassium ion at a concentration of at least about 50 mM and (ii) sodium ion at a concentration of less than about 115 mM.
  • the medium is hypotonic. In some aspects, the medium is isotonic.
  • the medium comprises potassium ion at a concentration between 5 mM and 40 mM and NaCl at a concentration between 70 mM and 135 mM, wherein the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.
  • the medium comprises potassium ion at a concentration between 40 mM and 80 mM and NaCl at a concentration between 30 mM and 100 mM, wherein the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.
  • the medium comprises (i) potassium ion at a concentration between
  • the medium comprises (i) potassium ion at a concentration between 40 mM and 80 mM and NaCl at a concentration between 30 mM and 100 mM, wherein the total concentration of potassium ion and NaCl is between 110 mM and 140 mM; and (ii) about 15 pM or about 12 pM phosphoAKT inhibitor VIII.
  • the medium comprises (i) potassium ion at a concentration between 40 mM and 80 mM and NaCl at a concentration between 30 mM and 100 mM, wherein the total concentration of potassium ion and NaCl is between 110 mM and 140 mM; and (ii) about 15 pM or about 12 pM phosphoAKT inhibitor VIII.
  • the medium comprises (i) potassium ion at a concentration between
  • the medium comprises (i) potassium ion at a concentration between 40 mM and 80 mM and NaCl at a concentration between 30 mM and 100 mM, wherein the total concentration of potassium ion and NaCl is between 110 mM and 140 mM; and (ii) about 100 pM or about 200 pM metformin.
  • the medium comprises (i) potassium ion at a concentration between 40 mM and 80 mM and NaCl at a concentration between 30 mM and 100 mM, wherein the total concentration of potassium ion and NaCl is between 110 mM and 140 mM; and (ii) about 1 pM or about 5 pM rapamycin.
  • the medium comprises (i) potassium ion at a concentration between 40 mM and 80 mM and NaCl at a concentration between 30 mM and 100 mM, wherein the total concentration of potassium ion and NaCl is between 110 mM and 140 mM; and (ii) about 45 mM 2DG.
  • the medium further comprises glucose.
  • the concentration of glucose is more than about 10 mM.
  • the concentration of glucose is from about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 15 mM to about 19 mM, about 15 mM to about 18 mM, about 15 mM to about 17 mM, about 15 mM to about 16 mM, about 16 mM to about 20 mM, about 16 mM to about 19 mM, about 16 mM to about 18 mM, about 16 mM to about 17 mM, about 17 mM to about 20 mM, about 17 mM to about 19 mM, or about 17 mM to about 18 mM.
  • the concentration of glucose is about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM.
  • the concentration of glucose is about 15.4 mM, about 15.9 mM, about 16.3 mM, about 16.8 mM, about 17.2 mM, or about 17.7 mM.
  • the medium further comprises calcium ion.
  • the concentration of calcium ion is more than about 0.4 mM.
  • the concentration of calcium ion is from about 0.4 mM to about 2.5 mM, about 0.5 mM to about 2.0 mM, about 1.0 mM to about 2.0 mM, about 1.1 mM to about 2.0 mM, about 1.2 mM to about 2.0 mM, about 1.3 mM to about 2.0 mM, about 1.4 mM to about 2.0 mM, about 1.5 mM to about 2.0 mM, about 1.6 mM to about 2.0 mM, about 1.7 mM to about 2.0 mM, about 1.8 mM to about 2.0 mM, about 1.2 to about 1.3 mM, about 1.2 to about 1.4 mM, about 1.2 to about 1.5 mM, about 1.2 to about 1.6 mM, about 1.2 to about 1.7 mM, about 1.2 to about 1.8
  • the concentration of calcium ion is about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, or about 2.0 mM.
  • the medium comprises about 50 mM potassium ion and (i) about
  • the medium further comprises (iv) about 15 pM or about 12 pM phosphoAKT inhibitor VIII. In some aspects, the medium further comprises (iv) about 100 pM or about 200 pM metformin. In some aspects, the medium further comprises (iv) about 1 pM or about 5 pM rapamycin. In some aspects, the medium further comprises (iv) about 45 mM 2DG.
  • the medium comprises about 55 mM potassium ion and (i) about 76 mMNaCl; (ii) about 17.2 mM glucose; (iii) about 1.7 mM calcium ion; or (iv) any combination of (i)-(iii).
  • the medium further comprises (iv) about 15 pM or about 12 pM phosphoAKT inhibitor VIII.
  • the medium further comprises (iv) about 100 pM or about 200 pM metformin.
  • the medium further comprises (iv) about 1 pM or about 5 pM rapamycin.
  • the medium further comprises (iv) about 45 mM 2DG.
  • the medium comprises about 60 mM potassium ion and (i) about 72.2 mM NaCl; (ii) about 16.8 mM glucose; (iii) about 1.6 mM calcium ion; or (iv) any combination of (i)-(iii).
  • the medium further comprises (iv) about 15 pM or about 12 pM phosphoAKT inhibitor VIII.
  • the medium further comprises (iv) about 100 pM or about 200 pM metformin.
  • the medium further comprises (iv) about 1 pM or about 5 pM rapamycin.
  • the medium further comprises (iv) about 45 mM 2DG.
  • the medium comprises about 65 mM potassium ion and (i) about
  • the medium comprises about 70 mM potassium ion and (i) about 63.9 mM NaCl; (ii) about 15.9 mM glucose; (iii) about 1.4 mM calcium ion; or (iv) any combination of (i)-(iii).
  • the medium comprises about 75 mM potassium ion and (i) about 59.3 mM NaCl; (ii) about 15.4 mM glucose; (iii) about 1.3 mM calcium ion; or (iv) any combination of (i)-(iii). In some aspects, the medium comprises about 80 mM potassium ion and (i) about 55.6 mM NaCl; (ii) about 15 mM glucose; (iii) about 1.2 mM calcium ion; or (iv) any combination of (i)-(iii). In some aspects, the medium further comprises (iv) about 15 pM or about 12 pM phosphoAKT inhibitor VIII.
