WO2018229163A1 - Méthodes d'activation des lymphocytes t v delta 2 négatifs gamma delta - Google Patents

Méthodes d'activation des lymphocytes t v delta 2 négatifs gamma delta Download PDF

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WO2018229163A1
WO2018229163A1 PCT/EP2018/065730 EP2018065730W WO2018229163A1 WO 2018229163 A1 WO2018229163 A1 WO 2018229163A1 EP 2018065730 W EP2018065730 W EP 2018065730W WO 2018229163 A1 WO2018229163 A1 WO 2018229163A1
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cells
tigit
antibody
agent
inhibitor
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PCT/EP2018/065730
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Maria Luisa IANNITTO
Oliver Nussbaumer
Adrian HAYDAY
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King's College London
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2304—Interleukin-4 (IL-4)
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2321—Interleukin-21 (IL-21)
    • 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
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
    • 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
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the invention relates to methods for activating tissue-infiltrating gamma delta T cells, particularly V52 ⁇ gamma-delta T cells ( ⁇ T cells), in a mammalian subject by antagonizing TIGIT activity of V52 ⁇ ⁇ T cells.
  • ⁇ T cells intra-tumorai gamma delta T cells
  • TCR ⁇ T-cell receptor
  • ⁇ T cells represent a subset of T cells that express on their surface a distinct, defining ⁇ T-cell receptor (TCR).
  • the ⁇ TCR is made up of one gamma ( ⁇ ) and one delta ( ⁇ ) chain.
  • Human ⁇ T cells can be broadly classified into two types: peripheral blood-resident ⁇ T cells and non- hematopoietic, tissue-resident, ⁇ T cells.
  • ⁇ T cells Most blood-resident ⁇ T cells express a ⁇ 2 TCR; however, a smaller population of ⁇ 2 " ⁇ T cells can be detected among ⁇ T cells in blood. Conversely, tissue-resident ⁇ T cells more frequently use ⁇ and/or other ⁇ chains ( ⁇ 2 ⁇ ⁇ T cells); however, a smaller population of ⁇ 2 + ⁇ T cells can be detected among ⁇ T cells in tissue. Therefore, there is a need for novel methods for the specific activation and/or derepression of ⁇ T cells, and in particular, the ⁇ 2 ⁇ ⁇ T cells.
  • the application herein discloses methods of activating and/or de-repressing ⁇ 2 " gamma-delta T cells ( ⁇ T cells) in a mammalian subject, comprising antagonizing TIGIT activity of ⁇ 2 " ⁇ T cells present in the subject to an extent sufficient to activate the ⁇ 2 " ⁇ T cells in vivo.
  • the ⁇ 2 " ⁇ T cells are ⁇ 1 + cells.
  • the ⁇ 2 " ⁇ T cells are ⁇ ⁇ 2 " double negative (DN) cells.
  • antagonizing TIGIT activity comprises administering at least one TIGIT inhibitor to the subject.
  • the at least one TIGIT inhibitor is administered adjunctively with ⁇ 2 " ⁇ T cells that have been cultured ex vivo.
  • the at least one TIGIT inhibitor is added to the ⁇ 2 " ⁇ T cells during culturing ex vivo.
  • the cultured ⁇ 2 " ⁇ T cells have been expanded ex vivo.
  • the at least one TIGIT inhibitor is administered in a composition comprising the cultured ⁇ T cells.
  • the at least one TIGIT inhibitor is administered separately from the ex vivo expanded ⁇ T cells.
  • the methods further comprise administering to the subject at least one additional agent in an amount sufficient to activate ⁇ 2 " ⁇ T cells.
  • the at least one additional agent is an agonist of DNAM-1.
  • the at least one TIGIT inhibitor is selected from the group consisting of an antagonist of TIGIT activity, an antagonist of TIGIT interaction with Polio virus receptor (PVR), an agent that inhibits and/or blocks the interaction of TIGIT with PVR, an agent that inhibits and/or blocks the interaction of TIGIT with PVRL2, an agent that inhibits and/or blocks the interaction of TIGIT with PVRL3, an agent that inhibits and/or blocks the intracellular signaling mediated by PVR binding to TIGIT, an agent that inhibits and/or blocks the intracellular signaling mediated by PVRL2 binding to TIGIT, an agent that inhibits and/or blocks the intracellular signaling mediated by PVLR3 binding to TIGIT, and combinations thereof.
  • PVR Polio virus receptor
  • the TIGIT inhibitor is an antibody. In an aspect, the antibody binds specifically to TIGIT. In an aspect, the antibody binds to PVR to specifically inhibit PVR interaction with TIGIT. In an aspect, the antibody is a full length IgG antibody. In an aspect, the antibody is an antigen-binding antibody fragment. In an aspect, the antibody is a single domain antibody. In an aspect, the antibody is fully human. In an aspect, the antibody is humanized. In an aspect, the TIGIT inhibitor is a small molecule inhibitor.
  • the ex vivo expanded V52 " ⁇ T cells were obtained by culturing lymphocytes obtained from non-hematopoietic tissue of humans or non-human animals in the presence of interleukin-2 (IL-2) and/or interleukin-15 (IL-15), and not in direct contact with stromal or epithelial cells during culture.
  • the culturing step comprises culturing the lymphocytes obtained from human or non-human animal non- hematopoietic tissue in the presence of IL-2.
  • the culturing step comprises culturing the lymphocytes obtained from human or non-human animal non-hematopoietic tissue in the presence of interleukin-15 (IL-15).
  • the culturing step comprises culturing the lymphocytes obtained from human or non-human animal non-hematopoietic tissue in the presence of IL-2 and IL-15. In an aspect, the culturing step comprises culturing the lymphocytes in the absence of TCR activation or co-stimulation signals. In an aspect, the culturing step comprises culturing the lymphocytes in the absence of a T cell receptor pathway agonist. In an aspect, the culturing step comprises culturing the lymphocytes in the absence of stromal or epithelial cells. In an aspect, stromal or epithelial cells are removed prior to culture. In an aspect, the lymphocytes are cultured in the absence of fibroblasts.
  • the lymphocytes have been obtained from skin, the gastrointestinal tract ⁇ e.g. colon), mammary gland tissue, lung, liver, pancreas or prostate.
  • the ex vivo expanded V52 " ⁇ T cells were obtained by culturing lymphocytes obtained from non-hematopoietic tissue of humans or non-human animals in the presence of IL-2, IL-15, and a factor selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL- ⁇ , human platelet lysate (HPL), and stromal cell-derived factor- 1 (SDF-1) for at least 5 days to produce an expanded population of ⁇ T cells.
  • IL-2 IL-15
  • the expanded population of ⁇ T cells comprises at least 20-fold the number of ⁇ T cells as the ⁇ T cells obtained from a non-hematopoietic tissue. In an aspect, within 7 days of culture, the expanded population of ⁇ T cells comprises at least 2-fold the number of ⁇ T cells as the ⁇ T cells obtained from a non-hematopoietic tissue. In an aspect, the expanded population of ⁇ T cells is at least 50% ⁇ 1 + cells. In an aspect, the expanded population of ⁇ T cells is at least 70% ⁇ 1 + cells. In an aspect, the expanded population of ⁇ T cells is at least 90%> ⁇ 1 + cells. In an aspect, the subject has cancer.
  • the cancer has high infiltration of ⁇ T cells.
  • the application further discloses a TIGIT inhibitor as disclosed in any of the aspects and embodiments described herein for use in any of the methods described herein of activating and/or de-repressing ⁇ 2 " gamma-delta T cells ( ⁇ T cells) in a mammalian subject.
  • the application discloses herein a method of treating cancers having high infiltration of ⁇ T cells, comprising administering at least one TIGIT inhibitor to a patient with a cancer having high infiltration of ⁇ T cells, in an amount sufficient to activate tissue-infiltrating ⁇ T cells in the patient.
  • the cancer-infiltrating ⁇ T cells are ⁇ 2 " cells.
  • the ⁇ 2 " cells are ⁇ 1 + or DN cells.
  • the at least one TIGIT inhibitor is selected from the group consisting of an antagonist of TIGIT activity, an antagonist of TIGIT interaction with PVR, an agent that inhibits and/or blocks the interaction of TIGIT with PVR, an agent that inhibits and/or blocks the interaction of TIGIT with PVRL2, an agent that inhibits and/or blocks the interaction of TIGIT with PVRL3, an agent that inhibits and/or blocks the intracellular signaling mediated by PVR binding to TIGIT, an agent that inhibits and/or blocks the intracellular signaling mediated by PVRL2 binding to TIGIT, an agent that inhibits and/or blocks the intracellular signaling mediated by PVLR3 binding to TIGIT, and combinations thereof.
  • the TIGIT inhibitor is an antibody. In an aspect, the antibody binds specifically to TIGIT. In an aspect, the antibody binds to PVR to specifically inhibit PVR interaction with TIGIT. In an aspect, the antibody is a full length IgG antibody. In an aspect, the antibody is an antigen-binding antibody fragment. In an aspect, the antibody is a single domain antibody. In an aspect, the antibody is fully human. In an aspect, the antibody is humanized. In an aspect, the TIGIT inhibitor is a small molecule inhibitor. In an aspect, the methods further comprise the antecedent step of selecting for treatment a patient whose cancer shows high levels of ⁇ T cell infiltration.
  • the ⁇ T cell infiltration is detected by analysis of a tumor biopsy by a method comprising an assay selected from the group consisting of immunohistochemistry, polymerase chain reaction, in situ hybridization, and combinations thereof.
  • the ⁇ T cell infiltration comprises ⁇ 1 + or double negative (DN) cells.
  • at least one additional therapeutic agent is administered.
  • the at least one or more additional agents comprises an agonist of DNAM-1.
  • the at least one or more additional agents comprises an anti-cancer agent.
  • the anti-cancer agent is selected from the group consisting of radiation, a chemotherapeutic or growth inhibitory agent, a targeted therapeutic agent, a small molecule inhibitor, a T cell expressing a chimeric antigen receptor, an antibody or antigen-binding fragment thereof, an antibody-drug conjugate, an angiogenesis inhibitor, an antineoplastic agent, a cancer vaccine, an adjuvant, and combinations thereof.
  • the anti-cancer agent is a chemotherapeutic.
