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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
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  • A61K40/421—Immunoglobulin superfamily
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  • A61P35/02—Antineoplastic agents specific for leukemia
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/7051—T-cell receptor (TcR)-CD3 complex
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70517—CD8
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  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • 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
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
• • A—HUMAN NECESSITIES
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  • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
  • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • C—CHEMISTRY; METALLURGY
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  • C07K2319/00—Fusion polypeptide
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  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
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  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
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  • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
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  • C12N2510/00—Genetically modified cells

Definitions

• TCRs tumor-associated molecules with T-cell receptors
• CARs chimeric antigen receptors
• vectors, nucleic acids, and transgenic cells that can be used to improve the clinical efficacy of adoptive cell therapy by improving expansion and/or persistence of the infused cells.
• Described herein is a vector that includes a nucleic acid.
• the nucleic acid encodes an erythropoietin (Epo) receptor; a self-cleaving peptide or internal ribosome entry site; and a cell surface protein.
• Epo erythropoietin
• the Epo receptor can have at least 90% sequence identity to any of SEQ ID NOS: 2, 4, 6, and 8. In some embodiments, the Epo receptor can have at least 90% sequence identity to SEQ ID NOS: 2. In some embodiments, the Epo receptor can have at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the Epo receptor can have at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the Epo receptor can have at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, the Epo receptor is a mutant Epo receptor. In some embodiments, the nucleic acid has a mutation that encodes a stop codon within exon 8 of the Epo receptor.
• the signal peptide can be a signal peptide of a surface protein, such as CD8a signal peptide.
• the cell surface receptor can be a chimeric antigen receptor (CAR).
• the CAR can include a signal peptide; an extracellular receptor domain that binds a target cell antigen; a hinge and transmembrane domain that anchors the extracellular receptor domain on the surface of a cell; and an effector domain.
• the extracellular domain is typically a single-chain variable fragment (scFv).
• the extracellular receptor domain can include a variable immunoglobulin light chain domain and a variable immunoglobulin heavy chain domain joined by a linker domain.
• the linker domain can be (G4S)x (SEQ ID NO: 24), wherein x is an integer from 1 to 100.
• the linker domain can be (G4S)3 (SEQ ID NO: 25).
• the cell surface receptor can be a T-cell receptor.
• the extracellular receptor domain can include an immunoglobulin Fc receptor, such as CD 16, CD32 or CD64.
• the extracellular receptor domain can include a cytokine, such as IL-13, IL-4, IL-7, or IL-3.
• the cell surface receptor can activate immune cells.
• the cell surface receptor can include NKG2D, NKG2C, NCR1, NCR2, NCR3, CD137, CD28, or ICOS.
• the cell surface receptor can include a fragment of NKG2D, NKG2C, NCR1, NCR2, NCR3, CD137, CD28, or ICOS.
• the cell surface receptor can include a ligand of NKG2D, NKG2C, NCR1, NCR2, NCR3, CD137, CD28, or ICOS.
• the cell surface receptor can inhibit immune cells.
• the cell surface receptor can include NKG2A, PD-l, or CTLA-4.
• the cell surface receptor can include a fragment of NKG2A, PD-l, or CTLA-4.
• the cell surface receptor can include a ligand of NKG2A, PD-l, or CTLA-4.
• the cell surface receptor can be a receptor for a cytokine.
• the cell surface receptor can be a receptor for IL-6, IL-l, or TNF alpha.
• the target cell antigen can be a tumor associated antigen or a tumor specific antigen.
• the target cell antigen can be a viral, bacterial, fungal, or parasite associated antigen.
• the target cell antigen is CD19, CD20, CD22, CD123, CD33, B-cell maturation antigen (BCMA), mesothelin, human epidermal growth factor receptor 2 (Her2), prostate-specific membrane antigen (PSMA), or disialoganglioside (GD)-2.
• BCMA B-cell maturation antigen
• Her2 human epidermal growth factor receptor 2
• PSMA prostate-specific membrane antigen
• GD disialoganglioside
• the target cell antigen can be CD 19.
• the extracellular domain can be an anti-CD 19 single-chain variable fragment (scFv).
• the hinge and transmembrane domain can be a CD8a hinge and transmembrane domain.
• the hinge can include a plurality of amino acid residues.
• the transmembrane domain can be a transmembrane domain from CD4, CD8P, CD 16, CD28, CD32, CD34, CD64, CD 137, FceRIy, 0X40, O ⁇ 3z, CD3e, CD3y, CD35, TCRa, VEGFR2, FAS, or FGFR2B.
• the effector domain can include 4-1BB and CD3z.
• the CAR can be anti-CDl9- 41BB-O ⁇ 3z.
• the vector can be a retrovirus, such as a murine stem cell virus (MSCV) retroviral vector.
• the vector can further encode a fluorescent protein.
• the vector can encode an internal ribosomal entry site (IRES).
• the vector can further encode at least one regulatory element for expression of the nucleic acid.
• the method can include introducing into a mammalian host cell any of the vectors described herein.
• the mammalian host cell can be an immune cell, such as a natural killer (NK) cell, a monocyte/macrophage cell, a dendritic cell, or a T cell.
• the T cell can be a human peripheral blood T lymphocyte.
• the T cell can be a CD4+ T cell.
• the T cell can be a CD8+ T cell.
• the T cell can further expresses a T-cell receptor (TCR) that binds a tumor antigen or a viral antigen.
• TCR is endogenous.
• the T cell can be a tumor-infiltrating lymphocyte (TIL), and the method can further include extracting the tumor-infiltrating lymphocyte from a tumor and expanding the TIL ex vivo.
• TIL tumor-infiltrating lymphocyte
• the TCR can be exogenous.
• the method can further include introducing into the T cell a vector that expresses the exogenous TCR.
• the mammalian immune cell can be a natural killer (NK) cell, a monocyte/macrophage cell, a dendritic cell, or a T cell.
• the T cell can be a human T cell.
• the T cell can be a human peripheral blood T lymphocyte.
• the T cell can be as described in the preceding paragraph or as otherwise described herein.
• the method can include introducing mammalian T cells into the subject.
• the mammalian T cells can include any of the vectors described herein.
• the mammal can be a human.
• the mammalian T cells can be autologous cells isolated from the mammal.
• the mammalian T cells can be allogenic cells isolated from a donor.
• the method can further include administering Epo to the subject.
• the method can further include administering IL-2 to the subject. Reducing the number of CD 19+ cells in the mammal can treat acute lymphoblastic leukemia (ALL).
• ALL acute lymphoblastic leukemia
• Described herein is use of any of the vectors described herein in the manufacture of a medicament for treating or preventing cancer, a viral infection, a bacterial infection, a fungal infection, or a parasite in a mammal in need thereof.
• Described herein is use of any of the mammalian immune cells described herein for reducing the number of CD19+ cells in a mammal.