  • the medium further comprises (iv) about 100 pM or about 200 pM metformin. In some aspects, the medium further comprises (iv) about 1 pM or about 5 pM rapamycin. In some aspects, the medium further comprises (iv) about 45 mM 2DG.
  • the media for redirection can further comprise an immunomodulatory molecule such as a cytokine.
  • immunomodulatory molecules are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian-inhibiting substance; mouse gonadotropin- associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-beta; platelet-
  • growth hormones such as
  • the medium further comprises one or more cytokines.
  • the one or more cytokines comprise Interleukin-2 (IL-2), Interleukin-7 (IL-2), Interleukin- 21 (IL-21), Interleukin- 15 (IL- 15), or any combination thereof.
  • the one or more cytokines comprise IL-2, IL-7, and IL-15.
  • the one or more cytokines comprise IL-2.
  • the medium comprises IL-2 at a concentration from about 0.1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about 1 ng/mL to about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3
  • the concentration of IL-2 is about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 4 ng/mL, about 5 ng/mL, about 6 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, or about 20 ng/mL.
  • the medium comprises IL-2 at a concentration at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 1100, at least about 1200, at least about 1300, at least about 1400, at least about 1500, at least about 1600, at least about 1700, at least about 1800, at least about 1900, at least about 2000, at least about 2100, at least about 2200, at least about 2300, at least about 2400, at least about 2500, at least about 2600, at least about 2700, at least about 2800, at least about 2900, at least about 3000, at least about 3100, at least about 3200, at least about 3300, at least about 3400, at least about 3500, at least about 3600, at least about 3700, at least about 3800, at least about 3900, at least about 4000, at least about 4100, at least about 4200
  • the medium comprises IL-2 at a concentration between about 1000 and about 8000, about 1500 and about 8000, about 2000 and about 8000, about 2500 and about 8000, about 3000 and about 8000, about 3500 and about 8000, about 4000 and about 8000, about 4500 and about 8000, about 5000 and about 8000, about 5500 and about 8000, about 6000 and about 8000, about 6500 and about 8000, about 7000 and about 8000, about 1000 and about 7000, about 1500 and about 7000, about 2000 and about 7000, about 2500 and about 7000, about 3000 and about 7000, about 3500 and about 7000, about 4000 and about 7000, about 4500 and about 7000, about 5000 and about 7000, about 5500 and about 7000, about 6000 and about 7000, about 6500 and about 7000, about 1000 and about 6000, about 1500 and about 6000, about 2000 and about 6000, about 2500 and about 6000, about 3000 and about 6000, about 3500 and about 7000, about 3500 and
  • the medium comprises IL-2 at a concentration of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3100, about 3200, about 3300, about 3400, about 3500, about 3600, about 3700, about 3800, about 3900, about 4000, about 4100, about 4200, about 4300, about 4400, about 4500, about 4600, about 4700, about 4800, about 4900, about 5000, about 5100, about 5200, about 5300, about 5400, about 5500, about 5600, about 5700, about 5800, about 5900, about 6000, about 6100, about 6200, about 6
  • the concentration of IL-2 is about 60 lU/mL.
  • the medium further comprises calcium ion, glucose, or any combination thereof.
  • T cell Activating or Stimulating Agents and Costimulatory agents T cell Activating or Stimulating Agents and Costimulatory agents.
  • the cells herein are contacted with a T cell activating or stimulating agent, and/or a costimulatory agent, that provides a primary activation signal and an agent that provides a costimulatory signal.
  • a T cell activating or stimulating agent and/or the costimulatory agent are provided in the medium (either control medium, medium comprising at least 5 mM potassium concentration, or standard immune cell (e.g., T cell) culture medium).
  • the medium further comprises a T cell activating agent.
  • the T cell activating or stimulating agent and/or the costimulatory agent is absorbed to a surface.
  • the origin immune cells, the partially reprogrammed cells and the redirected cells can all be contacted with the T cell activating or stimulating agents and/or costimulatory agents. In some aspects, the origin immune cells and the partially reprogrammed cells can all be contacted with the T cell activating or stimulating agents and/or costimulatory agents. In some aspects, the origin immune cells and the redirected cells can all be contacted with the T cell activating or stimulating agents and/or costimulatory agents. In some aspects, the partially reprogrammed cells and the redirected cells can all be contacted with the T cell activating or stimulating agents and/or costimulatory agents. In some aspects, the origin immune cells, the partially reprogrammed cells and/or the redirected cells can be contacted with the same or different T cell activating or stimulating and/or costimulatory agents.
  • T cell costimulatory agent includes, but is not limited to CD28, CD27, CD137 (4-1BB), 0X40, or ICOS, including antibodies such as those present on the surface of a solid support, such as a bead (for example, TRANSACT, Miltenyi).
  • the T cell activating agent comprises an antibody or antigen-binding fragment thereof that binds CD3.
  • the T cell activating agent is anti-CD3, anti-CD28, one or more cytokines such as IL2, IL7, IL15.
  • the medium further comprises a T cell costimulatory agent.
  • the T cell costimulatory agent comprises an antibody that binds CD28.
  • Agents that provide a primary activation signal include for example antibodies or antigen-binding fragments thereof or ligands, or target binding fragments thereof, that bind to the CD3 cell surface receptor (e.g., an anti-CD3 antibody) expressed on T cells.
  • the agent that provides a primary activation signal is an antigen (e.g., a tumor antigen).
  • the medium further comprises a T cell activating agent.
  • the activating and/or costimulatory agents herein may be coated or adsorbed onto one or more surfaces.
  • Such surfaces include, for example, solid surfaces, porous surfaces, semi-porous surfaces, spherical surfaces, non-spherical surfaces, rod-like surfaces, and polymeric surfaces.
  • the T cell activating agent is capable of inhibiting the expression or activity of the at least one reprogramming factor.
  • the cells can be activated and expanded generally using methods as described, for example, in U.S. Pats. 5,858,358; 5,883,223; 6,352,694; 6,534,055; 6,797,514; 6,867,041; 6,692,964; 6,887,466; 6,905,680; 6,905,681; 6,905,874; 7,067,318; 7,144,575; 7,172,869; 7,175,843; 7,232,566; 7,572,631; and 10,786,533.