  • the anti-cancer agent is a small molecule inhibitor.
  • the anti-cancer agent is an antibody.
  • the TIGIT inhibitor is administered continuously. In an aspect, the TIGIT inhibitor is administered intermittently.
  • the anti-cancer agent is administered continuously. In an aspect, the anti-cancer agent is administered intermittently. In an aspect, wherein the TIGIT inhibitor is administered before the anti-cancer agent. In an aspect, the TIGIT inhibitor is administered simultaneous with the anti-cancer agent. In an aspect, the TIGIT inhibitor is administered after the anticancer agent.
  • the application further discloses a TIGIT inhibitor as disclosed in any of the aspects and embodiments described herein for use in any of the methods described herein of treating cancers having high infiltration of ⁇ T cells.
  • the application discloses herein a method of increasing the number of tissue infiltrating ⁇ T cells in a mammalian subject recipient, comprising obtaining ⁇ T cells from tissue comprising ⁇ T cells form a donor; expanding the ⁇ T cells ex vivo; transplanting the expanded ⁇ T cells into a recipient, and administering at least one TIGIT inhibitor to the recipient.
  • the obtained and/or expanded ⁇ T cells are ⁇ 2 ⁇ .
  • the ⁇ 2 " cells are ⁇ 1 + or DN.
  • the at least one TIGIT inhibitor is selected from the group consisting of an antagonist of TIGIT activity, an antagonist of TIGIT interaction with PVR, an agent that inhibits and/or blocks the interaction of TIGIT with PVR, an agent that inhibits and/or blocks the interaction of TIGIT with PVRL2, an agent that inhibits and/or blocks the interaction of TIGIT with PVRL3, an agent that inhibits and/or blocks the intracellular signaling mediated by PVR binding to TIGIT, an agent that inhibits and/or blocks the intracellular signaling mediated by PVRL2 binding to TIGIT, an agent that inhibits and/or blocks the intracellular signaling mediated by PVLR3 binding to TIGIT, or combinations thereof.
  • the at least one TIGIT inhibitor is an antibody.
  • the antibody binds specifically to TIGIT.
  • the antibody binds to PVR.
  • the antibody is a full length IgG antibody.
  • the antibody is an antigen- binding antibody fragment.
  • the antibody is a single domain antibody.
  • the antibody is fully human.
  • the antibody is humanized.
  • the at least one TIGIT inhibitor comprises a small molecule inhibitor.
  • the ex vivo expanded ⁇ 2 " ⁇ T cells were obtained by culturing lymphocytes obtained from non- hematopoietic tissue of humans or non-human animals in the presence of interleukin-2 (IL-2) and/or interleukin-15 (IL-15), and not in direct contact with stromal or epithelial cells during culture.
  • the culturing step comprises culturing the lymphocytes obtained from human or non-human animal non-hematopoietic tissue in the presence of IL-2.
  • the culturing step comprises culturing the lymphocytes obtained from human or non-human animal non-hematopoietic tissue in the presence of interleukin-15 (IL-15).
  • the culturing step comprises culturing the lymphocytes obtained from human or non-human animal non-hematopoietic tissue in the presence of IL-2 and IL-15. In an aspect, the culturing step comprises culturing the lymphocytes in the absence of TCR activation or co-stimulation signals. In an aspect, the culturing step comprises culturing the lymphocytes in the absence of a T cell receptor pathway agonist. In an aspect, the culturing step comprises culturing the lymphocytes in the absence of stromal or epithelial cells. In an aspect, stromal or epithelial cells are removed prior to culture. In as aspect, the lymphocytes are cultured in the absence of fibroblasts.
  • the lymphocytes have been obtained from skin, the gastrointestinal tract (e.g. colon), mammary gland tissue, lung, liver, pancreas or prostate.
  • the ex vivo expanded V52 " ⁇ T cells were obtained by culturing lymphocytes obtained from non- hematopoietic tissue of humans or non-human animals in the presence of IL-2, IL-15, and a factor selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL- ⁇ , human platelet lysate (HPL), and stromal cell-derived factor-1 (SDF-1) for at least 5 days to produce an expanded population of ⁇ T cells.
  • the expanded population of ⁇ T cells comprises at least 20-fold the number of ⁇ T cells as the ⁇ T cells obtained from a non-hematopoietic tissue. In an aspect, within 7 days of culture, the expanded population of ⁇ T cells comprises at least 2-fold the number of ⁇ T cells as the ⁇ T cells obtained from a non-hematopoietic tissue. In an aspect, the expanded population of ⁇ T cells is at least 50% ⁇ 1 + cells. In an aspect, the expanded population of ⁇ T cells is at least 70% ⁇ 1 + cells. In an aspect, the expanded population of ⁇ T cells is at least 90% ⁇ 1 + cells.
  • the administration of the at least one TIGIT inhibitor is performed prior to, during, after the transplantation, or combinations thereof.
  • at least one additional agent is administered to the recipient.
  • the at least one additional agent is an anti-cancer agent.
  • the anti-cancer agent is selected from the group consisting of radiation, a chemotherapeutic or growth inhibitory agent, a targeted therapeutic agent, a small molecule inhibitor, a T cell expressing a chimeric antigen receptor, an antibody or antigen-binding fragment thereof, an antibody-drug conjugate, an angiogenesis inhibitor, an antineoplastic agent, a cancer vaccine, an adjuvant, and combinations thereof.
  • the recipient has cancer.
  • the ⁇ T cells are obtained from an autologous donor.
  • the non-hematopoietic tissue is tumor tissue.
  • the non-hematopoietic tissue is skin tissue.
  • the method further comprises separating ⁇ 1 + ⁇ T cells or DN cells from the ⁇ T cells prior to transplantation.
  • at least one additional checkpoint inhibitor is administered.
  • the additional checkpoint inhibitor does not inhibit PD1.
  • the additional checkpoint inhibitor inhibits TIM-3.
  • the additional checkpoint inhibitor inhibits LAG-3.
  • the administration of the TIGIT inhibitor results in elevated release of cytokines from the ⁇ T cells, selected from the group consisting of IFN- ⁇ , TNF-a, interleukins, and combinations thereof.
  • the application further discloses expanded ⁇ T cells obtained from tissue comprising ⁇ T cells from a donor as disclosed in any of the aspects and embodiments described herein and at least one TIGIT inhibitor as disclosed in any of the aspects and embodiments described herein for use in any of the methods described herein of increasing the number of tissue infiltrating ⁇ T cells in a mammalian subject recipient.
  • described herein are methods of treating cancer, comprising administering a therapeutically effective amount of a TIGIT inhibitor to a cancer patient who has been determined to have an elevated representation of ⁇ 1 + cells in a sample of peripheral blood.
  • the methods further comprise the prior step of determining the representation of ⁇ 1 + cells in the peripheral blood sample.
  • the methods further comprise the step, after determining the representation of ⁇ 1 + cells and before administering the TIGIT inhibitor, of selecting the patient for TIGIT inhibitor treatment if the patient's sample has been determined to have an elevated representation of ⁇ 1 + cells.
  • the elevated representation is an increase in ⁇ 1 + cells as a percentage of total lymphocytes in the sample.
  • greater than 0.5% of total lymphocytes in the sample are determined to be ⁇ 1 + cells. In certain aspects, greater than 1.0% of total lymphocytes are determined to be ⁇ 1 + cells. In certain aspects, greater than 2.0% of total lymphocytes are determined to be ⁇ 1 + cells. In certain aspects, wherein the elevated representation is an increase in ⁇ 1 + cells as a percentage of total ⁇ T cells in the sample. In certain aspects, greater than 10% of total ⁇ T cells in the sample are ⁇ 1 + cells. In certain aspects, greater than 15% of total ⁇ T cells in the sample are ⁇ 1 + cells. In certain aspects, the elevated representation is an increase in the ratio of ⁇ 1 + cells to ⁇ 2 + cells in the sample.
  • the ratio of ⁇ 1 + : ⁇ 2 + cells is greater than 1 :9. In certain aspects, the ratio of ⁇ 1 + : ⁇ 2 + cells is greater than 1 :8. In certain aspects, the ratio of ⁇ 1 + : ⁇ 2 + cells is greater than 1 :7. In certain aspects, the ratio of V51 + :V52 + cells is greater than 1 :6.
  • the TIGIT inhibitor is an antibody or antigen-binding antibody fragment that binds specifically to TIGIT.
  • the methods further comprise administration of at least one additional anti-cancer therapy. In certain aspects, at least one of the at least one additional anti-cancer therapy is administration of a non-TIGIT checkpoint inhibitor.
  • the non-TIGIT checkpoint inhibitor is an antibody that binds specifically to PD-1. In certain aspects, the non-TIGIT checkpoint inhibitor is an antibody that binds specifically to PDL1.
  • the cancer is a solid, non-hematopoietic, cancer. In certain aspects, the cancer is a breast cancer. In certain aspects, the cancer is a hematological cancer.
  • the application further discloses a TIGIT inhibitor as disclosed in any of the aspects and embodiments described herein for use in any of the methods described herein of treating cancer in a patient who has been determined to have an elevated representation of V51 + cells in a sample of peripheral blood.
  • Figure 1A provides a conceptual schema of sub-populations of T cells.
  • Figure IB shows results of flow cytometry performed on human peripheral blood mononuclear cells ("human blood”) and human skin-derived lymphocytes (“human tissue”) stained with antibodies specific for CD3, V51, and V52.
  • FIG. 1 illustrates PVR expression on Antigen Presenting Cells (APC) and antagonistic action on T Cells through DNAM1 and TIGIT.
  • APC Antigen Presenting Cells
  • FIG. 3 shows results of flow cytometry ("FACS") analysis for DNAM 1 expression of ⁇ T cells derived from human peripheral blood mononuclear cells (“PBMCs”) and human skin lymphocytes.
  • the shaded histogram is the signal of the Fluorescence minus one (FMO) control.
  • Percent of Max refers to the percentage of maximum count.
  • Figure 4 shows results of FACS analysis for TIGIT expression of ⁇ T cells derived from human PBMCs, both unstimulated (left panel) and stimulated for 72 hours with PHA.
  • Figure 5 shows results of FACS analysis for TIGIT expression on ⁇ T cells derived from human skin.