• Described herein is a vector for use in a method for reducing the number of CD19+ cells in a mammal.
• the vector can be any of the vectors described herein.
• Described herein is a mammalian immune cell for use in a method for reducing the number of CD19+ cells in a mammal.
• the mammalian immune cell can be any of the mammalian immune cells described herein.
• a vector that includes a nucleic acid encoding a mutant erythropoietin (Epo) receptor.
• the mutant Epo receptor can have at least 90% sequence identity to SEQ ID NO: 6.
• a method of making a transgenic mammalian host cell by introducing into a mammalian host cell the vector that includes a nucleic acid encoding a mutant erythropoietin (Epo) receptor.
• a mammalian immune cell that includes the vector that includes a nucleic acid encoding a mutant erythropoietin (Epo) receptor.
• the mammalian immune cell can be a T cell, natural killer (NK) cell, a monocyte/macrophage cell, or a dendritic cell.
• Epo receptor can be expressed in T cells and delivers signals.
• EpoRm is expressed at higher levels than EpoR.
• EpoRm induces stronger and more durable signals.
• FIGs. 1 A-E show expression of EpoR in human peripheral blood T cells confers survival signal.
• FIG. 1 A is flow cytometric dot-plots that illustrate surface EpoR expression in Jurkat cells, as detected by a PE-conjugated anti-EpoR antibody (R&D Systems); cells transduced with GFP only are shown as control. Percentage of cells in each quadrant is shown.
• FIG. 1B is flow cytometric histograms that illustrate Epo binding to Jurkat cells. Cells were labelled with biotin-conjugated Epo (R&D Systems) and streptavidin-PE (Jackson ImmunoResearch). FIG.
• FIG. 1C is flow cytometric dot-plots that illustrate surface EpoR expression in T lymphocytes transduced with GFP only or GFP plus EpoR.
• FIG. 1D is representative flow cytometric dot-plots that illustrate phosphorylation of STAT5 Y647, detected with an AF647-conjugated antibody (BD Biosciences), after stimulation with 10 IU/mL Epo for 15 minutes; results with cells treated with 10 mM ruxolitinib for 1 hour prior to Epo stimulation are also shown.
• FIGs. 2A-F show that EpoRm has higher and more stable expression than EpoR.
• FIG. 2A is a western blot analysis of EpoR expression in 293T cells. Cell lysates of 293T cells transduced with EpoR, EpoRm or GFP only were separated on a 10% polyacrylamide gel under reducing condition. The blotted membrane was probed with mouse anti-Flag antibody (9 A3; Cell Signaling Technology) followed by goat anti-mouse IgG conjugated to horseradish peroxidase (HRP, R&D Systems); rabbit anti-human GAPDH (EPR16891;
• FIG. 2B is flow cytometric analysis of T lymphocytes transduced with EpoR, EpoRm or GFP only. Flow cytometric dot- plots illustrate EpoR expression as detected by a PE-conjugated anti-EpoR antibody (R&D Systems). Percentage of cells in each quadrant is shown.
• FIG. 2C is charts showing the percentage of GFP+ T cells expressing EpoR (left) or mean fluorescent intensity (MFI) of EpoR (right) of T cells transduced with EpoR or EpoRm. ****, p ⁇ 0.0001.
• FIG. 2D is charts showing the percentage of CD4+ (left) or CD8+ (right) T cells from 6 donors expressing EpoR. ****, P ⁇ 0.0001.
• FIG. 2E is graphs showing the relation between MFI of GFP and MFI of EpoR in T cells from 3 donors transduced with either EpoR or EpoRm. Each panel shows results with T cells of 1 donor. ****, p ⁇ 0.0001.
• 2F is graph showing T lymphocytes from 3 donors that were transduced with EpoR or EpoRm and then stimulated with 10 IU/mL Epo. Expression of surface EpoR was assessed by flow cytometry at the indicated time points after stimulation. Each panel shows results with T cells of 1 donor.
• 3C is plots showing percentage of GFP+ cells expressing pSTAT5 in T cells transduced with EpoR, EpoRm or GFP only after stimulation with 10 IU/mL Epo or treated with 10 mM ruxolitinib prior to Epo stimulation. Each symbol represents results of one experiment. ****, p ⁇ 0.0001; **, P ⁇ 0.01.
• FIG. 3D is a graph showing percentage of GFP+ cells expressing pSTAT5 following stimulation with different concentration of Epo for 15 minutes. Each symbol indicates the average value of 2 experiments.
• FIGs. 4A-D show proliferation and survival signals induced by EpoRm in T cells.
• FIG. 4A are flow cytometry dot-plots illustrate cell cycle analysis of T lymphocytes transduced with EpoR, EpoRm or GFP only, unstimulated (top row) or stimulated with 10 IU/mL Epo (bottom row) after 3 days of culture in cytokine-free medium.
• DNA content detected by FxCycle staining, is shown in the x axes;
• DNA synthesis, shown by Edu incorporation, is shown on the y axes.
• Edu+ cells are shown in box, with their percentage.
• FIG. 4B is graphs showing survival of T lymphocytes transduced with EpoR, EpoRm or GFP-only cultured in absence of exogenous cytokines (no Epo) or in presence of Epo (10 IU/mL) for 3 weeks. Symbols indicate mean ( ⁇ SD) percentage of cell recovery relative to the number of input cells in triplicate measurements. **** P ⁇ 0.0001; ** P ⁇ 0.01.
• 4D is graphs showing survival of T lymphocytes transduced with EpoR, EpoRm or GFP only cultured with 100 IU/mL IL-2 in the absence or presence of Epo (10 IU/mL). Percentage of T cell recovery relative to input cells at the indicated days is shown.
• FIG. 5C are plots showing percentage of GFP+ cells expressing CAR (left) and EpoR (right) among T lymphocytes transduced with CAR, EpoRm-CAR or GFP only. Each symbol corresponds to a measurement for one transduction. Horizontal bars indicate median value.
• FIG. 5D are plots showing percentage of GFP+ cells expressing pSTAT5 in T lymphocytes transduced with CAR, EpoRm-CAR or GFP only after 15 minutes stimulation with 10 IU/mL Epo, or 1 hour pre-treatment with 10 mM ruxolitinib prior to Epo stimulation. Each symbol corresponds to a measurement for one transduction. Horizontal bars indicate median value.
• FIG. 5C are plots showing percentage of GFP+ cells expressing CAR (left) and EpoR (right) among T lymphocytes transduced with CAR, EpoRm-CAR or GFP only. Each symbol corresponds to a measurement for one transduction. Horizontal bars indicate median value.
• FIG. 5E is charts showing cytotoxicity of T lymphocytes transduced with CAR, EpoRm-CAR or GFP only against the CD19+ cell lines OP-l, RS4;l 1 and Nalm6. Bars represent mean ( ⁇ SD) of triplicate experiments in a 4-hour cytotoxicity assay at a 1 :1 E:T ratio.