  • Agents that provide a costimulatory signal include, but are not limited to, antibodies or ligands that bind to CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, etc.
  • the activating or stimulating agents include one or more agents, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex.
  • the agent turns on or initiates a primary TCR/CD3 intracellular signaling cascade in a T cell, such as agents suitable to deliver a primary signal, e.g., to initiate activation of an IT AM- induced signal, such as those specific for a TCR component.
  • the activating or stimulating agent is provided in combination with or at the same time as an agent that promotes a costimulatory signal.
  • Suitable agents that promote a costimulatory signal include antibodies or ligands that promote signaling of CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, inducible T cell costimulator (ICOS), lymphocyte function-associated antigen- 1 (LFA-1 (CD1 la/CD18), CD247, CD276 (B7-H3), Ig alpha (CD79a), DAP- 10, Fc gamma receptor or any combination thereof.
  • the stimulatory and costimulatory agents for the activation steps of the methods here comprise an anti-CD3, anti-CD28, anti-41-BB, or anti-CD27 antibody for example, bound to solid support such as a bead, and/or one or more cytokines.
  • the agents comprise a ligand for an activating receptor or a costimulatory receptor, e.g., a peptide/MHC complex and/or the CD27 ligand (e.g., CD70 or trimerized versions thereof), CD80, CD86 and the like.
  • the stimulating and costimulatory agents are anti-CD3/anti-CD28 beads (e.g., TRANSACT, DYNABEADS M-450 CD3/CD28 T Cell Expander, and/or ExpACT beads).
  • the stimulation and/or activation may comprise adding anti-CD3 and/or anti CD28 antibody to the culture medium.
  • the activating or stimulating agents include IL-2, IL-7 and/or IL-15.
  • a T cell activating or stimulating agents is an antigen.
  • An antigen for use herein can be any antigen which is associated with a condition.
  • the antigen may be, but is not limited to, a cancer antigen (also termed a tumor antigen or a tumor-associated antigen) or a viral antigen, a bacterial antigen, a fungal antigen.
  • Viral antigens are known in the art and include, for example, any viral protein, e.g., env, gag, pol, gpl20, thymidine kinase, and the like).
  • a tumor antigen is neo-antigen.
  • the antigen is a tumor-associated antigen derived or associated with any of the cancers listed herein.
  • the antigen is or is derived from CD 19, TRAC, TCRCE ⁇ BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL- 13Ra2, mesothelin, IL-1 IRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-
  • the T cell media or media comprising at least about 5mM potassium ion, or media comprising a phosphoAKT inhibitor, metformin, an mTOR inhibitor, 2DG, or any combination thereof, used for activation of the origin cells, partially reprogrammed cells or redirected T cells may further comprise an immunomodulatory molecule such as a cytokine.
  • immunomodulatory molecules are lymphokines, monokines, and traditional polypeptide hormones.
  • growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoin
  • FSH follicle
  • the methods herein comprise formulating the redirected T cells for administration.
  • compositions comprising the redirected T cells herein.
  • Some aspects of the present disclosure are directed to a population of T cells or redirected T cells prepared by a method disclosed herein.
  • Some aspects of the present disclosure are directed to a population of T cells or redirected T cells prepared by a method disclosed herein.
  • Some aspects of the present disclosure are directed to a method of preventing or treating a disease or a condition in a subject in need thereof comprising administering to the subject a composition comprising the cells produced by a method or population of cells disclosed herein.
  • the disease or condition comprises a cancer, a viral condition or an automimmune disorder.
  • the cancer is acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, head and neck cancers (e.g., cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity), cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian
  • Some aspects of the present disclosure are directed to a population of redirected cells, e.g., redirected immune cells (e.g., T cells, NK cells, and/or TILs), cultured according to any of the methods disclosed herein.
  • Some aspects of the present disclosure are directed to a composition comprising a population of redirected cells, e.g., redirected immune cells (e.g., T cells, NK cells, and/or TILs), cultured according to any of the methods disclosed herein.
  • the cells produced using the methods described herein comprise a CAR.
  • redirected immune cell e.g., T cells, NK cells, and/or TILs
  • the redirected T cell is a CD8+ T cell or CD4+ T cell.
  • the T cell is a Th 1, Th2, Th 17, or Tcl7 cell.
  • a CAR-expressing cell is a CAR T cell, e.g., a mono CAR T cell, a genome-edited CAR T cell, a dual CAR T cell, or a tandem CAR T cell. Examples of such CAR T cells are provided in International Application No. PCT/US2019/044195.
  • a cell composition described herein is administered to a subject in need thereof.
  • the cell e.g., a redirected cell prepared using the methods disclose herein is administered to a subject to treat a cancer, e.g., a tumor.
  • the method of treating comprises administering to the subject an effective amount of a cell composition of the disclosure, e.g., a cell prepared according to the methods disclosed herein.
  • the present disclosure also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue in a subject, comprising administering an effective amount of a cell composition of the disclosure, e.g., a cell prepared according to the methods disclosed herein.
  • the present disclosure also provides a method of providing an anti-tumor immunity in a subject in need thereof, the method comprising administering a cell composition of the disclosure, e.g., a cell prepared according to the methods disclosed hereinto the subject.
  • a cell composition of the disclosure e.g., a cell prepared according to the methods disclosed hereinto the subject.
  • the cell administered in the cell composition of the disclosure is a T cell.
  • the cell is an autologous T cell.
  • the cell is an allogenic T cell.
  • administering the cell composition of the disclosure e.g., a redirected immune cell (e.g., a redirected T cell), prepared according to the methods disclosed herein) reduces a tumor volume in the subject compared to a reference tumor volume.
  • the reference tumor volume is the tumor volume in the subject prior to the administration of the engineered cell.
  • the reference tumor volume is the tumor volume in a corresponding subject that did not receive the administration.
  • the tumor volume in the subject is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after the administration compared to the reference tumor volume.
  • treating a tumor comprises reducing a tumor weight in the subject.
  • administering the cell composition of the disclosure e.g., a redirected immune cell e.g. T cell
  • the tumor weight is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after the administration compared to a reference tumor weight.