  • Figure 6 shows results of F ACS analysis for DNAM 1 and TIGIT expression on V51 + , V52 + , and DN ⁇ T cells derived from a representative donor of human PBMCs from peripheral blood (PB).
  • PB peripheral blood
  • FIG. 7 shows results of FACS analysis for DNAM 1 and TIGIT expression on V5T
  • V52 + , and DN ⁇ T cells derived from multiple donors of human PBMCs from PB are also known.
  • Figure 8 shows results of FACS analysis for DNAM 1 and TIGIT expression on ⁇ and DN ⁇ T cells derived from a representative donor of human skin leukocytes.
  • Figure 9 shows results of FACS analysis for DNAM 1 and TIGIT expression on V5T and DN ⁇ T cells derived from multiple donors of human skin leukocytes.
  • Figure 10 shows interferon gamma (IFNy) production in anti-CD3 stimulated V51 + .
  • PVR Fc Chimera Protein or an isotype control antibody (iso), demonstrating that PVR inhibits TCR stimulation in DN/ ⁇ T cells from human PBMCs.
  • FIG 11 shows results from FACS analysis of Interferon gamma (IFNy) and Tumor Necrosis Factor alpha (TNFa) production in anti-CD3 stimulated V51 + , V52 + , and DN ⁇ T cells derived from human PBMCs and human skin leukocytes upon exposure to PVR or an isotype control antibody (iso), demonstrating that PVR inhibits TCR stimulation in DN/V51 + T cells.
  • n 6 peripheral blood-derived ⁇ cell samples from different donors and 8 skin-derived ⁇ cell samples (2 out of 8 are sorted ⁇ cells).
  • n 3 peripheral blood-derived ⁇ cell samples and 1 skin-derived ⁇ cell samples.
  • the graphs for ⁇ 1 + and DN cell samples are generated from exactly all the same donors, while the graphs for V ⁇ 2 + are generated with a different set of donors.
  • the donors used for the V ⁇ 2 + graphs are all included among the donors for V ⁇ 1 + and DN samples.
  • Figure 12 shows results of FACS analysis for proliferation indicator/dye retention of anti-CD3 stimulated ⁇ 1 + and DN ⁇ T cells derived from human skin leukocytes upon exposure to PVR or an isotype control antibody, demonstrating the PVR inhibits TCR- induced proliferation in DN/V01 + T cells from human skin.
  • Figure 13 shows quantitation of results from FACS analysis for proliferation indicator/dye retention of anti-CD3 stimulated ⁇ 1 + and DN ⁇ T cells derived from human skin leukocytes upon exposure to PVR or an isotype control antibody, demonstrating that PVR inhibits TCR-induced proliferation in DN/V01 + T cells from human skin.
  • Figure 14 shows quantification of results from FACS analysis of Interferon gamma (IFNy) and Tumor Necrosis Factor alpha (TNFa) production in anti-CD3 stimulated V51 + and DN ⁇ T cells derived from human skin leukocytes upon exposure to an IgG control (mlgG), anti-CD3 (aCD3), and anti-CD3 plus PVR, demonstrating that PVR specifically inhibits TCR signaling.
  • IFNy Interferon gamma
  • TNFa Tumor Necrosis Factor alpha
  • Figure 15 shows quantification of results from FACS analysis of Interferon gamma (IFNy) production in anti-CD3 stimulated V51 + and DN ⁇ T cells derived from human skin leukocytes upon exposure to PVR and/or MICA, anti-DNAMl or an IgG control.
  • IFNy Interferon gamma
  • Figure 16 shows quantification of results from FACS analysis of Tumor Necrosis Factor alpha (TNFa) production in anti-CD3 stimulated V51 + and DN ⁇ T cells derived from human skin leukocytes upon exposure to PVR and/or MICA, anti-DNAMl or an IgG control, demonstrating that PVR specifically inhibits TCR signaling.
  • TNFa Tumor Necrosis Factor alpha
  • FIG. 17 shows quantification of results from FACS analysis of granulocyte- macrophage colony-stimulating factor (GMCSF), Interferon gamma (IFNy) and Tumor Necrosis Factor alpha (TNFa) production of ⁇ T cells from human skin lymphocytes of 5 different donors sorted with a pan ⁇ T cell antibody upon exposure to an IgG control (mlgG), anti-CD3 (aCD3), and anti-CD3 plus PVR, demonstrating that PVR is a general inhibitor of TCR stimulation.
  • GMCSF granulocyte- macrophage colony-stimulating factor
  • IFNy Interferon gamma
  • TNFa Tumor Necrosis Factor alpha
  • Figure 18 shows quantification of results from FACS analysis for TIGIT expression of tissue-derived V51 + and V53 + cells cultured in the presence of IL-2 and IL-15 (culture method 1), or in the presence of IL-2, IL-4, IL-15 and IL-21 (culture method 2), demonstrating that cells grown using culture method 2 express low levels of TIGIT.
  • Figure 19 are graphs showing quantification of results from FACS analysis of tissue- derived V51 + and V53 + cells from an individual donor cultured in the presence of IL-2, IL-4, IL-15 and IL-21 upon exposure to an IgG control (mlgG), anti-CD3 (aCD3), and anti-CD3 plus PVR, demonstrating that the PVR inhibition of INFy and TNFa was lost in V51 + and V53 + cells expressing low levels of TIGIT.
  • IgG control mlgG
  • aCD3 anti-CD3 plus PVR
  • Figure 20 are graphs showing quantification of results from FACS analysis of tissue- derived V51 + and V53 + cells from an individual donor cultured in the presence of IL-2, IL-4, IL-15 and IL-21 upon exposure to an IgG control (mlgG), anti-CD3 (aCD3), and anti-CD3 plus PVR, demonstrating that the PVR inhibition of INFy and TNFa was lost in V51 + and V53 + cells expressing low levels of TIGIT. 6.
  • IgG control mlgG
  • aCD3 anti-CD3 plus PVR
  • tissue-infiltrating ⁇ T cells particularly V52 " ⁇ T cells
  • methods for activating tissue-infiltrating ⁇ T cells, particularly V52 " ⁇ T cells in mammalian subjects, by antagonizing TIGIT activity of the V52 " ⁇ T cells, and methods for the treatment of cancer comprising administration of at least one TIGIT inhibitor.
  • V62 ⁇ T cells refers to any ⁇ T cell that does not express V52.
  • DN Double negative
  • DN cells can express, but are not limited to those that express, V53, V54, V55, V56, V57, and
  • activate a T cell includes activation, de-repression, and both activation and de-repression of the T cell.
  • TIGIT inhibitor refers to any agent or compound capable of disrupting TIGIT activity or disrupting TIGIT interaction with PVR.
  • a “TIGIT inhibitor” can increase PVR interaction with DNAMl .
  • a “TIGIT inhibitor” may block the interaction of PVR with TIGIT, leading to sequestration of TIGIT and increased binding of PVR to DNAMl.
  • a “TIGIT inhibitor” can be an antagonist of TIGIT activity, an antagonist of TIGIT interaction with PVR, an agent that inhibits and/or blocks the interaction of TIGIT with PVR, an agent that inhibits and/or blocks the interaction of TIGIT with PVRL2, an agent that inhibits and/or blocks the interaction of TIGIT with PVRL3, an agent that inhibits and/or blocks the intracellular signaling within a V52 " ⁇ T cell mediated by PVR binding to TIGIT, an agent that inhibits and/or blocks the intracellular signaling within a V52 " ⁇ T cell mediated by PVRL2 binding to TIGIT, an agent that inhibits and/or blocks the intracellular signaling within a V52 " ⁇ T cell mediated by PVLR3 binding to TIGIT, and combinations thereof.
  • an “expanded population of ⁇ cells” refers to a population of cells that has been cultured under conditions and for a duration sufficient to cause proliferation of ⁇ cells within that population.
  • an “expanded population of V61 + T cells,” as used herein, refers to a population of cells that has been cultured under conditions and for a duration sufficient to cause proliferation of V51 + T cells within that population.
  • tissue-resident refers to cells that are located at non-vascular anatomical sites.
  • Antigen binding fragment of an antibody or “antigen binding antibody fragment” refers to any single chain or multiple chain portion of an antibody that is capable of binding specifically to an epitope of an antigen.
  • Antigen binding fragment includes, without limitation, Fab, F(ab)2, and single-chain Fv (scFv).
  • Human antibody unless otherwise indicated is one whose sequences correspond to (i.e., are identical in sequence to) an antibody that could be produced by a human and/or consists entirely of amino acid sequences that are encoded in the human genome.
  • a "human antibody” as used herein can be produced using various techniques known in the art, including phage-display libraries and by administering the antigen (e.g., TIGIT) to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized Xenomice.
  • Humanized antibodies refer to non-human (e.g., murine) antibodies that are chimeric antibodies and contain minimal sequence derived from non-human immunoglobulin.
  • a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • donor antibody such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • FR residues of the human immunoglobulin variable domain are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. In an embodiment, the humanized antibodies do not comprise residues that are not found in the recipient antibody or in the donor antibody.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In an embodiment where the humanized antibodies do comprise residues that are not found in the recipient antibody or in the donor antibody, the Fc regions of the antibodies are modified.
  • Binds specifically refers to the property of an antibody or fragment of binding to the (specified) antigen with a dissociation constant that is ⁇ 1 ⁇ , preferably ⁇ 1 nM and most preferably ⁇ 10 pM.
  • the Kd of the antibody that binds specifically to TIGIT is 250-500 pM.
  • An epitope that "binds specifically "or "binds preferentially” (used
  • an antibody or a polypeptide is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art.
  • a molecular entity is said to exhibit "specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances.
  • An antibody "specifically binds” or “preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.
  • an antibody that specifically or preferentially binds to a TIGIT conformational epitope is an antibody that binds this epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other TIGIT epitopes or non-TIGIT epitopes. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding.
  • cancers or tissues with "high ⁇ T cell infiltration” refers to tissues (e.g., tumor from tumor biopsies) wherein the fraction of ⁇ T cells to total leukocytes, either measured or estimated, is greater than the fraction of ⁇ T cells in a given reference tissue, such as non-tumor tissue of a similar origin.