• FIGs. 6A-F show that Epo supports the proliferation of EpoRm-CAR T cells.
• FIG. 6A is charts that show survival and expansion of T lymphocytes from 3 donors transduced with CAR, EpoRm-CAR or GFP only co-cultured with Streck-treated or irradiated OP-l cells at 1 : 1 ratio, in the absence or presence of 10 IU/mL Epo. Each symbol indicates percentage of cell recovery as compared with the number of input cells. Mean ( ⁇ SD) of triplicate cultures is shown, except for the day 14 measurement for 1 donor, where only the mean of 2 measurements is shown.
• FIG. 6A is charts that show survival and expansion of T lymphocytes from 3 donors transduced with CAR, EpoRm-CAR or GFP only co-cultured with Streck-treated or irradiated OP-l cells at 1 : 1 ratio, in the absence or presence of 10 IU/mL Epo. Each symbol indicates percentage of cell recovery as compared with the number of input cells. Mean
• FIG. 6B is a plot showing recovery of EpoRm-CAR-transduced T cells relative to input cells after 7 days of culture with or without 1 mM ruxolitinib in the presence of 10 IU/mL Epo. *** P ⁇ 0.00l.
• FIG. 6C is charts showing percentage of T lymphocytes transduced with CAR, EpoRm-CAR or GFP only recovered 7 days after co culture with irradiated CD 19+ OP-l at 1 : 1 ratio in the presence of 10 IU/mL Epo with 10 IU/mL or 100 IU/mL of IL-2. Each symbol indicates the mean of triplicate cultures.
• FIG. 6C is charts showing percentage of T lymphocytes transduced with CAR, EpoRm-CAR or GFP only recovered 7 days after co culture with irradiated CD 19+ OP-l at 1 : 1 ratio in the presence of 10 IU/mL Epo with 10 IU/mL or 100 IU/mL of
• FIG. 6D is plots showing MFI of pSTAT5 in EpoRm-CAR-transduced T cells after stimulation with 10 IU/mL Epo and/or 100 IU/mL IL-2 at the indicated time points.
• FIGs. 7A-E show in vivo tumor killing and expansion of EpoRm-CAR T cells in immunodeficient mice.
• FIG. 7A are representative images of NSG mice infused i.v. with 5 x 10 5 Nalm6-luciferase cells. Two days later, 4 mice were injected i.v. with 2 x 10 7 EpoRm- CAR T cells; 2 of them received i.p. injections of 100 IU Epo 3 times a week for 2 weeks; 1 mouse received no T cells. Bioluminescence ventral images on day 2 are shown with enhanced sensitivity to document Nalm6 engraftment.
• FIG. 7B is plots showing leukemia cell growth expressed as photons per second in mice shown in Panel 7A. Symbols corresponds to total bioluminescence by ventral and dorsal imaging. Mice were euthanized when the aggregate ventral and dorsal bioluminescence signal reached 1 x 10 10 photons per second.
• FIG. 7D is representative flow cytometric contour plots that illustrate the presence of human CD45+ GFP+ (top) and human CD45+ (bottom) T cells in mouse peripheral blood.
• FIG. 7E is a chart showing pSTAT5 MFI in T cells transduced with CAR, EpoRm-CAR, or GFP only following stimulation with 80 ng/mL of either mouse or human Epo for 15 minutes.
• FIGs. 8A-C show degranulation and cytokine release profile of EpoRm-CAR T cells.
• FIG. 8A is a chart showing percentage of GFP+ cells expressing CDl07a in T cells transduced with CAR, EpoRm-CAR, or GFP only, after co-culture with OP-l cells at 1 : 1 E:T ratio for 4 hours. Bars represent mean ( ⁇ SD) triplicate measurements with cells from 3 donors.
• FIG. 8B is graphs showing percentage of GFP+ cells expressing IFNy (left) and TNFa (right) in T cells transduced as in FIG. 8 A after 6 hours of co-culture with OP-l cells at 1 : 1 E:T ratio.
• FIG. 8C is plots of Luminex analysis of cytokines production by EpoRm-CAR-transduced T cells after co-culture with OP-l cells at 1 :4 E:T ratio for 24 hours in absence or presence of 10 IU/mL Epo.
• FIGs. 9A-F show anti -leukemic activity of EpoRm-CAR-T cells in xenograft models.
• FIG. 9A is images of NOD/scid IL2RGnull mice injected i.v. with 1 x 10 7 T cells transduced with CAR, EpoRm-CAR or GFP only; one group of mice received no T cells. Two weeks later, mice were injected i.v. with 2.5 x 10 5 Nalm6 cells expressing luciferase. Ventral images illustrate Nalm6 cell engraftment as measured by luminescence after i.p. injection of aqueous D-luciferin potassium salt (150 pg/g body weight).
• FIG. 9A is images of NOD/scid IL2RGnull mice injected i.v. with 1 x 10 7 T cells transduced with CAR, EpoRm-CAR or GFP only; one group of mice received no T cells. Two weeks later, mice were
• FIG. 9B is charts showing luminescence measurements in the groups of mice shown in FIG. 9A. Shown is the number of mice in each group with signals below l0 8 photons/second on day 59.
• FIG. 9C is a chart showing persistence of infused T cells in the mice shown in FIG. 9A.
• Mouse blood was collected by cheek prick and cells were stained with APC (2D1; Biolegend) or PE/Cy7- conjugated anti-human CD45 (HI30; BD Pharmingen), and PE-conjugated anti-mouse CD45 (30-F11; BD Pharmingen). Percentage of hCD45+ GFP+ cells among all CD45+ (human and mouse) lymphoid cells is shown.
• FIG. 9B is charts showing luminescence measurements in the groups of mice shown in FIG. 9A. Shown is the number of mice in each group with signals below l0 8 photons/second on day 59.
• FIG. 9C is a chart showing persistence of infused T cells
• FIG. 9D is images where Nalm6 cells transduced with luciferase were injected i.v. in NOD/scid IL2RGnull mice (5 x 10 5 cells per mouse). Four days later, tumor engraftment was assessed and 1-2 x 10 7 T cells were injected i.v.. Ventral images illustrate Nalm6 cell engraftment as measured by luminescence after i.p. injection of aqueous D-luciferin potassium salt (150 pg/g body weight).
• FIG. 9E is charts showing luminescence measurements in the groups of mice shown in FIG. 9D. The number of mice in each group with signals below l0 8 photons/second on day 55 is shown.
• FIG. 9F is a chart showing aggregate long-term survival of mice included in the two sets of experiments.
• Epo receptors can be expressed in immune cells (e.g., T cells) and are functional. Compared to normal Epo receptor, a mutant Epo receptor exhibited higher and more durable expression, higher signal intensity, and greater stimulation of T cell activity. Expression and function of Epo receptor in T cells are unexpected, as is the superiority of the mutant EpoR. Acute Lymphoblastic Leukemia and CD 19
• Acute lymphoblastic leukemia is a cancer of lymphoid blood cells. ALL progresses rapidly and is fatal if untreated. Standard treatment includes chemotherapy and hematopoietic stem cell transplant.