  • the reference tumor weight is the tumor weight in the subject prior to the administration of the cell composition of the disclosure.
  • the reference tumor weight is the tumor weight in a corresponding subject that did not receive the administration.
  • administering the cell composition of the disclosure e.g., a redirected immune cell (e.g., T cell), prepared according to the methods disclosed herein
  • a subject e.g., suffering from a tumor
  • TILs e.g., CD4 + or CD8 +
  • TEE tumor microenvironment
  • the number and/or percentage of TILs in a tumor and/or TME is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, or at least about 300% or
  • administering the cell composition of the disclosure e.g., a redirected immune cell (e.g., T cell), prepared according to the methods disclosed herein
  • a subject e.g., suffering from a tumor
  • administering the cell composition of the disclosure can increase the duration of an immune response in a subject relative to the duration of an immune response in a subject administered a similar cell therapy comprising cells prepared according to conventional methods.
  • the duration of the immune response is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 1000% or more compared to a reference (e.g, a subject administered a similar cell therapy comprising cells prepared according to conventional methods).
  • a reference e.g, a subject administered a similar cell therapy comprising cells prepared according to conventional methods.
  • the duration of the immune response is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold or more compared to a reference (e.g., a subject administered a similar cell therapy comprising cells prepared according to conventional methods).
  • a reference e.g., a subject administered a similar cell therapy comprising cells prepared according to conventional methods.
  • administering the cell composition of the disclosure can have other effects which are conducive for the treatment of a tumor.
  • a redirected immune cell e.g., T cell
  • Some aspects of the present disclosure are directed to a method of preventing or treating a disease or a condition in a subject in need thereof comprising administering to the subject a population of redirected T cells produced by a method or population of cells disclosed herein.
  • the disease or condition comprises a cancer, a viral condition or an automimmune disorder.
  • the cancer is acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, head and neck cancers (e.g., cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity), cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian
  • the cell composition of the disclosure can be used to treat variety of cancer types, e.g., a tumor derived from a cancer comprising a breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, renal cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (gastric) cancer, gastrointestinal cancer, ovarian cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof.
  • a redirected immune cell e.g., T cell
  • the cell composition of the disclosure e.g., a redirected immune cell (e.g., T cell), prepared according to the methods disclosed herein
  • other therapeutic agents e.g., anti-cancer agents and/or immunomodulating agents.
  • a method of treating a tumor disclosed herein comprises administering the cell composition of the disclosure in combination with one or more additional therapeutic agents.
  • the cell composition of the disclosure e.g., a redirected immune cell (e.g., T cell), prepared according to the methods disclosed herein
  • a redirected immune cell e.g., T cell
  • an anti-cancer agent comprises an immune checkpoint inhibitor (i.e., blocks signaling through the particular immune checkpoint pathway).
  • Non-limiting examples of immune checkpoint inhibitors that can be used in the present methods comprise a CTLA-4 antagonist (e.g., anti-CTLA-4 antibody), PD-1 antagonist (e.g., anti-PD-1 antibody, anti-PD-Ll antibody), TIM-3 antagonist (e.g., anti-TIM-3 antibody), or combinations thereof.
  • the checkpoint inhibitor is a PD-1 antagonist.
  • the checkpoint inhibitor is an anti-PD-1 antibody.
  • the checkpoint inhibitor is an anti-PD-Ll antibody.
  • the cell composition of the disclosure (e.g., a redirected immune cell (e.g., T cell), prepared according to the methods disclosed herein) is administered to the subject prior to or after the administration of the additional therapeutic agent.
  • the cell composition of the disclosure e.g., a redirected immune cell (e.g., T cell), prepared according to the methods disclosed herein) is administered to the subject concurrently with the additional therapeutic agent.
  • the cell composition of the disclosure (e.g., a redirected immune cell (e.g., T cell), prepared according to the methods disclosed herein) and the additional therapeutic agent can be administered concurrently as a single composition in a pharmaceutically acceptable carrier.
  • the cell composition of the disclosure e.g., a redirected immune cell (e.g., T cell), prepared according to the methods disclosed herein) and the additional therapeutic agent are administered concurrently as separate compositions.
  • a cell composition disclosed herein e.g., a redirected immune cell (e.g., T cell), prepared according to the methods disclosed herein
  • can be used in combination with other therapeutic agents e.g., anti-cancer agents and/or immunomodulating agents.
  • a method of treating a tumor disclosed herein comprises administering a cell composition of the present disclosure (e.g., a redirected immune cell (e.g., T cell), prepared according to the methods disclosed herein) in combination with one or more additional therapeutic agents to a subject.
  • Such agents can include, for example, chemotherapeutic drug, targeted anticancer therapy, oncolytic drug, cytotoxic agent, immune-based therapy, cytokine, surgical procedure, radiation procedure, activator of a costimulatory molecule, immune checkpoint inhibitor, a vaccine, a cellular immunotherapy, or any combination thereof.
  • a cell composition disclosed herein e.g., a redirected immune cell (e.g., T cell), prepared according to the methods disclosed herein
  • a standard of care treatment e.g., surgery, radiation, and chemotherapy.
  • Methods described herein can also be used as a maintenance therapy, e.g. , a therapy that is intended to prevent the occurrence or recurrence of tumors.
  • a cell composition of the present disclosure e.g., a redirected immune cell (e.g., T cell), prepared according to the methods disclosed herein
  • a redirected immune cell e.g., T cell
  • Non-limiting of such combinations include: a therapy that enhances tumor antigen presentation (e.g., dendritic cell vaccine, GM-CSF secreting cellular vaccines, CpG oligonucleotides, imiquimod); a therapy that inhibits negative immune regulation e.g., by inhibiting CTLA-4 and/or PD-1/PD-L1/PD-L2 pathway and/or depleting or blocking Tregs or other immune suppressing cells (e.g., myeloid-derived suppressor cells); a therapy that stimulates positive immune regulation, e.g., with agonists that stimulate the CD-137, OX-40, and/or CD40 or GITR pathway and/or stimulate T cell effector function; a therapy that increases systemically the frequency of anti-tumor T cells; a therapy that depletes or inhibits Tregs, such as Tregs in the tumor, e.g., using an antagonist of CD25 (e.g., daclizumab) or by ex vivo anti-CD25
  • an anti-cancer agent comprises an immune checkpoint inhibitor (i.e., blocks signaling through the particular immune checkpoint pathway).