  • tissues with "high ⁇ T cell infiltration” refers to tissues wherein the fraction of ⁇ T cells to total leukocytes is greater than 0.0001 , greater than 0.001 , greater than 0.01 , greater than 0.1 , greater than 0.2, greater than 0.3, greater than 0.4, or greater than 0.5.
  • tissues with "high ⁇ T cell infiltration” refers to tissues in which the fraction of ⁇ T R A to total leukocyte R A is greater than 0.0001, greater than 0.001, greater than 0.01 , or greater than 0.1.
  • T-cell checkpoints or “checkpoints” refers to any protein or gene that functions to inhibit or suppress the activation of a T-cell.
  • T-cell checkpoints include, but are not limited to: A2A , B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3 and VISTA.
  • T-cell checkpoint inhibitor or "checkpoint inhibitor” refers to any molecule that inhibits the activity of a T-cell checkpoint.
  • “Sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate ⁇ T cell activity.
  • Therapeutically effective amount is an amount that is effective to ameliorate a symptom of a disease.
  • a therapeutically effective amount can be a “prophylactically effective amount” as prophylaxis can be considered therapy.
  • adjunctive administration or “administered to the subject adjunctively” are used interchangeably herein to mean administering a second therapeutic agent in therapeutically effective temporal proximity to a first therapeutic agent.
  • adjunctive administration improves the effectiveness of the second therapeutic agent, the first therapeutic agent, or both the first and the second therapeutic agent as compared to administration of the agent alone.
  • ⁇ T cells refers to gamma delta T cells
  • TGIT refers to T-Cell-IG and ITIM domain
  • TCR refers to the T-cell Receptor
  • PVR refers to Poliovirus receptor
  • IL-2 refers to native or recombinant inter leukin 2
  • IL-15 refers to native or recombinant interleukin 15, etc.
  • PB refers to peripheral blood and "PBMC” refers to peripheral blood mononuclear cells.
  • antagonizing TIGIT activity comprises administering at least one TIGIT inhibitor to the subject. TIGIT inhibitors suitable for use in the methods are described below in Section 6.6.
  • the at least one TIGIT inhibitor is administered to the subject adjunctively with V52 " ⁇ T cells that have been cultured ex vivo.
  • the at least one TIGIT inhibitor is administered in a
  • composition that also comprises the cultured ⁇ T cells.
  • the at least one TIGIT inhibitor is added to the V52 " ⁇ T cells during culturing ex vivo. In certain embodiments, the at least one TIGIT inhibitor is added to the V52 " ⁇ T cells after culturing ex vivo.
  • the at least one TIGIT inhibitor is administered to the subject separately from, but adjunctively with, the ex vivo expanded ⁇ T cells.
  • ⁇ 2 + T cells predominate among the ⁇ cells in hematopoietic tissues
  • ⁇ 2 " cells are also present, albeit in lower numbers.
  • the ⁇ T cells are obtained for culture from hematopoietic tissues, such as peripheral blood.
  • ⁇ 2 " ⁇ T cells are obtained for culture from the hematopoietic tissues.
  • ⁇ T cells from hematopoietic tissues are stimulated to reduce ⁇ 2 expression and/or induce ⁇ expression and/or selectively increase proliferation of ⁇ 2 " cells.
  • the ⁇ T cells are stimulated according to a protocol described in WO 2012/156,958, the contents of which are incorporated herein by reference in their entirety.
  • ⁇ T cells obtained from hematopoietic tissues are stimulated with a ⁇ TCR antibody to expand ⁇ 2 " ⁇ T cells and/or ⁇ 1 + ⁇ cells ex-vivo.
  • ⁇ T cells obtained from hematopoietic tissues are stimulated with a ⁇ TCR antibody to increase TIGIT expression of the ⁇ T cells ex-vivo.
  • ⁇ T cells are obtained for culture from non-hematopoietic tissues.
  • Detailed methods for the isolation, culturing and expansion of ⁇ T cells from non- hematopoietic tissues are disclosed in WO 2017/072367, which is incorporated herein by reference in its entirety.
  • V52 " ⁇ T cells for culture are obtained from any human or non-human mammal non-hematopoietic tissue that can be removed from a subject.
  • the non-hematopoietic tissue from which the V52 " ⁇ T cells are obtained for culture is skin (e.g., human skin), which can be obtained by methods known in the art.
  • the skin is obtained by punch biopsy.
  • the V52 " ⁇ T cells are obtained for culture from the gastrointestinal tract (e.g., colon), mammary gland, lung, prostate, liver, spleen, and pancreas.
  • the V52 " ⁇ T cells are obtained for culture from non-neoplastic tissues.
  • the V52 " ⁇ T cells are obtained for culture from human cancer tissues, e.g., tumors of the breast, the prostate, or other solid tumors.
  • the ⁇ T cells that are dominant in the non-hematopoietic tissues are primarily V51 + T cells, such that V51 + T cells comprise about 70-80% of the non-hematopoietic tissue-resident ⁇ T cell population.
  • the V52 " ⁇ T cells that have been cultured ex vivo are V51 + .
  • V52 T cells that are resident in non-hematopoietic tissues express neither V51 nor V52 TCR and we have named them double negative (DN) ⁇ T cells.
  • V52 " ⁇ T cells that have been cultured ex vivo are V51 " V52 " double negative (DN) cells.
  • DN ⁇ T cells are likely to be mostly V53 -expressing with a minority of V55 -expressing T cells.
  • the cultured V52 " ⁇ T cells have been expanded ex vivo.
  • the ex vivo expanded V52 " ⁇ T cells are obtained by culturing lymphocytes obtained from non-hematopoietic tissue of humans or non-human animals in the presence of inter leukin-2 (IL-2) and/or interleukin-15 (IL-15), and not in direct contact with stromal or epithelial cells during culture.
  • IL-2 inter leukin-2
  • IL-15 interleukin-15
  • the culturing step comprises culturing the lymphocytes obtained from human or non-human animal non-hematopoietic tissue in the presence of IL-2. In certain embodiments, the culturing step comprises culturing the lymphocytes obtained from human or non-human animal non-hematopoietic tissue in the presence of interleukin-15 (IL- 15). In particular embodiments, the culturing step comprises culturing the lymphocytes obtained from human or non-human animal non-hematopoietic tissue in the presence of IL-2 and IL-15.
  • IL-2 interleukin-15
  • the culturing step comprises culturing the lymphocytes in the absence of TCR activation or co-stimulation signals. In some embodiments, the culturing step comprises culturing the lymphocytes in the absence of a T cell receptor pathway agonist. In some embodiments, the culturing step comprises culturing the lymphocytes in the absence of stromal or epithelial cells. In some embodiments, stromal or epithelial cells are removed prior to culture. In some embodiments, the lymphocytes are cultured in the absence of fibroblasts. In various embodiments, the lymphocytes have been obtained from skin, the gastrointestinal tract (e.g., colon), mammary gland tissue, lung, liver, pancreas or prostate.
  • the gastrointestinal tract e.g., colon
  • mammary gland tissue e.g., lung, liver, pancreas or prostate.
  • the ex vivo expanded V52 " ⁇ T cells are obtained by culturing lymphocytes obtained from non-hematopoietic tissue of humans or non-human animals in the presence of IL-2, IL-15, and a factor selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL- ⁇ , human platelet lysate (HPL), and stromal cell-derived factor- 1 (SDF-1) for at least 5 days to produce an expanded population of ⁇ T cells.
  • IL-2 lymphocytes obtained from non-hematopoietic tissue of humans or non-human animals in the presence of IL-2, IL-15, and a factor selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL- ⁇ , human platelet lysate
  • the ex vivo expanded V52 " ⁇ T cells are obtained by culturing lymphocytes obtained from non-hematopoietic tissue of humans or non-human animals under conditions in which, within 14 days of culture, the expanded population of ⁇ T cells comprises at least 20-fold the number of ⁇ T cells as the ⁇ T cells obtained from a non- hematopoietic tissue.
  • the ex vivo expanded ⁇ 2 " ⁇ T cells were obtained by culturing lymphocytes obtained from non-hematopoietic tissue of humans or non- human animals under conditions in which, within 7 days of culture, the expanded population of ⁇ T cells comprises at least 2-fold the number of ⁇ T cells as the ⁇ T cells obtained from a non-hematopoietic tissue.
  • the expanded population of ⁇ T cells is at least 50% ⁇ 1 + cells. In certain embodiments, the expanded population of ⁇ T cells is at least 70% ⁇ 1 + cells. In select embodiments, the expanded population of ⁇ T cells is at least 90%> ⁇ 1 + cells.
  • the method further comprises expanding non-hematopoietic tissue-resident ⁇ T cells (e.g., skin-derived ⁇ T cells and/or ⁇ - ⁇ 2 T cells, e.g., ⁇ T cells and/or DN T cells).
  • the non-hematopoietic tissue-resident ⁇ T cells are expanded from a population of ⁇ T cells that has been separated from non- hematopoietic tissue according to methods described above.
  • non-hematopoietic tissue-resident ⁇ T cells are capable of spontaneously expanding upon removal of physical contact with stromal cells (e.g., skin fibroblasts).
  • the scaffold-based culture methods described above can be used to induce such separation, resulting in de-repression of the ⁇ T cells to trigger expansion. Accordingly, in some embodiments, no substantial TCR pathway activation is present during the expansion step (e.g., no exogenous TCR pathway activators are included in the culture). In various embodiments, the methods of expanding non- hematopoietic tissue-resident ⁇ T cells omit contact with feeder cells, tumor cells, and/or antigen-presenting cells.
  • the amount of each of the above cytokines required to produce an expanded population of ⁇ T cells will depend of the concentrations of one or more of the other cytokines. For example, if the concentration of IL-2 is increased or decreased, the concentration of IL-15 may be accordingly decreased or increased,
  • the amount effective to produce an expanded population refers herein to composite effect of all factors on cell expansion.
  • the ⁇ T cells are exposed to certain factors prior to culture with other factors.
  • the expansion culture can be gradually supplied with additional factors over the course of expansion, or, alternatively, the ⁇ T cells can be transferred from a culture of one factor or group of factors to another.
  • the ⁇ T cells are expanded in culture for a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, or 21 hours) to about 35 days (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 days).
  • the ⁇ T cells are expanded for a period of 14 to 21 days.