• CD 19 is a B-cell-specific antigen that is expressed on all leukemic cells in the majority of cases of ALL.
• the vectors described herein can be used to generate modified T cells, which, in turn, can be used for targeted treatment of ALL.
• the processes described herein can be used to create transgenic T cells that can target CD 19+ B-cells for destruction, thereby decreasing the risk and/or severity of ALL.
• nucleotide and“nucleotide monomer” refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as non-naturally occurring derivatives and analogs thereof. Accordingly, nucleotides can include, for example, nucleotides comprising naturally occurring bases (e.g, adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or
• naturally occurring bases e.g, adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or
• deoxycytidine and nucleotides comprising modified bases known in the art.
• sequence identity refers to the extent to which two nucleotide sequences, or two amino acid sequences, have the same residues at the same positions when the sequences are aligned to achieve a maximal level of identity, expressed as a percentage.
• sequence alignment and comparison typically one sequence is designated as a reference sequence, to which a test sequences are compared.
• sequence identity between reference and test sequences is expressed as the percentage of positions across the entire length of the reference sequence where the reference and test sequences share the same nucleotide or amino acid upon alignment of the reference and test sequences to achieve a maximal level of identity.
• two sequences are considered to have 70% sequence identity when, upon alignment to achieve a maximal level of identity, the test sequence has the same nucleotide or amino acid residue at 70% of the same positions over the entire length of the reference sequence.
• Alignment of sequences for comparison to achieve maximal levels of identity can be readily performed by a person of ordinary skill in the art using an appropriate alignment method or algorithm.
• the alignment can include introduced gaps to provide for the maximal level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computerized
• test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
• sequence comparison algorithm calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
• a commonly used tool for determining percent sequence identity is Protein Basic Local Alignment Search Tool (BLASTP) available through National Center for Biotechnology Information, National Library of Medicine, of the LTnited States National Institutes of Health. (Altschul et al., JMol Biol. 215(3):403-10 (1990)).
• two nucleotide sequences, or two amino acid sequences can have at least, e.g, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity.
• sequences described herein are the reference sequences.
• the terms“vector”,“vector construct” and“expression vector” mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
• Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA encoding a protein is inserted by restriction enzyme technology.
• a common type of vector is a“plasmid”, which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell.
• plasmid which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell.
• a large number of vectors, including plasmid and fungal vectors have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts.
• the terms“express” and“expression” mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence.
• a DNA sequence is expressed in or by a cell to form an“expression product” such as a protein.
• the expression product itself e.g. the resulting protein, may also be said to be “expressed” by the cell.
• a polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter.
• Gene delivery vectors generally include a transgene (e.g., nucleic acid encoding an enzyme) operably linked to a promoter and other nucleic acid elements required for expression of the transgene in the host cells into which the vector is introduced.
• a transgene e.g., nucleic acid encoding an enzyme
• Suitable promoters for gene expression and delivery constructs are known in the art.
• Recombinant plasmids can also comprise inducible, or regulatable, promoters for expression of an enzyme in cells.
• viral vectors suitable for gene delivery include, e.g., vector derived from the herpes virus, baculovirus vector, lentiviral vector, retroviral vector, adenoviral vector, adeno- associated viral vector (AAV), and murine stem cell virus (MSCV).
• the viral vector can be replicating or non-replicating.
• Such vectors may be introduced into many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.
• the vector comprises an internal ribosome entry site (IRES).
• the vector includes a selection marker, such as an ampicillin resistance gene (Amp).
• the nucleic acid encodes a fluorescent protein, such as green fluorescent protein (GFP) or mCherry.
• the nucleic acid is suitable for subcloning into pMSCV-IRES-GFP between EcoRI and Xhol.
• the vector contains a multiple cloning site (MCS) for the insertion of the desired gene.
• MCS multiple cloning site
• the vector includes a nucleotide sequence that has been optimized for expression in a particular type of host cell (e.g ., through codon optimization).
• Codon optimization refers to a process in which a polynucleotide encoding a protein of interest is modified to replace particular codons in that polynucleotide with codons that encode the same amino acid(s), but are more commonly used/recognized in the host cell in which the nucleic acid is being expressed.
• the polynucleotides described herein are codon optimized for expression in T cells.
• Epo receptor refers to a protein that binds
• erythropoietin which is a glycoprotein cytokine.
• Epo receptors and mutants thereof are described in the Exemplification.
• mutant Epo receptors include truncated Epo receptors, which can be formed by several different types of mutations, including frameshifts, insertions, and deletions. Those lacking the C-terminal negative regulatory domain exhibit hypersensitivity to Epo stimulation in red cells.
• One example is represented by a nucleic acid encoding an Epo receptor that has nonsense mutations within exon 8 of the Epo receptor gene that encode premature stop codons.
• Such mutants can produce a truncated form of EpoR with augmented Epo signaling in erythrocyte progenitors.
• An EpoR mutant has a mutation at nucleotide 6002 so that codon 439 encodes a stop codon (TAG) instead of tryptophan (TGG).
• FIG. 5 A illustrates a particular construct that is an anti-CD 19 single-chain variable fragment (anti-CD 19 scFv) coupled to a hinge and transmembrane domain.
• the hinge and transmembrane domain are a CD8a hinge and
• the anti-CD 19 scFv includes an anti-CD 19 variable light chain domain, an anti-CD 19 variable heavy chain domain, and a linker domain joining the variable light chain domain and the variable heavy chain domain.
• the relative positions of the variable light and variable heavy chain domain can be reversed, but they are both N’ terminal to a transmembrane domain, illustrated in FIG. 5A as a CD8a hinge and transmembrane domain.
• the construct can also include an N-terminal signal peptide, such as a CD8a signal peptide (see SEQ ID NOS: 21 and 22). Signal peptides of surface proteins are generally suitable.
• linker domains are suitable.
• the linker domain can be (G4S)x (SEQ ID NO: 24), wherein x is an integer from 1 to 100; preferably, x is an integer from 1 to 10; even more preferably, x is an integer from 2 to 5.
• x is an integer from 1 to 100; preferably, x is an integer from 1 to 10; even more preferably, x is an integer from 2 to 5.
• the linker domain can be (G4S)3 (SEQ ID NO: 25).
• the linker domain can be one or more glycine residues (e.g., (G)y (SEQ ID NO: 26), where y is an integer from 2 to 100.
• the linker domain can be (EAAAK) 3 (SEQ ID NO: 27).
• (G4S) X (SEQ ID NO: 24), (G4S) 3 (SEQ ID NO: 25), and (G) y (SEQ ID NO: 26) are examples of flexible linkers, while (EAAAK) 3 (SEQ ID NO: 27) is an example of a more rigid linker.