  • immune checkpoint inhibitors that can be used in the present methods comprise a CTLA-4 antagonist (e.g., anti-CTLA-4 antibody), PD-1 antagonist (e.g., anti-PD-1 antibody, anti-PD-Ll antibody), TIM-3 antagonist (e.g., anti-TIM-3 antibody), or combinations thereof.
  • Non-limiting examples of such immune checkpoint inhibitors include the following: anti-PDl antibody (e.g., nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®; MK-3475), pidilizumab (CT-011), PDR001, MEDI0680 (AMP-514), TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380), PF- 06801591, BGB-A317, BI 754091, SHR-1210, and combinations thereof); anti-PD-Ll antibody (e.g., atezolizumab (TECENTRIQ®; RG7446; MPDL3280A; RO5541267), durvalumab (MEDI4736, IMFINZI®), BMS-936559, avelumab (BAVENCIO®), LY3300054, CX-072 (Proclaim-CX-072), FAZ053, KN035, MDX-1105,
  • an anti-cancer agent comprises an immune checkpoint activator (i.e., promotes signaling through the particular immune checkpoint pathway).
  • immune checkpoint activator comprises 0X40 agonist (e.g., anti-OX40 antibody), LAG-3 agonist (e.g. anti-LAG-3 antibody), 4-1BB (CD137) agonist (e.g., anti-CD137 antibody), GITR agonist (e.g., anti-GITR antibody), TIM3 agonist (e.g., anti-TIM3 antibody), or combinations thereof.
  • a cell composition disclosed herein e.g., a redirected immune cell (e.g., T cell), prepared according to the methods disclosed herein
  • a cell composition disclosed herein is administered to the subject prior to or after the administration of the additional therapeutic agent.
  • cell composition disclosed herein e.g., a redirected immune cell (e.g., T cell), prepared according to the methods disclosed herein
  • the additional therapeutic agent can be administered concurrently as a single composition in a pharmaceutically acceptable carrier.
  • the cell composition disclosed herein e.g., a redirected immune cell (e.g., T cell), prepared according to the methods disclosed herein
  • the additional therapeutic agent and the cell composition disclosed herein are administered sequentially.
  • Some aspects of the present disclosure are directed to methods of treating an autoimmune disease, comprising administering a cell, e.g., a Treg cell, cultured according to any of the methods disclosed herein. Some aspects of the present disclosure are directed to methods of treating an inflammatory pathology, comprising administering a cell, e.g., a Treg cell, cultured according to any of the methods disclosed herein.
  • the inflammatory pathology comprises cytokine release syndrome.
  • the inflammatory pathology comprises sepsis.
  • the inflammatory pathology comprises graft-versus host disease.
  • the cell, e.g., the Treg cell is engineered.
  • the isolated T cells can be genetically modified following isolation using known methods, or the T cells can be partially reprogrammed, redirected and expanded in vitro prior to being genetically modified.
  • the T cells are genetically modified with a recombinant TCR or CAR and then are redirected using the partial reprogramming and reactivation methods described herein.
  • the redirected cells described herein are further activated and/or expanded in vitro.
  • Methods for activating and expanding immune cells are known in the art and are described, for example, in U.S. Pat. Nos. 6,905,874; 6,867,041; 6,797,514; and W02012/079000.
  • such methods include contacting the T cells with a stimulatory (e.g., activating) agent (in some aspects, referred to as a T cell activating compound) (e.g., an agent that stimulates a CD3/TCR complex (for example, an anti-CD3 antibody or a CD3 agonist) and a costimulatory agent (an agent such as an antibody or ligand that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, CD2, CD226 or any combination thereof).
  • a stimulatory agent e.g., activating
  • a T cell activating compound e.g., an agent that stimulates a CD3/TCR complex (for example, an anti-CD3 antibody or a CD3 agonist) and a costimulatory agent (an agent such as an antibody or ligand that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1
  • the stimulatory agent and the costimulatory agent are attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2.
  • the redirected T cell may be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Pat. Nos. 6,040,177; 5,827,642; and WO/2012129514.
  • the disclosure provides a method of storing the redirected T cells described herein. This involves cryopreserving the T cells such that the cells remain viable upon thawing. A fraction of the T cells can be cryopreserved by methods known in the art to provide a permanent source of such cells for the future treatment of patients afflicted with a malignancy. When needed, the cryopreserved T cells can be thawed, grown and expanded for more such cells. [0522] As used herein, “cryopreserve” refers to the preservation of cells by cooling to subzero temperatures, such as (typically) 77 Kelvin or 196° C. (the boiling point of liquid nitrogen).
  • Cryoprotective agents are often used at sub-zero temperatures to prevent the cells being preserved from damage due to freezing at low temperatures or warming to room temperature. Cryopreservative agents and optimal cooling rates can protect against cell injury.
  • Cryoprotective agents which can be used in accordance with the disclosure include but are not limited to: dimethyl sulfoxide (DMSO) (Lovelock & Bishop, Nature, 1959, 183, 1394-1395; Ashwood- Smith, Nature, 1961, 190, 1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, Ann. N.Y. Acad. Sci., 1960, 85, 576), and polyethylene glycol (Sloviter & Ravdin, Nature, 1962, 196, 48).
  • the preferred cooling rate is l°-3° C/minute.
  • the term, “substantially pure,” is used to indicate that a given component is present at a high level.
  • the component is desirably the predominant component present in a composition. Preferably it is present at a level of more than 30%, of more than 50%, of more than 75%, of more than 90%, or even of more than 95%, said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration. At very high levels (e.g.
  • the component can be regarded as being in “pure form.”
  • Biologically active substances of the present disclosure can be provided in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated.
  • the contaminant will be at a low level (e.g., at a level of less than 10%, less than 5%, or less than 1% on the dry weight/dry weight basis set out above).