  • the separation and expansion steps in some embodiments, can last between 28 and 56 days, or about 41 days.
  • Methods of expansion provide an expanded population of ⁇ T cells that is greater in number than a reference population.
  • the expanded population of ⁇ T cells is greater in number than the separated population of ⁇ T cells prior to the expansion step (e.g., at least 2-fold in number, at least 3-fold in number, at least 4-fold in number, at least 5-fold in number, at least 6-fold in number, at least 7-fold in number, at least 8-fold in number, at least 9-fold in number, at least 10-fold in number, at least 15-fold in number, at least 20-fold in number, at least 25-fold in number, at least 30-fold 35 in number, at least 35-fold in number, at least 40-fold in number, at least 50-fold in number, at least 60- fold in number, at least 70- fold in number, at least 80-fold in number, at least 90-fold in number, at least 100-fold in number, at least 200-fold in number, at least 300-fold in number, at least 400-fold in number, at least 500-
  • the invention provides a means to produce large populations of non-hematopoietic tissue-derived ⁇ T cells (e.g., skin-derived ⁇ T cells and/or ⁇ - ⁇ 2 T cells, e.g., ⁇ T cells and/or DN T cells) at high rates (e.g., by removing stromal cell contact and/or TCR stimulation, or by culturing in the presence of an effective amount of factors).
  • the expansion step described herein expands the ⁇ T cells at a low population doubling time, which is given by the following equation:
  • non-hematopoietic tissue-derived ⁇ T cells e.g., skin-derived ⁇ T cells and/or ⁇ - ⁇ 2 T cells, e.g., ⁇ T cells and/or DN T cells
  • a population doubling time of less than 5 days e.g., less than 4.5 days, less than 4.0 days, less than 3.9 days, less than 3.8 days, less 15 than 3.7 days, less than 3.6 days, less than 3.5 days, less than 3.4 days, less than 3.3 days, less than 3.2 days, less than 3.1 days, less than 3.0 days, less than 2.9 days, less than 2.8 days, less than 2.7 days, less than 2.6 days, less than 2.5 days, less than 2.4 days, less than 2.3 days, less than 2.2 days, less than 2.1 days, less than 2.0 days, less than 46 hours, less than 42 hours, less than 38 hours, less than 35 hours, less than 32
  • the expanded population of ⁇ T cells comprises at least 10-fold the number of ⁇ T cells relative to the separated population of ⁇ T-cells prior to expansion (e.g., at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, or at least 8,000- fold the number of ⁇ T cells relative to the separated population of ⁇ T cells prior to
  • the expanded population of ⁇ T cells comprises at least 20-fold the number of ⁇ T cells relative to the separated population of ⁇ T cells prior to expansion (e.g., at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000- fold, or at least 10,000-fold the number of ⁇ T cells relative to
  • the expanded population of ⁇ T cells comprises at least 50-fold the number of ⁇ T cells relative to the separated population of ⁇ T cells prior to expansion (e.g., at least 60- fold, at least 70-fold, at least 80-fold, at least 90- fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or least 10,000- fold the number of ⁇ T cells relative to the separated population of ⁇ T cells prior to expansion).
  • at least 50-fold the number of ⁇ T cells relative to the separated population of ⁇ T cells prior to expansion e.g., at least 60- fold, at least 70
  • the expanded population of ⁇ T cells comprises at least 100-fold the number of ⁇ T cells relative to the separated population of ⁇ T cells prior to expansion (e.g., at least 110-fold, at least 120-fold, at least 130-fold, at least 140-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, at least 10,000-fold, at least 12,000-fold, or at least 15,000-fold the number of ⁇ T cells relative to the separated population of ⁇ T cells prior
  • Non-hematopoietic tissue-derived ⁇ T cells expanded by the methods provided herein can have a phenotype well-suited for anti-tumor efficacy.
  • the expanded population of ⁇ T cells e.g., skin-derived ⁇ T cells
  • the expanded population of ⁇ T cells has a mean expression of CD27 that is at least 2-fold relative to the separated population of ⁇ T cells (e.g., at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900- fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 20,000-fold, or more, relative to the separated population of ⁇ T cells).
  • An increase or decrease in expression of other markers can be additionally or alternatively used to characterize one or more expanded populations of non-hematopoietic tissue-derived ⁇ T cells (e.g., skin-derived ⁇ T cells and/or ⁇ - ⁇ 2 T cells, e.g., ⁇ T cells and/or DN T cells), including CD124, CD215, CD360, CTLA4, CDlb, BTLA, CD39, CD45RA, Fas Ligand, CD25, ICAM-1, CD31, KLRG1,CD30, CD2, NKp44, NKp46, ICAM- 2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64.
  • non-hematopoietic tissue-derived ⁇ T cells e.g., skin-derived ⁇ T cells and/or ⁇ - ⁇ 2 T cells, e.g., ⁇ T cells and/or DN T cells
  • the expanded population of ⁇ T cells has a greater mean expression of one or more of the markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CDlb, BTLA, CD39, CD45RA, Fas Ligand, CD25, ICAM-1, CD31 , KLRG1, CD30, and CD2, relative to the separated population of ⁇ T cells, e.g., prior to expansion.
  • the expanded population of ⁇ T cells may have a greater frequency of cells expressing one or more of the markers selected from the group consisting of CD 124, CD215, CD360, CTLA4, CDlb, BTLA, CD39, CD45RA, Fas Ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2, relative to the separated population of ⁇ T cells.
  • the expanded population of ⁇ T cells (e.g., skin-derived ⁇ T cells and/or non- V52 T cells, e.g., V51 T cells and/or DN T cells) has a lower mean expression of one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64, relative to the separated population of ⁇ T cells.
  • the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 relative to the separated population of ⁇ T cells.
  • the expanded population may similarly have a lower frequency of cells expressing one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64, relative to the separated population of ⁇ T cells.
  • a non-hematopoietic tissue-resident ⁇ T cell produced by the method of the invention may thus have one or more of the following properties: (i) displays the phenotype CD69high, TIM3high and CD281ow/absent; (ii) upregulates of one or more of CCR3, CD39, CDl lb, and CD9; (iii) produces IFN- ⁇ in response to an NKG2D ligand in the absence of TCR agonists; (iv) produces IL-13 in the absence of TCR agonists; (v) produces one or more of IFN- ⁇ , TNF- ⁇ and GM-CSF in response to TCR activation; (vi) produces no or substantially no IL-17 in response to TCR activation; (vii) grows in culture medium containing IL-2 without additional growth factors; (viii) displays a cytotoxic T cell response in the absence of TCR agonists; and/or (ix) displays selective cytotoxicity for tumor cells over normal cells.
  • a non-hematopoietic tissue-resident ⁇ T cell produced by the method of the invention produces IL-13 in the absence of TCR agonists and/or produces IFN- ⁇ in response to an NKG2D ligand in the absence of TCR agonists.
  • basal culture media suitable for use in the proliferation of ⁇ T cells are available, in particular complete media, such as AIM-V, Iscoves medium and RPMI-1640 (Life Technologies).
  • the medium may be supplemented with other media factors, such as serum, serum proteins and selective agents, such as antibiotics.
  • RPMI-1640 medium containing 2 mM glutamine, 10% FBS, 10 mM HEPES, pH 7.2, 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies), nonessential amino acids (e.g.
  • cells are cultured at 37°C in a humidified atmosphere containing 5% C02 in a suitable culture medium.
  • the ⁇ T cells may be cultured as described herein in any suitable system, including stirred tank fermenters, airlift fermenters, roller bottles, culture bags or dishes, and other bioreactors, in particular hollow fiber bioreactors. The use of such systems is well-known in the art. General methods and techniques for culture of lymphocytes are well-known in the art.
  • the methods described herein can include more than one selection step, e.g., more than one depletion step.
  • Enrichment of a T cell population by negative selection can be accomplished, e.g., with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method is cell sorting and/or selection via negative magnetic immuno adherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • the ACT methods comprise, T cell immunotherapy wherein subjects are infused with heterologous, autologous or allogeneic ⁇ T cells to treat disease.
  • the ACT methods comprise infusion of subjects of V52 " ⁇ T cells to treat disease.
  • the ACT methods comprise infusion of subjects of V52 " ⁇ T cells that have been expanded ex vivo to treat disease.
  • the ACT methods comprise infusion of subjects of V52 " ⁇ T cells that have been expanded ex vivo to treat cancer.
  • V52 " ⁇ T cells are selected and expanded ex vivo to enrich for V52 " ⁇ T cells from a mixed population of immune cells.
  • ⁇ T cells are selected and/or engineered ex vivo to target specific antigens, such as tumor-associated antigens.
  • immune cells comprising ⁇ T cells are obtained from tissue of donor subjects.
  • ⁇ T cells are obtained from non-hematopoietic tissue of donor subjects.
  • ⁇ T cells are obtained from blood of donor subjects.
  • tumor infiltrating lymphocytes (“TIL"s) comprising ⁇ T cells are obtained from donor subjects.
  • TIL tumor infiltrating lymphocytes
  • ⁇ T cells are expanded in culture and selected for antigen specificity without altering their native specificity.
  • T lymphocytes comprising ⁇ T cells obtained from the donor are engineered ex vivo by transduction with viral expression vectors, to express chimeric antigen receptors ("CAR"s) of predetermined specificity.
  • CAR chimeric antigen receptors
  • Concentrations of cytokines and growth factors can range from about 0.1 ng/mL to about 500 ng/mL, from about 1 ng/mL to about 200 ng/mL, from about 10 ng/ml to 100 ng/ml, depending on the type of cytokine and growth factor. Appropriate combinations and concentrations of cytokines are disclosed in WO 2017/072367, which is incorporated herein by reference in its entirety.
  • the methods further comprise administering to the subject at least one additional agent in an amount sufficient to activate V52 " ⁇ T cells.
  • the at least one additional agent is an agonist of DNAM-1.
  • the mammalian subject has cancer.
  • the mammalian subject has cancer.
  • the cancer has high infiltration of ⁇ T cells.
  • the mammalian subject is a human patient.
  • the subject from whom ⁇ 2 " T cells are obtained for culture is the same individual as the ultimate recipient of the cells, i.e., an autologous donor.