• hinge and transmembrane domains are suitable. In some embodiments,
• the hinge domain can be a CD8a hinge domain.
• the transmembrane domain can be a CD8a transmembrane domain.
• the hinge and transmembrane domain can be a CD8a hinge and transmembrane domain.
• the hinge can be a plurality of amino acid residues.
• the transmembrane domain can be a transmembrane domain from CD4, CD8P, CD 16, CD28, CD32, CD34, CD64, CD137, FceRIy, 0X40, O ⁇ 3z, CD3e, CD3y, CD35, TCRa, VEGFR2, FAS, or FGFR2B.
• FIG. 5 A is an anti-CD 19 construct
• a similar approach can be applied to generate constructs for other target antigens, such as CD20, CD22, CD 123, CD33, B-cell maturation antigen (BCMA), mesothelin, human epidermal growth factor receptor 2 (Her2), prostate-specific membrane antigen (PSMA), or disialoganglioside (GD)- 2.
• BCMA B-cell maturation antigen
• mesothelin mesothelin
• human epidermal growth factor receptor 2 Her2
• PSMA prostate-specific membrane antigen
• GD disialoganglioside
• the constructs also encode an Epo receptor.
• the Epo receptor is a mutant (EpoRm) that was generated using site-directed mutagenesis polymerase chain reaction (PCR) to alter the codon for amino acid 439 from TGG (Trp) to TAG (stop).
• PCR site-directed mutagenesis polymerase chain reaction
• Other mutants of Epo receptor are known in the art, and other Epo receptors variants not existing in nature can be created.
• One example is an Epo receptor lacking the
• ITIM immunoreceptor tyrosine-based inhibition motif
• Another example is an Epo receptor with multiple JAK2 -binding domains in the intracellular portion.
• the Epo receptor is joined with the chimeric antigen receptor (CAR) by a 2A peptide, which is a self-cleaving peptide.
• CAR chimeric antigen receptor
• 2A peptide which is a self-cleaving peptide.
• P2A SEQ ID NOS: 13 and 14
• T2A SEQ ID NOS: 15 and 16
• E2A SEQ ID NOS: 17 and 18
• F2A SEQ ID NOS: 19 and 20
• transgenic host cells such as transgenic T cells.
• the transgenic host cells can be made, for example, by introducing one or more of the vector embodiments described herein into the host cell.
• the method comprises introducing into a host cell a vector that includes a nucleic acid that encodes an Epo receptor and a chimeric antigen receptor (CAR), such as an anti-CD19-41BB-CD3z.
• a nucleic acid such as a bicistronic vector, expresses Epo receptor and the CAR.
• two separate vectors can be used to create a transgenic cell, such as a transgenic T cell, that expresses Epo receptor and the CAR.
• one or more of the nucleic acids are integrated into the genome of the host cell.
• the nucleic acids to be integrated into a host genome can be introduced into the host cell using any of a variety of suitable methodologies known in the art, including, for example, homologous recombination, CRISPR-based systems (e g., CRISPR/Cas9; CRISPR/Cpfl) and TALEN systems.
• the T cell can also expresses a T-cell receptor (TCR) that binds a tumor antigen or a viral antigen.
• TCR can be endogenous.
• the T cell can be a tumor-infiltrating lymphocyte (TIL) that is extracted from a tumor and expanded ex vivo.
• TIL tumor-infiltrating lymphocyte
• the TCR can be exogenous.
• the TCR can be expressed in the T cell by viral transduction or other means.
• the TCR can be specific for a viral peptide, such as a peptide derived from hepatitis B virus, hepatitis C virus, Epstein-Barr virus, cytomegalovirus, or from a tumor cell, such as melanoma-associated antigen (MAGE), NY-ESO-l, telomerase reverse transcriptase (TERT).
• a viral peptide such as a peptide derived from hepatitis B virus, hepatitis C virus, Epstein-Barr virus, cytomegalovirus
• MAGE melanoma-associated antigen
• NY-ESO-l telomerase reverse transcriptase
• the leukemia cell lines Jurkat, Nalm6, and RS4; 11 were obtained from the American Type Culture Collection (ATCC; Rockville, MD).
• the CD 19+ B-lineage ALL cell line OP-l was developed in our laboratory.
• 19 A murine stem cell virus (MSCV) retroviral vector, containing green fluorescent protein (GFP) or mCherry and an internal ribosomal entry site (IRES) was used to express the firefly luciferase gene in Nalm6, and mCherry in OP-l, respectively.
• Cell lines were maintained in RPMI-1640 (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin- streptomycin.
• FBS fetal bovine serum
• penicillin- streptomycin Human embryonic kidney fibroblast 293T (HEK 293T) cells were cultured in DMEM (HyClone, GE Life Sciences, Logan, Utah) supplemented
• EpoRm-2A-CAR was generated by fusion PCR, combining EpoRm and anti-CD19-41BB-CD3z through 2A peptide sequence. 22
• the constructs and expression cassette were subcloned into EcoRI and Xhol sites of the pMSCV-IRES-GFP vector.
• RetroNectin RetroNectin (Takara, Otsu, Japan)-coated polypropylene tubes; after centrifugation and removal of the supernatant, T cells (5 x 10 5 ) were added to the tubes and left at 37°C for 12 hours; fresh viral supernatant was added on two other successive days. T lymphocytes were then maintained in RPMI-1640 with FBS, antibiotics and 200 IU/mL IL-2 until the time of the experiments, 7-21 days after transduction.
• EpoR Surface expression of EpoR was detected with phycoerythrin (PE)-conjugated anti-human EpoR antibody (38409; R&D Systems, Minneapolis, MN). In some experiments, surface staining of EpoR was done on cells that had been cultured in cytokine-free media for 2 hours followed by incubation with 10 IU/mL of recombinant human Epo (Thermo Fisher Scientific) at 37°C for 15-60 minutes. Expression of CAR was detected using a biotin- conjugated goat anti-mouse F(ab’)2 antibody (Jackson ImmunoResearch, West Grove, PA) followed by streptavidin conjugated to allophycocyanin (APC; Jackson ImmunoResearch).
• PE phycoerythrin
• PE/Cy7-conjugated anti-CD4 (SK3) antibody was from BD Biosciences (San Jose, CA); APC-conjugated anti-CD8 (BW135/80) antibody was from Miltenyl Biotec (Bergisch Gladbach, Germany). In all tests, non-reactive isotype-matched antibodies were used as controls. Cell staining was analysed using Accuri C6 or Fortessa flow cytometers (BD Bioscience), with Diva (BD Biosciences) or FlowJo software (FlowJo, Ashland, OR).
• GPDH glyceraldehyde 3-phosphate dehydrogenase
• T cells were cultured in absence of exogenous cytokines, with or without Epo (4 - 10 IU/mL) in a flat bottom 96-well or 24-well plate (Cellstar).