  • the cells herein are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a “pharmaceutically acceptable” carrier) in a treatment-effective amount.
  • a medium and container system suitable for administration a “pharmaceutically acceptable” carrier
  • Suitable infusion media can be any isotonic medium formulation, typically normal saline, NormosolTM R (Abbott) or Plasma-LyteTM A (Baxter), but also 5% dextrose in water or Ringer’ s lactate can be utilized.
  • the infusion medium can be supplemented with human serum albumin.
  • Desired treatment amounts of cells in the composition is generally at least 2 cells or is more typically greater than 10 2 cells, and up to 10 6 , up to and including 10 8 or 10 9 cells and can be more than 10 10 cells.
  • the number of cells will depend upon the desired use for which the composition is intended, and the type of cells included therein.
  • the density of the desired cells is typically greater than 10 6 cells/ml and generally is greater than 10 7 cells/ml, generally 10 8 cells/ml or greater.
  • the clinically relevant number of immune cells e.g., T cells
  • T cells can be apportioned into multiple infusions that cumulatively equal or exceed 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 cells.
  • infused cells may be administered.
  • Redirected T cell treatments may be administered multiple times at dosages within these ranges.
  • the cells may be autologous, allogeneic, or heterologous to the patient undergoing therapy.
  • the redirected T cells of the present disclosure may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.
  • Pharmaceutical compositions of the present disclosure may comprise a redirected T cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins polypeptides or amino acids such as glycine
  • antioxidants e.g., chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • compositions of the present application can comprise, consist essentially of, or consist of, the components disclosed.
  • compositions of the disclosure may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylene-diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • An injectable pharmaceutical composition is preferably sterile.
  • adverse events may be minimized by transducing the immune cells (e.g., T cells) with a suicide gene.
  • Suitable “kill switches” are described for example in W02021/189008. It may also be desired to incorporate an inducible “on” or “accelerator” switch into the immune cells (e.g., T cells).
  • These techniques may employ the use of dimerization domains and optional activators of such domain dimerization. These techniques include, e.g., those described by Wu et al., Science 2014, 350(6258) utilizing FKBP/Rapalog dimerization systems in certain cells. Additional dimerization technology is described in, e.g., Fegan et al. Chem. Rev.
  • dimerization pairs may include cyclosporine- A/cyclophilin, receptor, estrogen/estrogen receptor (optionally using tamoxifen), glucocorticoids/glucocorticoid receptor, tetracycline/tetracycline receptor, vitamin D/vitamin D receptor.
  • dimerization technology can be found in e.g., WO 2014/127261, WO 2015/090229, US 2014/0286987, US 2015/0266973, US 2016/0046700, U.S. Patent No.
  • Suitable techniques for genetic modification of the redirected cells herein include use of inducible caspase-9 (U.S. Appl. Pub. No. 2011/0286980) or a thymidine kinase, before, after or at the same time, as the cells are genetically modified to express a CAR or other engineered TCR. Additional methods for introducing suicide genes and/or “on” switches include CRISPR, TALENS, MEGATALEN, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other techniques known in the art.
  • the methods of the disclosure can be used to redirect cells in culture (e.g., ex vivo or in vitro) to improve function and potency for use in cell therapy.
  • the cells used in treatment of a patient can be autologous or allogeneic.
  • the cells are derived from the patient or a matched donor.
  • cells are obtained directly from the patient to be treated, transfected with a polynuceotide encoding cellular reprogramming factors, as described herein, and reimplanted in the patient.
  • Such cells can be obtained, for example, from a biopsy or surgical procedure performed on the patient.
  • kits comprising one or more redirected T cells prepared by the present disclosure.
  • the present disclosure includes kits compising one or more partially reprogrammed cells and a cell culture medium comprising a phosphoAKT inhibitor, metformin, an mTOR inhibitor, 2DG, or any combination thereof.
  • the kits include at least one reprogramming factor for contacting one or more origin cells in vitro and a culture medium comprising a potassium ion concentration higher than 5 mM.
  • Kits typically include a label indicating the intended use of the contents of the kit and instructions for use.
  • label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
  • the disclosure provides a kit for preparing one or more origin cells for a cell therapy for a subject in need thereof, the kit comprising (i) at least one reprogramming factor selected from the group consisting of a molecule that increases expression of a KLF4 protein, a molecule that increases expression of an OCT4 protein, a molecule that increases expression of a SOX2 protein and a molecule that increases expression of a C-MYC protein, a molecule that increases expression of a LIN28 protein, a molecule that increases expression of a NANOG protein and optionally, a molecule that increases expression of a SV40 Large T Antigen and (ii) a cell culture medium comprising a phosphoAKT inhibitor, metformin, an mTOR inhibitor, 2DG, or any combination thereof.
  • a reprogramming factor selected from the group consisting of a molecule that increases expression of a KLF4 protein, a molecule that increases expression of an OCT4 protein, a molecule that increases expression of a SO
  • the disclosure provides a kit for preparing one or more cell vectors for a cell therapy for a subject in need thereof, the kit comprising (i) a Sendai Virus encoding a KLF4 protein, an OCT4 protein, a SOX2 protein, a C-MYC protein, a LIN28 protein, and/or a NANOG protein; and A Sendai virus encoding a SV40 Large T Antigen and (ii) a cell culture medium comprising a phosphoAKT inhibitor, metformin, an mTOR inhibitor, 2DG, or any combination thereof.
  • the kit further comprises a cell activating compound or agent. In some aspects, the kit comprises both an activating agent and a differentiating agent. In some aspects, the kit further comprises one or more cytokines. Such cytokines include but are not limited to IL-2, IL-7, IL-15 and IL-12.
  • Example 1 Redirected partially reprogrammed CD19-CAR T cells exhibit lower mitochondrial membrane potential ( 'l'm)
  • Mitochondria play a key role as the powerhouse of cells, they are crucial to catabolize nutrients for energy and generate biosynthetic precursors for formation of macromolecules. Therefore, properties of the mitochondria in redirected T cells and control cells were compared using flow cytometry to analyze mitochondrial membrane potential.