  • Autologous ⁇ T cells which are removed and in some instances stored are transplanted back into the subject.
  • the autologous ⁇ T cells are subject to additional treatments prior to transplantation back into the recipient.
  • autologous ⁇ T cells obtained from the subject are further treated or purified to remove any disease cells, such as cancer cells, prior to transplantation back into the subject.
  • treatments and purification also referred to as "purging," of cell preparations to remove disease cell can include, among others, use of antibodies directed against cell surface markers expressed in disease cells and anti-cancer (i.e., chemotherapeutic) treatments.
  • the autologous ⁇ T cells are obtained following treatment of the subject for the underlying disease, thereby reducing the risk of presence of disease cells in the autologous ⁇ T cell preparations.
  • the subject can be treated with an antibody therapeutic targeting the disease or treated with anti-cancer agent that does not destroy or suppress the hematopoietic system prior to obtaining the ⁇ T cells for transplantation.
  • the purged preparation of ⁇ T cells can be further purified, such as by FACS or affinity selection (e.g., chromatography), to select ⁇ T cells or ⁇ 2 " ⁇ T cells from other cells (i.e., other immune cells or diseased cells).
  • the donor subject is not the same individual as the ultimate recipient of the cells, i.e., a heterologous or allogeneic donor.
  • the cells obtained from the donor subject are not screened or matched for the recipient.
  • ⁇ T cells are non-MHC restricted, they do not recognize a host into which they are transferred as foreign, which means that they are less likely to cause graft- versus-host disease. This means that they can be used "off the shelf and transferred into any recipient, e.g., for heterologous or allogeneic adoptive T cell therapy.
  • allogeneic immune cells comprising ⁇ T cells are obtained from donors who are selected based on matching at loci of the human lymphocyte antigen (HLA) complex.
  • the allogeneic immune cells comprising ⁇ T cells can be further isolated or purified, and/or subject to further manipulation.
  • the allogeneic immune cells comprising ⁇ T cells are subject to additional treatments to expand a population of immune cells or manipulated by recombinant methods to introduce heterologous genes or additional functionality to the allogeneic immune cells prior to transplantation into the recipient subject.
  • ⁇ T cells obtained from a donor can be subject to additional treatments prior to transplantation into a recipient subject.
  • the immune cells comprising ⁇ T cells are treated to expand the population of ⁇ T cells, for example by culturing in a suitable medium.
  • the method further comprises the step of detecting ⁇ T cell infiltration, or ⁇ 2 " ⁇ T cell infiltration, of tissues in a mammalian subject and selecting subjects who have ⁇ T cell infiltrates.
  • ⁇ T cell infiltration or ⁇ 2 " ⁇ T cell infiltration is detected in tumors of a mammalian subject. Detection of ⁇ T cell or ⁇ 2 " ⁇ T-cells may be performed by any method known in the art for the detection of subpopulations of immune cells in tissue. T cell infiltration in tissues ⁇ e.g., tumor from tumor biopsies) can be determined by
  • An increase or decrease in expression of other markers can be additionally or alternatively used to characterize ⁇ T cells from tissue or ⁇ T cells from tissue which have been expanded ex-vivo ⁇ e.g., skin-derived ⁇ T cells and/or ⁇ - ⁇ 2 T cells, e.g., ⁇ T cells and/or DN T cells), including CD124, CD215, CD360, CTLA4, CDlb, BTLA, CD39, CD45RA, Fas Ligand, CD25, ICAM-1 , CD31 , KLRG1 , CD30, CD2, NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1 , and CD64.
  • ⁇ T cells from tissue or ⁇ T cells from tissue which have been expanded ex-vivo ⁇ e.g., skin-derived ⁇ T cells and/or ⁇ - ⁇ 2 T cells, e.g., ⁇ T cells and/or DN T cells
  • the expanded population of ⁇ T cells has a greater mean expression of one or more of the markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CDlb, BTLA, CD39, CD45RA, Fas Ligand, CD25, ICAM-1 , CD31 , KLRG1 , CD30, and CD2, relative to the separated population of ⁇ T cells, e.g., prior to expansion.
  • the expanded population of ⁇ T cells may have a greater frequency of cells expressing one or more of the markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CDlb, BTLA, CD39, CD45RA, Fas Ligand, CD25, ICAM-1 , CD31 , KLRG1 , CD30, and CD2, relative to the separated population of ⁇ T cells.
  • the expanded population of ⁇ T cells (e.g., skin-derived ⁇ T cells and/or non- ⁇ 2 T cells, e.g., ⁇ T cells and/or DN T cells) has a lower mean expression of one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1 , and CD64, relative to the separated population of ⁇ T cells.
  • the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1 , and CD64 relative to the separated population of ⁇ T cells.
  • the expanded population may similarly have a lower frequency of cells expressing one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1 , and CD64, relative to the separated population of ⁇ T cells.
  • the composition is co-administered with an additional agent for treating cancer (e.g., an anti-cancer agent).
  • an additional agent for treating cancer e.g., an anti-cancer agent.
  • the composition is coadministered with an as yet to be described therapeutic for treating cancer.
  • the agents may be administered concurrently or sequentially.
  • agents described herein are administered prior to the other active agent(s).
  • the pharmaceutical formulations and modes of administration may be any of, but not limited to, those described below. It is also anticipated that there could be two or more co-administered chemical agents, biological agents or radiation that may each be administered using different modes or different formulations. 6.3.1.4.2. Selection of subjects with high V62 " ⁇ T cells in peripheral blood
  • the method further comprises the step of detecting V52 " ⁇ in the blood of a mammalian subject, and selecting subjects who have high ⁇ 2 ⁇ ⁇ T cells in the blood.
  • the method comprises determining the ratio of ⁇ 1 + and ⁇ 2 + in a blood sample from a potential mammalian subject.
  • the method comprises determining the total number of ⁇ 1 + and ⁇ 2 + in a blood sample from a potential mammalian subject.
  • the method further comprises comparing the ratio of ⁇ 1 + and ⁇ 2 + in a blood sample from a subject with cancer to a healthy individual.
  • the method further comprises comparing the total number of ⁇ 1 + and ⁇ 2 + in a blood sample from a subject with cancer to a healthy individual.
  • the cancer has high infiltration of ⁇ T cells.
  • the methods comprise administering at least one TIGIT inhibitor to a patient with a cancer having high infiltration of ⁇ T cells, in an amount sufficient to activate tissue-infiltrating ⁇ T cells in the patient.
  • the cancer-infiltrating ⁇ T cells are ⁇ 2 " cells, and the amount of TIGIT inhibitor is sufficient to activate ⁇ 2 " cells.
  • the amount of TIGIT inhibitor is sufficient to activate ⁇ 1 + cells.
  • the amount of TIGIT inhibitor is sufficient to activate double negative (DN) cells.
  • the TIGIT inhibitor is an inhibitor as described in
  • the TIGIT inhibitor is an antibody.
  • the antibody binds specifically to TIGIT.
  • the method further comprises the earlier step of selecting for treatment a patient whose cancer shows high levels of ⁇ T cell infiltration.
  • the ⁇ T cell infiltration is detected by analysis of a tumor biopsy.
  • the analysis comprises an assay selected from
  • the cancer shows high levels of V51 + or double negative (DN) cell infiltrate.
  • the method further comprises the prior step of detecting ⁇ T cell infiltration, or V52 " ⁇ T cell infiltration, of tissues in a mammalian subject and selecting subjects who have ⁇ T cell infiltrates.
  • ⁇ T cell infiltration or V52 " ⁇ T cell infiltration is detected in tumors of a mammalian subject. Detection of ⁇ T cell or ⁇ 2 " ⁇ T-cells may be performed by any method known in the art for the detection of subpopulations of immune cells in tissue. T cell infiltration in tissues (e.g., tumor from tumor biopsies) can be determined by
  • An increase or decrease in expression of other markers can be additionally or alternatively used to characterize ⁇ T cells from tissue or ⁇ T cells from tissue which have been expanded ex-vivo (e.g., skin-derived ⁇ T cells and/or ⁇ - ⁇ 2 T cells, e.g., ⁇ T cells and/or DN T cells), including CD124, CD215, CD360, CTLA4, CDlb, BTLA, CD39, CD45RA, Fas Ligand, CD25, ICAM-1, CD31, KLRG1, CD30, CD2, NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64.
  • tissue or ⁇ T cells from tissue which have been expanded ex-vivo e.g., skin-derived ⁇ T cells and/or ⁇ - ⁇ 2 T cells, e.g., ⁇ T cells and/or DN T cells
  • the expanded population of ⁇ T cells has a greater mean expression of one or more of the markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CDlb, BTLA, CD39, CD45RA, Fas Ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2, relative to the separated population of ⁇ T cells, e.g., prior to expansion.
  • the expanded population of ⁇ T cells may have a greater frequency of cells expressing one or more of the markers selected from the group consisting of CD 124, CD215, CD360, CTLA4, CDlb, BTLA, CD39, CD45RA, Fas Ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2, relative to the separated population of ⁇ T cells.
  • the expanded population of ⁇ T cells (e.g., skin-derived ⁇ T cells and/or non- ⁇ 2 T cells, e.g., ⁇ T cells and/or DN T cells) has a lower mean expression of one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64, relative to the separated population of ⁇ T cells.
  • the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 relative to the separated population of ⁇ T cells.
  • the expanded population may similarly have a lower frequency of cells expressing one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64, relative to the separated population of ⁇ T cells.
  • the TIGIT inhibitor is co-administered with an additional agent for treating cancer (e.g., an anti-cancer agent).
  • the composition is coadministered with an as yet to be described therapeutic for treating cancer.
  • the agents may be administered concurrently or sequentially.
  • agents described herein are administered prior to the other active agent(s).
  • the pharmaceutical formulations and modes of administration may be any of, but not limited to, those described below. It is also anticipated that there could be two or more co-administered chemical agents, biological agents or radiation that may each be administered using different modes or different formulations.
  • the method of treating cancer comprises administering a therapeutically effective amount of a TIGIT inhibitor to a cancer patient who has been determined to have an elevated representation of V51 + cells in a sample of peripheral blood.