• T cells were co-cultured with target cells (OP-l cells) at 1 : 1 effector-to- target (E:T) ratio in a flat bottom 96-well plate; Epo (10 IU/mL) was added every two days.
• Target cells irradiated (100 Gy) or treated with Streck cell preservative (Streck Laboratories, Omaha, NE) to inhibit growth, were added at the beginning of the cultures, and every 7 days thereafter.
• low dose (10 IU/mL) or high dose (100 IU/mL) IL-2 was added to the culture as well.
• the number of GFP+ T cells was measured by flow cytometry.
• CD19+ target cells (OP-l, RS4; 11, and Nalm6) were labelled with calcein red-orange AM (Thermo Fisher Scientific) and placed into a 96-well round bottom plate (Coming Costar, Coming, NY). T cells were added at E:T ratio of 1 : 1 and co cultured with target cells for 4 hours at 37°C and 5% C02 incubator. Viable target cells were counted by flow cytometry. 25 For long-term cytotoxicity, OP-l mCherry cells were placed into a 96-well flat bottom plate, T cells were added at different E:T ratios and cultured for 3 days with 10 IU/mL of Epo. Plates were placed in IncuCyte Zoom System (Essen
• BioScience set to collect data collection (whole-well imaging) every 4 hours.
• T cells were co-cultured with OP-l cells at 1 : 1 E:T ratio for 4 hours in a 96-well round bottom plate.
• PE-conjugated anti-human CD 107a antibody H4A3; BD Biosciences was added at the beginning of the cultures and monensin (BD Golgi Stop) 1 hour later.
• interferon-g interferon-g
• TNF-a tumor necrosis factor-a
• target and effector cells at a 1 : 1 E:T ratio were plated as above.
• brefeldin A BD GolgiPlug
• intracellular staining with PE-conjugated anti-IFN-g (clone 25723.11; BD Biosciences) or anti-TNF-a (clone 6401.1111; BD Biosciences) was done prior to analysis by flow cytometry.
• target and effector cells were co-cultured at 1 :4 E:T ratio in absence or presence of 10 IU/mL Epo for 24 hours. Culture supernatant was collected to be analysed by Luminex Multiplex Assay (Bio-Rad).
• NOD.Cg-Prkdcscid ⁇ L2rgtmlWjl/SzJ mice (The Jackson Laboratory, Bar Harbor, ME) were injected intravenously (i.v.) with 1 x 10 7 T cells transduced with GFP alone, CAR, or EpoRm-CAR.
• Epo Epo mice
• 100 IU of Epo were injected intraperitoneally (i.p) every two days for two weeks.
• blood cells were counted with a cell counter (Beckman Coulter, Miami, FL).
• red blood cell lysis solution Sigma-Aldrich
• cells were stained with APC-conjugated anti-human CD45 (2D1; Biolegend) and PE-conjugated anti-mouse CD45 (30-F11; BD Pharmingen).
• NOD-scid-IL2RGnull mice were injected with lxlO 7 T cells i.v., followed 2 weeks later by 2.5xl0 5 Nalm6 cells expressing luciferase i.v.. ALL cell engraftment was determined by measuring luminescence signal with the Xenogen IVIS-200 System (Perkin Elmer, Waltham, MA), after i.p. injection of aqueous D-luciferin potassium salt (150 pg/g body weight; Perkin Elmer); signals were analyzed with Living Image 3.0 software. In another model, 5xl0 5 Nalm6-luciferase cells were injected i.v.
• EpoR can be expressed in T cells and mediates Epo survival signals
• FIG. 1 A Retroviral transduction of Jurkat cells with the EpoR gene resulted in high expression of the receptor (FIG. 1 A).
• Cells expressing EpoR could bind Epo, while there was no detectable binding in cells transduced with GFP alone (FIG. 1B).
• FIG. 1B we determined whether EpoR could also be expressed in peripheral blood T lymphocytes.
• the percentage of transduced T lymphocytes (GFP+) expressing EpoR was 74.9% ⁇ 4.8%; EpoR was undetectable in cells transduced with GFP only (FIG. 1C).
• EpoR mutant enhances EpoR expression in T cells
• EpoR and EpoRm in activated peripheral blood T lymphocytes, and observed that levels of expression of EpoRm were consistently higher than those measured for the wild-type EpoR construct in the same T cells (FIG. 2B).
• the percentage of GFP+ T cells expressing EpoR was 71.9% ⁇ 14.1% as compared to 93.1% ⁇ 6.1% for EpoRm (P ⁇ 0.0001 by paired t test); mean fluorescence intensity (MFI) was 3,620 ⁇ 2,449 and 8,773 ⁇ 5,851, respectively (P ⁇ 0.0001) (Fig. 2C).
• EpoRm expression was higher regardless of whether cells were CD4+ or CD8+ (Fig. 2D).
• EpoRm Higher expression of EpoRm was associated with a longer persistence after exposure to Epo. In experiments with T cells from 3 donors, reduction in percentage of EpoR-positive cells was clearly higher in cells transduced with EpoR than in those transduced with EpoRm (FIG. 2F).
• EpoRm T cells had higher pSTAT5 Y647 phosphorylation than EpoR T cells, which was suppressed by exposure to the JAK1/2 inhibitor ruxolitinib (10 mM) (FIG. 3A, B, C).
• the higher STAT5 phosphorylation caused by EpoRm was corroborated by an analysis of pSTAT5 Y647 MFI according to levels of GFP.
• EpoRm in a bicistronic vector also containing the gene encoding an anti-CD 19-41BB-O ⁇ 3z CAR developed in our laboratory (FIG. 5 A).
• 21 Transduction of this construct in T lymphocytes resulted in expression of both EpoRm and CAR (FIG. 5B), and either gene was expressed at levels which were similar to those of cells transduced with a single-gene vector (FIG. 5C).
• the functionality of EpoRm was maintained regardless of whether it was expressed alone or in combination with the CAR (FIG. 5D).
• CAR-T cell function was also retained in cells expressing EpoRm. Thus, there were no differences in exocytosis of cytotoxic granules, as measured by CD 107a expression after 4 hours of co-culture with the CD19+ ALL cell line OP-l cells, regardless of whether CAR-T cells expressed EpoRm or whether there was Epo in the cultures (FIG. 8A).
• EpoRm-CAR-T cells secreted more TNFoc in the presence of Epo than CAR-T cells lacking EpoRm (P ⁇ 0.001) (FIG. 8B).
• EpoRm-CAR T cells The higher killing exerted by EpoRm-CAR T cells in long-term cultures might be explained by a higher rate of proliferation of these cells, thereby creating a higher E:T ratio.
• Epo 10 IU/mL
• FIG. 6A the expansion of T cells from 3 donors was higher when CAR-T cells expressed EpoRm, indicating that the CAR-driven cell proliferation is enhanced by EpoRm signaling.