  • PBMC Peripheral blood mononuclear cell
  • TCM T cell culture medium
  • CTS SR Immune Cell Serum Replacement
  • L-Glutamine 200 mM LOOx
  • GlutaMAX 200 mM LOOx
  • Penicillin- Streptomycin 10,000 units/mL
  • IL-2 60 lU/ml The next day (day -1), cells were transduced with a lentivirus encoding an anti-CD19-CAR in the presence of Lentiboost 1/50.
  • reprogramming factors (Yamanaka factors + SV40) were introduced into the T cells by Sendai virus transduction. Activation of T cells generally increases the efficiency of transduction of the Sendai virus.
  • the Sendai virus (Cytotune iPS 2.0 Sendai Reprogramming Kit, ThermoFisher) was composed of three vectors: 1) encoding KLF4-OCT3/4- SOX2 (KOS); 2) encoding KLF4; 3) encoding C-MYC.
  • TRJ transfected T cells
  • AMSBIO iMatrix
  • Control cells were kept in culture in TCM + IL-2 T cell culture medium (TCM) with 60 lU/ml IL2 (TCM: OpTmizer Basal Medium; OpTmizer Cell Supplement; Immune Cell Serum Replacement (CTS SR); L-Glutamine 200 mM (lOOx); GlutaMAX 200 mM (lOOx); Penicillin-Streptomycin 10,000 units/mL (lOOx); IL-2 60 lU/ml).
  • TCM T cell culture medium
  • CTS SR Immune Cell Serum Replacement
  • Samples were normalized to CD19-CAR + percentage cell numbers and cultured in a ratio 1 :4 effector to target ratio (5e4 CD19-CAR + T cells and 2e5 Nalm6 cells), for 3 sequential rounds, by adding to the next culture 1 :4 of the previous co-culture as shown in FIG. 1C.
  • a ratio 1 :4 effector to target ratio 5e4 CD19-CAR + T cells and 2e5 Nalm6 cells
  • Example 2 Redirected partially reprogrammed CD19-CAR T cells showed decreased expression of glucose transporter 1 (Glutl) and similar carnitine palmitoyltransferase 1A (CPTla) expression
  • Glutl glucose transporter 1
  • CPTla carnitine palmitoyltransferase 1A
  • Glucose transporter 1 (Glutl) is conventionally associated with increased glycolytic metabolism, given the role that it plays in transporting glucose molecules inside the cell as a substrate for glycolysis.
  • CPTla carnitine palmitoyltransferase 1 A
  • FEO fatty acid oxidation
  • OXPHOS oxidative phosphorylation
  • PBMC derived CD8 + T cells from 3 independent donors (#6, #8, #889) were transduced with a lentivirus codifying for CD19-CAR, and partially reprogrammed, as described in Example 1.
  • PBMC derived CD8 T cells from donor #27035 were transduced with a lentivirus codifying for CD19-CAR, and partially reprogrammed, as described in Example 1.
  • control T cells TCT
  • TRJ redirected T cells
  • AKT inhibition improved TCT antitumor activity in vitro, while TRJ retained similar activity in the presence or absence of AKT inhibition.
  • a low dose of Rapamycin improved TCT antitumor activity in vitro, while TRJ retained similar activity in the presence or absence of low dose Rapamycin.
  • a high dose of Rapamycin greatly impaired both TCT and TRJ antitumor activity in vitro.
  • FIG. 7 shows that low and high doses of Metformin (100 uM or 200 uM) improved TCT antitumor activity in vitro, while TRJ retained similar activity in the presence or absence of metformin regardless of the concentration provided, indicating that 100 uM is sufficient to exert its activity.
  • Example 4 Redirected partially reprogrammed NY-ESO-1 T cells showed increased expression of CCR7 after in vitro pretreatment with selected small molecules and show lower mitochondrial membrane potential, and lower Glutl expression after serial in vitro antigen recognition
  • each of the cell types, TCT and TRJ were collected and either left untreated in TCM + IL-2, or treated with small molecules including AKT inhibitor VIII, 12 uM, Rapamycin, 1 nM (Selleckchem), Metformin, 100 uM (Sigma- Aldrich), or 2-Deoxy-D-Glucose 45 nM (2-DG, Sigma-Aldrich).
  • 2-DG is a glucose analog in which the 2-hydroxyl group is replaced by hydrogen. This chemical modification leads to 2DG not being able to enter glycolysis and therefore cannot contribute to ATP production.
  • Samples were normalized to NY-ESO-1 + total cell numbers, and cultured in a ratio 1 :4 effector to target ratio (5e4 NY-ESO-1 + T cells and 2e5 A375-NLR cells), for 5 sequential rounds, by adding to the next culture a 1 :4 of the previous co-culture to a new 24-well plate seeded with fresh 2e5 A375-NLR cells every 3-4 days after co-culture, without addition of the test compounds to the run.
  • AKT inhibition improved NY-ESO-1 TRJ cells in vitro antitumor efficacy in donor #12.
  • Rapamycin 1 uM impaired NY-ESO-1 TRJ antitumor effect in both donors.
  • FIGs 13 A-13B show Metformin 100 uM improved NY-ESO-1 TRJ in both donors. In particular, for donor #11 the effect was evident until the 5 th round, and for donor #12 this effect was observed until the 4 th round of killing.
  • FIG. 15 A NY-ESO-1 TRJ showed higher CD3 + .
  • donor #11 showed higher % of CCR7 + cells, while #12 showed lower percentage CCR7 + cells. Both donors exhibited lower expression of Glutl, see FIG. 15C, and lower mitochondrial membrane potential, as shown in FIG. 15D.
  • Example 5 Redirected partially reprogrammed CD4 + and CD8 + T cells exhibit modified metabolism
  • TCM T cell culture medium
  • CTS SR Immune Cell Serum Replacement
  • L-Glutamine 200 mM LOOx
  • GlutaMAX 200 mM LOOx
  • Penicillin- Streptomycin 10,000 units/mL
  • reprogramming factors (Yamanaka factors + SV40) were introduced into the T cells by Sendai virus transduction.