  • the method further comprising the prior step of determining the representation of V51 + cells in the peripheral blood sample. In some embodiments, the method further comprises the step, after determining the representation of V51 + cells and before administering the TIGIT inhibitor, of selecting the patient for TIGIT inhibitor treatment if the patient's sample has been determined to have an elevated representation of V51 + cells.
  • the method comprises determining the total number of V51 + and V52 + in a blood sample from a potential mammalian subject. In particular embodiments, the method comprises determining the ratio of V51 + and V52 + in a blood sample from a potential mammalian subject. In certain embodiments, the method further comprises comparing the total number of V51 + and V52 + in a blood sample from a subject with cancer to a healthy individual. In certain embodiments, the method further comprises comparing the ratio of V51 + and V52 + in a blood sample from a subject with cancer to a healthy individual. [0108] In some embodiments, the elevated representation is an increase in V51 + cells as a percentage of total lymphocytes in the sample.
  • greater than 0.2% of total lymphocytes in the sample are determined to be V51 + cells. In particular embodiments, greater than 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% of total lymphocytes in the sample are determined to be V51 + cells. In some embodiments, greater than 1.5%, 2.0%, 2.5%), 3.0%), 3.5%), 4.0%), or 5.0% of total lymphocytes in the sample are determined to be V51 + cells.
  • the elevated representation is an increase in V51 + cells as a percentage of total ⁇ T cells in the sample. In certain embodiments, greater than 10% of total ⁇ T cells in the sample are V51 + cells. In particular embodiments, greater than 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of total ⁇ T cells in the sample are V51 + cells.
  • the elevated representation is an increase in the ratio of V51 + cells to V52 + cells in the sample.
  • the ratio of V51 + :V52 + cells is greater than 1 :9.
  • the ratio of V51 + :V52 + cells is greater than 1 :8, 1 :7, 1 :6, or 1 :5.
  • the TIGIT inhibitor is an antibody or antigen-binding antibody fragment that binds specifically to TIGIT.
  • the method further comprises administering at least one additional anti-cancer therapy.
  • at least one of the at least one additional anti-cancer therapy is a non-TIGIT checkpoint inhibitor.
  • the non-TIGIT checkpoint inhibitor is an antibody that binds specifically to PD- 1.
  • the non-TIGIT checkpoint inhibitor is an antibody that binds specifically to PDL 1.
  • the cancer is a solid, non-hematopoietic, cancer.
  • the cancer is a breast cancer.
  • the cancer is a hematological cancer.
  • the method further comprises administering at least one additional therapeutic agent.
  • the at least one additional therapeutic agent is an agonist of DNAM-1.
  • the at least one or more additional agent is an anti-cancer agent.
  • the anti-cancer agent is selected from the group consisting of radiation, a chemotherapeutic or growth inhibitory agent, a targeted therapeutic agent, a small molecule inhibitor, a T cell expressing a chimeric antigen receptor, an antibody or antigen- binding fragment thereof, an antibody-drug conjugate, an angiogenesis inhibitor, an
  • the anti-cancer agent is a chemotherapeutic agent.
  • the anti-cancer agent is a targeted therapeutic agent.
  • the anti-cancer agent is an antibody.
  • the anti-cancer agent is administered continuously. In some embodiments, the anti-cancer agent is administered intermittently.
  • the TIGIT inhibitor is administered before the anti-cancer agent. In some embodiments, the TIGIT inhibitor is administered simultaneous with the anticancer agent. In some embodiments, the TIGIT inhibitor is administered after the anti-cancer agent.
  • the additional therapeutic agent is a checkpoint inhibitor.
  • methods of increasing the number of tissue-infiltrating ⁇ T cells in a mammalian subject recipient comprise (z) obtaining ⁇ T cells from tissue of a donor, (ii) expanding the ⁇ T cells ex vivo, ⁇ Hi) transplanting the expanded ⁇ T cells into a recipient, and (iv) administering at least one TIGIT inhibitor to the recipient.
  • the obtained and/or expanded ⁇ T cells are ⁇ 2 " .
  • the ⁇ 2 " cells are ⁇ 1 + or DN T cells.
  • ⁇ 2 " cells comprise both ⁇ 1 + and DN T cells.
  • the ⁇ T cells are obtained from hematopoietic tissue described herein in Section 6.3.1.1. [0124] In some embodiments, the ⁇ T cells are obtained from non-hematopoietic tissue, described herein in Section 6.3.1.2.
  • the TIGIT inhibitor is an inhibitor described herein in Section 6.6.
  • the ex vivo expanded V52 " ⁇ T cells were obtained by:
  • lymphocytes obtained from non-hematopoietic tissue of humans or non-human animals in the presence of interleukin-2 (IL-2) and/or interleukin-15 (IL-15), and not in direct contact with stromal or epithelial cells during culture.
  • IL-2 interleukin-2
  • IL-15 interleukin-15
  • the methods culture and expansion methods are selected from those described in Section 6.3 above.
  • the TIGIT inhibitor is selected from an antagonist of TIGIT activity, an antagonist of TIGIT interaction with PVR, an agent that inhibits and/or blocks the interaction of TIGIT with PVR, an agent that inhibits and/or blocks the interaction of TIGIT with PVRL2, an agent that inhibits and/or blocks the interaction of TIGIT with PVRL3, an agent that inhibits and/or blocks the intracellular signaling within a V52 " ⁇ T cell mediated by PVR binding to TIGIT, an agent that inhibits and/or blocks the intracellular signaling within a V52 " ⁇ T cell mediated by PVRL2 binding to TIGIT, an agent that inhibits and/or blocks the intracellular signaling within a V52 " ⁇ T cell mediated by PVLR3 binding to TIGIT, and combinations thereof.
  • the TIGIT inhibitor is an antagonist of TIGIT interaction with PVR.
  • the TIGIT inhibitor is an agent that inhibits and/or blocks the interaction of TIGIT with PVR.
  • the TIGIT inhibitor may block the interaction of PVR with TIGIT, leading to sequestration of TIGIT and increased binding of PVR to DNAM1.
  • the TIGIT inhibitor is an antibody that binds specifically to TIGIT or to PVR.
  • the TIGIT inhibitor is an antigen-binding fragment of an antibody that binds specifically to TIGIT or to PVR.
  • the TIGIT inhibitor is an antigen binding antibody fragment selected from Fab, Fab', F(ab') 2 , F d , F v , complementarity determining region (CDR) fragment, or single-chain antibody (scFv).
  • the TIGIT inhibitor is a single domain antibody or single-chain antibody.
  • the single domain antibody is a V H H domain.
  • the TIGIT inhibitor is an inhibitor described in
  • the TIGIT inhibitor is an inhibitor described in WO 2017/030823, WO 2016/028656,
  • the TIGIT inhibitor is a human antibody.
  • the human antibody has variable regions in which both the framework and CDR regions are sequences identical to those of human origin.
  • the TIGIT inhibitor is a humanized antibody.
  • the humanized antibody is a non-human (e.g., murine) antibody that is a chimeric antibody and contains minimal sequence derived from non-human immunoglobulin.
  • the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a HVR of a non- human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • donor antibody such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • FR residues of the human immunoglobulin variable domain are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. In an embodiment, the humanized antibodies do not comprise residues that are not found in the recipient antibody or in the donor antibody.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the Fc regions of the antibodies are modified.
  • the TIGIT inhibitor is a small molecule inhibitor. In certain aspects, the TIGIT inhibitor specifically binds TIGIT. In certain aspects, the TIGIT inhibitor binds PVR.
  • Methods of delivery include, but are not limited to, intra-arterial, intra-muscular, intravenous, intranasal, and oral routes.
  • Methods to enhance uptake can include, but are not limited to, encapsulation in liposomes, microparticles, microcapsules, receptor-mediated endocytosis, and the like.
  • the agents identified can be administered to subjects or individuals susceptible to or at risk of developing a variety of conditions benefiting from activation of ⁇ T cells.
  • the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject.
  • Therapeutic amounts are empirically determined and vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the agent. When delivered to an animal, the method is useful to further confirm efficacy of the agent.
  • in vivo administration is effected in one dose, continuously or intermittently throughout the course of treatment.
  • Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and vary with the composition used for therapy, the purpose of therapy, the target cell being treated, and the subject being treated. Single or multiple administrations are carried out with the dose level and pattern being selected by the treating physician.
  • Suitable dosage formulations and methods of administering the agents are readily determined by those of skill in the art.
  • the compounds are administered at about 0.01 mg/kg to about 200 mg/kg, more preferably at about 0.1 mg/kg to about 100 mg/kg, even more preferably at about 0.5 mg/kg to about 50 mg/kg.
  • the effective amount may be more or less than when the agent is used alone.
  • compositions can be administered orally, intranasally, parenterally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. They may also take the form of suspensions, solutions and emulsions of the active ingredient in aqueous diluents, nonaqueous diluents, syrups, granulates or powders.
  • the active ingredient in aqueous diluents, nonaqueous diluents, syrups, granulates or powders.
  • compositions can also contain other pharmaceutically active compounds or a plurality of compounds of the invention.
  • an agent of the present invention also referred to herein as the active ingredient, may be administered for therapy by any suitable route including, but not limited to, oral, rectal, nasal, topical (including, but not limited to, transdermal, aerosol, buccal and sublingual), vaginal, parental (including, but not limited to, subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It is also appreciated that the preferred route varies with the condition and age of the recipient, and the disease being treated.
  • the agent should be administered to achieve peak concentrations of the active compound at sites of disease. This may be achieved, for example, by the intravenous injection of the agent, optionally in saline, or orally administered, for example, as a tablet, capsule or syrup containing the active ingredient.
  • Desirable blood levels of the agent may be maintained by a continuous infusion to provide a therapeutic amount of the active ingredient within disease tissue.
  • the use of operative combinations is contemplated to provide therapeutic combinations requiring a lower total dosage of each component antiviral agent than may be required when each individual therapeutic compound or drug is used alone, thereby reducing adverse effects.
  • the present invention also includes methods involving co-administration of the TIGIT inhibitors described herein with one or more additional active agents. Indeed, it is a further aspect of this invention to provide methods for enhancing prior art therapies and/or pharmaceutical compositions by co-administering the agents of this invention.
  • the agents may be administered concurrently or sequentially.
  • the agents described herein are administered prior to the other active agent(s).
  • the pharmaceutical formulations and modes of administration may be any of those described above.