• EpoRm- CAR T cell proliferation was abrogated by ruxolotinib (1 mM) (FIG. 6B).
• ruxolotinib (1 mM)
• T cell recovery in co-culture with irradiated OP-l after 7 days, in the presence of either 10 IU/mL (4 donors) or 100 IU/mL of IL-2 (3 donors).
• CAR-stimulated EpoRm-CAR T cells cultured with both IL-2 and Epo had considerably higher expansion than those cultured with IL-2 alone (FIG. 6C).
• EpoRm signaling in CAR-T cells triggers a signaling cascade that is distinct from that of IL-2, and enhances the proliferative stimulus of IL-2.
• T lymphocytes transduced with CAR, or EpoRm- CAR of GFP alone were injected i.v. into NSG mice.
• peripheral blood was examined for the presence of cells expressing GFP and human CD45.
• levels of GFP+/hCD45+ cells in mice injected with EpoRm-CAR-T cells were higher than those measured in mice injected with cells transduced with GFP only, or with CAR lacking EpoRm; injection of 100 IU Epo i.p. 3 times a week for 2 weeks in the latter group did not significantly increase cell numbers.
• Levels of GFP+/hCD45+ cells were highest in mice injected with EpoRm-CAR-T cells who received Epo i.p. injections.
• mice were distributed in 4 groups with similar tumor load; three groups received either CAR-T cells, EpoRm-CAR-T cells, or T cells transduced with GFP only by i.v. injection; a 4 th group received only tissue culture medium. ALL cells rapidly expanded in the untreated mice, and in mice that received control T cells without CAR. Both CAR- and EpoRm-CAR-T cells markedly reduced leukemic signals (FIGs. 9D and 9E).
• Embodiment 1 A vector comprising a nucleic acid encoding: a) an erythropoietin (Epo) receptor; b) a self-cleaving peptide or internal ribosome entry site; and c) a cell surface receptor.
• Epo erythropoietin
• Embodiment 2 The vector of Embodiment 1, wherein the Epo receptor has at least 90% sequence identity to SEQ ID NO: 2.
• Embodiment 3 The vector of Embodiment 1, wherein the Epo receptor is a mutant Epo receptor.
• Embodiment 4 The vector of Embodiment 3, wherein the nucleic acid has a mutation that encodes a stop codon within exon 8 of the Epo receptor.
• Embodiment 5 The vector of Embodiment 3, wherein the Epo receptor has at least 90% sequence identity to SEQ ID NO: 6.
• Embodiment 6 The vector of any of Embodiments 1 through 5, wherein the nucleic acid further encodes a Flag tag (DYKDDDDK (SEQ ID NO: 23)) that is C terminal to the Epo receptor.
• DYKDDDDK SEQ ID NO: 23
• Embodiment 7 The vector of any of Embodiments 1 through 6, wherein the nucleic acid comprises a self-cleaving peptide.
• Embodiment 8 The vector of Embodiment 7, wherein the self-cleaving peptide is a 2A peptide.
• Embodiment 9 The vector of Embodiment 8, wherein the 2A peptide is a T2A peptide.
• Embodiment 10 The vector of any one of Embodiments 1 through 9, wherein the signal peptide is a CD8a signal peptide.
• Embodiment 11 The vector of Embodiment 10, wherein the cell surface receptor comprises an extracellular receptor domain that binds a target cell antigen.
• Embodiment 12 The vector of any one of Embodiments 1 through 10, wherein the cell surface receptor is a chimeric antigen receptor comprising: i) a signal peptide; ii) an extracellular receptor domain that binds a target cell antigen; iii) a hinge and transmembrane domain that anchors the extracellular receptor domain on the surface of a cell; and iv) an effector domain.
• Embodiment 13 The vector of Embodiment 11 or 12, wherein the extracellular receptor domain comprises a variable immunoglobulin light chain domain and a variable immunoglobulin heavy chain domain joined by a linker domain.
• Embodiment 14 The vector of Embodiment 13, wherein the linker domain is (G4S)x (SEQ ID NO: 24), wherein x is an integer from 1 to 100.
• Embodiment 15 The vector of Embodiment 13, wherein the linker domain is (G4S)3 (SEQ ID NO: 25).
• Embodiment 16 The vector of Embodiment 12, wherein the extracellular receptor domain is a single-chain variable fragment (scFv).
• Embodiment 17 The vector of any one of Embodiments 1 through 10, wherein the cell surface receptor is a T cell receptor.
• Embodiment 19 The vector of Embodiment 11, wherein the extracellular receptor domain comprises an immunoglobulin Fc receptor.
• Embodiment 20 The vector of Embodiment 19, wherein the immunoglobulin Fc receptor is CD 16, CD32 or CD64.
• Embodiment 23 The vector of Embodiment 11, wherein the cell surface receptor activates immune cells.
• Embodiment 24 The vector of Embodiment 23, wherein the cell surface receptor comprises NKG2D, NKG2C, NCR1, NCR2, NCR3, CD137, CD28, or ICOS, or a fragment or a ligand thereof.
• Embodiment 25 The vector of Embodiment 11, wherein the cell surface receptor inhibits immune cells.
• Embodiment 26 The vector of Embodiment 25, wherein the cell surface receptor comprises NKG2A, PD-l, or CTLA-4, or a fragment or ligand thereof.
• Embodiment 27 The vector of Embodiment 11, wherein the cell surface receptor is a receptor for a cytokine.
• Embodiment 29 The vector of any one of Embodiments 1 through 28, wherein the target cell antigen is a tumor associated antigen or a tumor specific antigen.
• Embodiment 31 The vector of any one of Embodiments 1 through 28, wherein the target cell antigen is CD19, CD20, CD22, CD123, CD33, B-cell maturation antigen (BCMA), mesothelin, human epidermal growth factor receptor 2 (Her2), prostate-specific membrane antigen (PSMA), or disialoganglioside (GD2).
• BCMA B-cell maturation antigen
• Her2 human epidermal growth factor receptor 2
• PSMA prostate-specific membrane antigen
• GD2 disialoganglioside
• Embodiment 33 The vector of any one of Embodiments 1 through 32, wherein the extracellular domain is an anti-CD 19 single-chain variable fragment (scFv).
• scFv single-chain variable fragment
• Embodiment 34 The vector of any one of Embodiments 1 through 33, wherein the hinge and transmembrane domain is a CD8a hinge and transmembrane domain.
• Embodiment 35 The vector of any one of Embodiments 1 through 34, wherein the hinge comprises a plurality of amino acid residues.
• Embodiment 36 The vector of any one of Embodiments 1 through 35, wherein the transmembrane domain is a transmembrane domain from CD4, CD8P, CD 16, CD28, CD32, CD34, CD64, CD137, FceRIy, 0X40, O ⁇ 3z, CD3e, CD3y, CD35, TCRa, VEGFR2, FAS, or FGFR2B.