  • the infected T cells were then transferred onto iMatrix (AMSBIO, 892021) coated dishes on day 1 and were cultured in iPS cell media. On day 3 fresh media was added, and on day 5 half of the media was carefully changed. Control cells were kept in culture in TCM + IL-2.
  • TCT an TRJ cells were collected and plated onto 96 well plate PDL-coated Seahorse cell plates, 3e5 cells per well, to facilitate T cell attachment.
  • T cell Metabolic Profiling Kit or Glycolytic Rate Assay Kit (Seahorse XF, Agilent) were performed following manufacturer’ s instructions.
  • Experiments with the Seahorse system used the following assay conditions: 3 cycles of 3min mixture; Imin wait; and 3min measurement.
  • CD4 + and CD8 + TRJ cells showed lower spare respiratory capacity, expressed as % of OCR (Oxygen Consumption Rate), while FIG. 16B shows that in both CD4 + and CD8 + cells the initial basal respiration of the cells at the beginning of the assay is greatly increased in redirected T cells, demonstrated by the higher oxygen consumption rate.
  • FIG.16C shows that CD4 + and CD8 + TRJ cells produced higher total ATP (pmol/min) from both mitochondrial-derived (e.g., from the oxidative phosphorylation pathway) and glycolysis-derived ATP energy sources.
  • CD4 + and CD8 + TRJ cells show higher basal glycolysis (glycoPER in pmol/min) (see FIG. 16D).
  • CD4 + and CD8 + TRJ cells showed higher compensatory glycolysis (glycoPER in pmol/min).
  • TILs Tumor infiltrating lymphocytes encompass diverse lymphocytic cell populations that invade the tumor tissue. Their presence has been documented in several solid tumors, and they are emerging as a significant biomarker for predicting efficacy and outcome of treatment.
  • TIL therapy is a form of cell-based immunotherapy that utilizes the patient's immune cells obtained from the tumor microenvironment. In this approach, lymphocytes, including TILs, are extracted, expanded ex vivo to increase their numbers, and then reintroduced into the patient.
  • the TILs described here were partially reprogrammed and redirected generally as described in Example 1-5 and tested for improved anti -tumor capacity.
  • the TILs in this example were derived from tumor tissues harvested from a lung adenocarcinoma donor. The tumor tissue was processed with mechanical separation and enzyme digestion to isolate TILs. The TILs were subjected to a pre-stimulation and expansion prior to the introduction of reprogramming factors.
  • the reprogramming factors (Yamanaka factors (OCT4, SOX2, KLF4, c-MYC) + LIN28 + NANOG) were introduced on day 0 using a Sendai virus vector at an MOI of 5.
  • the transfected TILs TRJ
  • TRJ transfected TILs
  • AMSBIO iMatrix
  • TCT Control cells
  • TCM T cell media
  • TRJ As described in Example 4, 2DG can inhibit the ATP production in glycolysis and thus influence cellular metabolism, which could improve the anti-tumor capacity of T cells.
  • TRJ was divided into 2 groups, TRJ and TRJ-2DG. TRJ in media without 2DG was used as the control and TRJ-2DG was cultured in media with 4 mM 2DG (Selleckchem). TRJ-2DG was kept in 2DG treatment until Day 21 and then cultured in media without 2DG.
  • the TRJ-2DG group showed more persistent killing capacity as compared to the TCT and TRJ groups in this assay.
  • TRJ-2DG was able to completely kill target cells up to 4 rounds while TRJ showed partial killing ability in 3 rounds and TCT stopped killing after 2 rounds. This result showed that the addition of 2DG during the redirection phase improved tumor killing persistence of the redirected TILs.
  • Example 7 In vitro pretreatment with 2DG with shorter treatment time improved the phenotype of redirected partially reprogrammed tumor infiltrating lymphocytes.
  • Example 6 The results in Example 6 showed that the 2DG treatment from Day 8 to Day 21 of the partial reprogramming and redirection process promoted a stemlike phenotype and tumor killing potency of partially reprogrammed TILs in a 28-day culture period. In this Example, the effect of a shorter 2DG treatment period on the phenotype of partially reprogrammed TILs was evaluated.
  • the TILs in this example were prepared and subjected to partial reprogramming as described in Example 6. The reprogramming lasted for 7 days. Briefly, the TILs were isolated from tumor tissue and expanded. Then, the reprogramming factors (Yamanaka factors (OCT4, SOX2, KLF4, c-MYC) + LIN28 + NANOG) were introduced to the TIL using a Sendai virus vector at an MOI of 5. The transfected TILs (TRJ) were then transferred onto iMatrix (AMSBIO, 892021) coated dishes on day 1 and were cultured in stem cell media.
  • AMSBIO iMatrix
  • TRJ was divided into 2 groups: TRJ in media without 2DG was used as the control; and TRJ-2DG was cultured in media with 4 mM 2DG (Selleckchem). TRJ-2DG was kept in 2DG treatment until Day 14. The cells were then cultured using culture media without 2DG.
  • the TRJ-2DG group also showed a larger central memory-like T cell (CD45RO+CCR7+) population (17.3%; FIG. 21B) in comparison with the TRJ group (3.52%; FIG. 21A).

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Abstract

La présente invention concerne des procédés de culture de cellules reprogrammées dans un milieu comprenant un inhibiteur de phosphoAKT, de la metformine, un inhibiteur de mTOR, du 2DG, ou toute combinaison de ceux-ci. Selon certains aspects, le milieu comprend en outre au moins environ 5 mM d'ion potassium. Selon certains aspects, les procédés selon l'invention augmentent le nombre de lymphocytes T réorientés comme des souches dans la population de cellules, tout en maintenant ou en améliorant l'activité d'élimination des cellules cibles. Selon certains aspects, les lymphocytes cultivés sont modifiés, par exemple pour comprendre un récepteur antigénique chimérique ou un récepteur T modifié. Selon certains aspects, les cellules sont administrées à un sujet en ayant besoin.
PCT/US2024/017729 2023-02-28 2024-02-28 Procédés pour la culture de cellules reprogrammées WO2024182539A1 (fr)

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