• Embodiment 37 The vector of any one of Embodiments 1 through 36, wherein the effector domain comprises 4-1BB and O03z.
• Embodiment 38 The vector of any one of Embodiments 1 through 37, wherein the CAR is anti-CD 19-41 BB -CD3 z.
• Embodiment 39 A vector comprising a nucleic acid encoding a mutant erythropoietin (Epo) receptor.
• Embodiment 40 The vector of Embodiment 39, wherein the nucleic acid has a mutation that encodes a stop codon within exon 8 of the Epo receptor.
• Embodiment 41 The vector of Embodiment 39, wherein the mutant Epo receptor has at least 90% sequence identity to SEQ ID NO: 6.
• Embodiment 42 The vector of any one of Embodiments 1 through 41, wherein the vector is a retrovirus.
• Embodiment 43 The vector of any one of Embodiments 1 through 42, wherein the vector is a murine stem cell virus (MSCV) retroviral vector.
• MSCV murine stem cell virus
• Embodiment 44 The vector of any one of Embodiments 1 through 43, wherein the vector further encodes a fluorescent protein.
• Embodiment 45 The vector of any one of Embodiments 1 through 44, wherein the vector encodes an internal ribosomal entry site (IRES).
• IRS internal ribosomal entry site
• Embodiment 46 The vector of any one of Embodiments 1 through 45, wherein the vector further encodes at least one regulatory element for expression of the nucleic acid.
• Embodiment 47 A method of making a transgenic mammalian host cell, the method comprising introducing into a mammalian host cell the vector of any of Embodiments 1 through 46.
• Embodiment 48 The method of Embodiment 47, wherein the mammalian host cell is an immune cell.
• Embodiment 49 The method of Embodiment 48, wherein the immune cell is a natural killer (NK) cell, a monocyte/macrophage cell, or a dendritic cell.
• NK natural killer
• Embodiment 50 The method of Embodiment 48, wherein the immune cell is a T cell.
• Embodiment 51 The method of Embodiment 50, wherein the T cell is a human peripheral blood T lymphocyte.
• Embodiment 54 The method of Embodiment 50, wherein the T cell further expresses a T-cell receptor (TCR) that binds a tumor antigen or a viral antigen.
• TCR T-cell receptor
• Embodiment 55 The method of Embodiment 54, wherein the TCR is endogenous.
• Embodiment 57 The method of Embodiment 54, wherein the TCR is exogenous.
• Embodiment 58 The method of Embodiment 57, wherein the method further comprises introducing into the T cell a vector that expresses the exogenous TCR.
• Embodiment 59 A mammalian immune cell comprising the vector of any one Embodiments 1 through 46.
• Embodiment 60 The mammalian immune cell of Embodiment 59, wherein the mammalian immune cell is a natural killer (NK) cell, a monocyte/macrophage cell, or a dendritic cell.
• NK natural killer
• Embodiment 61 The mammalian immune cell of Embodiment 59, wherein the mammalian immune cell is a T cell.
• Embodiment 63 The mammalian immune cell of Embodiment 61, wherein the T cell is a human peripheral blood T lymphocyte.
• Embodiment 64 A method of reducing the number of CD 19+ cells in a mammal, the method comprising introducing mammalian T cells into the subject, wherein the mammalian T cells comprise the vector of any of Embodiments 1 through 46.
• Embodiment 65 The method of Embodiment 64, wherein the mammal is a human.
• Embodiment 66 The method of any of Embodiments 64 through 65, wherein the mammalian T cells are autologous cells isolated from the mammal.
• Embodiment 68 The method of any of Embodiments 64 through 67, further comprising administering Epo to the subject.
• Embodiment 69 The method of any of Embodiments 64 through 68, further comprising administering IL-2 to the subject.
• Embodiment 70 The method of any of Embodiments 64 through 69, wherein reducing the number of CD 19+ cells in the mammal treats acute lymphoblastic leukemia (ALL).
• ALL acute lymphoblastic leukemia
• Embodiment 71 Lise of the vector according to any one of Embodiments 1 through 46 in the manufacture of a medicament for treating or preventing cancer, a viral infection, a bacterial infection, a fungal infection, or a parasite in a mammal in need thereof.
• Embodiment 72 ETse of a mammalian immune cell according to any one of Embodiments 59 through 63 for reducing the number of CD19+ cells in a mammal.
• Embodiment 73 A vector according to any one of Embodiments 1 through 46 for use in a method for reducing the number of CD19+ cells in a mammal.
• Embodiment 74 A mammalian immune cell according to any one of
• Embodiments 59 through 63 for use in a method for reducing the number of CD 19+ cells in a mammal.
• Embodiment 75 A method of making a transgenic mammalian host cell, the method comprising introducing into a mammalian host cell the vector of any of Embodiments 39 through 46.
• Embodiment 76 The method of Embodiment 75, wherein the mammalian host cell is an immune cell.
• Embodiment 77 The method of Embodiment 76, wherein the immune cell is a T cell.
• Embodiment 78 The method of Embodiment 77, wherein the T cell is a tumor- infiltrating lymphocyte (TIL).
• TIL tumor- infiltrating lymphocyte
• Embodiment 79 The method of Embodiment 76, wherein the immune cell is a natural killer cell.
• Embodiment 80 A mammalian immune cell comprising the vector of any one Embodiments 39 through 46.
• Embodiment 81 The mammalian immune cell of Embodiment 80, wherein the mammalian immune cell is a T cell, natural killer (NK) cell, a monocytic/macrophage cell, or a dendritic cell.
• NK natural killer
• SEQ ID NO: 1 Epo receptor (EpoR) cDNA:
• SEQ ID NO: 2 Epo receptor (EpoR) amino acid:
• SEQ ID NO: 3 EpoR with Flag tag cDNA:
• SEQ ID NO: 4 EpoR with Flag tag amino acid:
• SEQ ID NO: 7 EpoRm with Flag tag cDNA:
• SEQ ID NO: 9 anti-CDl9-4lBB-CD3C CAR cDNA:
• SEQ ID NO: 11 EpoRm-2A-CAR cDNA:
• SEQ ID NO: 13 P2A cDNA:
• SEQ ID NO: 14 P2A amino acid: GSGATNF SLLKQ AGD VEENPGP
• SEQ ID NO: 15 T2A cDNA:
• SEQ ID NO: 16 T2A amino acid: GSGEGRGSLLTCGD VEENPGP
• SEQ ID NO: 17 E2A cDNA:
• SEQ ID NO: 19 F2A cDNA:
• SEQ ID NO: 20 F2A amino acid: GSGVKQTLNFDLLKLAGDVESNPGP
• SEQ ID NO: 21 CD8a signal peptide cDNA:
• SEQ ID NO: 22 CD8a signal peptide amino acid:

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