  • the two or more co-administered chemical agents, biological agents or radiation may each be administered using different modes or different formulations.
  • one or more agents provided herein and one or more therapeutic agents, TIGIT inhibitors, T-cell checkpoint inhibitors or anti-cancer agents are administered to a subject under one or more of the following
  • the TIGIT inhibitor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-oxidethyl-N-oxidethyl
  • the TIGIT inhibitor is administered prior to an additional TIGIT inhibitor, T-cell checkpoint inhibitor, therapeutic or anti-cancer agent, e.g., 0.5, 1 , 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks prior to the administration of therapeutic or anti-cancer agent.
  • the TIGIT inhibitor is administered after an additional TIGIT inhibitor, T-cell checkpoint inhibitor, therapeutic or anti-cancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1 , 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks after the administration of an additional TIGIT inhibitor, T-cell checkpoint inhibitor, therapeutic or anti-cancer agent.
  • the TIGIT inhibitor and an additional TIGIT inhibitor, T-cell checkpoint inhibitor, therapeutic or anti-cancer agent are administered concurrently but on different schedules, e.g., the TIGIT inhibitor is administered daily while the additional TIGIT inhibitor, T-cell checkpoint inhibitor, therapeutic or anti-cancer agent, is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In other embodiments, the TIGIT inhibitor is administered once a week while the additional TIGIT inhibitor, T-cell checkpoint inhibitor, therapeutic or anti-cancer agent is administered daily, once a week, once every two weeks, once every three weeks, or once every four weeks.
  • TIGIT inhibitors are administered in combination with an additional T-cell checkpoint inhibitor.
  • the T-cell checkpoint inhibitor or checkpoint inhibitor is any molecule that inhibits the activity of a T-cell checkpoint.
  • the T-cell checkpoint inhibitor inhibits a protein or gene that functions to inhibit or suppress the activation of a T-cell.
  • the T-cell checkpoint inhibitor inhibits the expression or activity of, but not limited to: A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA and combinations thereof.
  • TIGIT inhibitors are administered in combination with anticancer agents.
  • suitable therapeutic or anticancer agents are contemplated for use in the methods provided herein. Indeed, the methods provided herein can include but are not limited to, administration of numerous therapeutic agents such as: agents that induce apoptosis; polynucleotides (e.g., anti-sense, ribozymes, siR A); polypeptides (e.g., enzymes and antibodies); biological mimetics; alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g., antibodies conjugated with anticancer drugs, toxins, defensins), toxins; radionuclides;
  • therapeutic agents such as: agents that induce apoptosis; polynucleotides (e.g., anti-sense, ribozymes, siR A); polypeptides (e.g., enzymes and antibodies); biological mimetics;
  • biological response modifiers e.g., interferons (e.g., IFN-a) and interleukins (e.g., IL-2)
  • adoptive immunotherapy agents hematopoietic growth factors; agents that induce tumor cell differentiation (e.g., all-trans-retinoic acid); gene therapy reagents (e.g., antisense therapy reagents and nucleotides); tumor vaccines; angiogenesis inhibitors; proteasome inhibitors: NF- KB modulators; anti-CDK compounds; HDAC inhibitors; and the like.
  • therapeutic agents such as chemotherapeutic compounds and anticancer therapies suitable for co-administration with the disclosed compounds are known to those skilled in the art.
  • the TIGIT inhibitors for use in the methods described herein can be formulated in pharmaceutical compositions.
  • the ex vivo cultured, and optionally expanded, ⁇ T cells for use in certain of methods described herein may formulated as a medicament.
  • compositions comprising TIGIT inhibitors can comprise, in addition to one or more of the TIGIT inhibitors, one or more additional checkpoint inhibitors, and a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material can depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • compositions for oral administration can be in tablet, capsule, powder or liquid form.
  • a tablet can include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen- free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives can be included, as required.
  • the pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a "therapeutically effective amount” or “prophylactically effective amount”(as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual.
  • a therapeutically effective amount or “prophylactically effective amount”(as the case can be, although prophylaxis can be considered therapy)
  • prophylaxis can be considered therapy
  • the actual amount administered, and rate and time-course of administration will depend on the nature and severity of the cancer being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.
  • a composition can be administered alone or in combination with other treatments (e.g., additional TIGIT inhibitors, T-Cell checkpoint inhibitors and/or anti-cancer agents), either simultaneously or sequentially dependent upon the cancer to be treated.
  • additional TIGIT inhibitors e.g., additional TIGIT inhibitors, T-Cell checkpoint inhibitors and/or anti-cancer agents
  • PB Peripheral Blood
  • PBMCs Peripheral Blood Mononuclear Cells
  • Ficoll Ficoll-Paque PLUS, GE Healthcare Life Sciences
  • centrifugation 400 g for 20 minutes to separate red blood cells, blood plasma, and white lymphocytes/monocytes.
  • White blood cells were carefully harvested through a stripette and washed four times in cold PBS.
  • the PBMCs were used either immediately for immunostaining and phenotypic analysis of PB- derived ⁇ + T cells or cultured to have PB-derived ⁇ + T cell line for functional assays.
  • cells were resuspended in RPMI-1640 medium (Life Technologies) with 10% heat-inactivated foetal bovine serum (Life Technologies), L-glutamine (292 ⁇ g/ml; Life Technologies), penicillin (100 units/ml; Life Technologies), streptomycin (100 ⁇ g/ml; Life Technologies), N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES; 0.01 M; Life Technologies), sodium pyruvate (1 mM; Life Technologies), minimal essential media (MEM) non-essential amino acids (IX; Life Technologies) at a density of 1 x 10 6 per mL and supplemented with human recombinant IL-2 (100 IU/ml; PROLEUKIN®; Novartis
  • IL-15 human recombinant IL-15 (20 ng/ml; Biolegend).
  • Cells were transferred into a 24 well plate that was coated with a-pan ⁇ TCR monoclonal Antibody (mAb) (20 ⁇ g/ml; clone Bl; Biolegend) 90 minutes prior to cell transfer.
  • mAb monoclonal Antibody
  • Cells were grown for 14 days, at 37°C and 5% C0 2 , media changed ever 2-3 days and fresh cytokines added. Upon reaching confluence, cells were split 1 : 1.
  • ⁇ + T cells Under these conditions, after 14 days, the original minor population of ⁇ + T cells is normally highly activated through their TCR (as indicated by upregulation if CD69 and CD25) and largely enriched consisting of mainly ⁇ 2 + T cells but also ⁇ T cells (up to 30% of all ⁇ + T cells).
  • lymphocytes were counted by either; (1) trypan blue stain (0.4%) (Life Technologies) and haemocytometer, or (2) CASY® Model TT cell counter and analyzer (Roche).
  • IMDM Iscove's Modified Dulbecco's Medium
  • L-glutamine (292 ⁇ g/ml; Life Technologies), penicillin (100 units/ml; Life Technologies), streptomycin (100 ⁇ g/ml; Life Technologies), N-2-hydroxyethylpiperazine-N- 2-ethane sulfonic acid (HEPES; 0.01 M; Life Technologies), sodium pyruvate (1 mM; Life Technologies), minimal essential media (MEM) non-essential amino acids (lx; Life
  • the matrices and media were transferred to a 50 ml centrifuge tube (Corning) containing 10 ml Hanks Balanced Salt Solution (HBSS; Life Technologies) with 0.01 mM HEPES (up to 12 matrices/tube).
  • the matrices were rinsed with the cell suspension using a 10 ml pipette, and the cell suspension was passed through a 70 ⁇ filter (BD Biosciences) into a fresh 50 ml centrifuge tube. The rinsing of the matrices was repeated two additional times.
  • the media from the culture well was also aspirated and passed through a 70 ⁇ filter into a fresh 50 ml centrifuge tube.
  • Lymphocytes (0.5xl0 6 cells/well) were plated in a 96-U bottom well plate and washed in PBS. First, a live/dead staining was done with the Fixable Blue Dead Cell Stain Kit (Life Technologies) or the Fixable Viability Dye eFluor 770 NIR (eBioscience) according the manufacture instructions.
  • BV510 conjugated a-CD3 clone OKT3; Biolegend
  • PC7-conjugated a-pan ⁇ IMMU510 clone; Beckman Coulter
  • FITC- conjugated a-V51 TS8.2 clone; Life Technologies
  • PE-conjugated a-V52 B6 clone;
  • PB-derived ⁇ + T cell line or Skin-derived lymphocytes or sorted pan ⁇ + T cells were plated in a 96-flat bottom well plate with a density of 0.2x10 6 cells/well.
  • the plate was previously coated with different combination of the following proteins: a-CD3 Ab (5 ⁇ g/ml) (HIT3a clone; Biolegend) or its isotype control (MOPC-173; Biolegend), recombinant human PVR Fc Chimera Protein (rhPVR) (20 ⁇ g/ml) (Life Technologies), recombinant human MICA Fc Chimera Protein (rhMICA) (10 ⁇ g/ml) (R&D Systems) or their hlgGl control (Biolegend).
  • a-CD3 Ab 5 ⁇ g/ml
  • HIT3a clone HIT3a clone
  • MOPC-173 isotype control
  • rhPVR recombinant human PVR Fc Chimer
  • the blocking a-DNAMl Ab (DX11 clone; BD Biosciences) was added to the cell suspension (10 ⁇ g/ml).
  • the cells were stimulated for 6 hours in RPMI 10%> FCS supplemented with the intracellular protein transport inhibitor Brefeldin A (20 ⁇ g/ml)(Sigma-Aldrich), at 37°C and 5% C0 2 .
  • the cells were harvested and moved to a 96-U bottom well plate, extracellular stained and fixed as previously described for the phenotypic analysis.
  • the intracellular staining was performed in Permeabilization Buffer (eBioscience) according the manufacture instructions and the cytokine production was assessed using the following mAbs: eFluor450-conjugated ⁇ -IFNy (4S.B3; eBioscience) APC-conjugated a-TNFa
  • Pan ⁇ + T cells were purified from Skin-derived lymphocytes using a a-pan ⁇ mAb (IMMU510 clone; Beckman Coulter) and a FACS Aria cell sorting. They were then stained with the CellTrace Violet (CTV) Cell Proliferation Kit (Life Technologies) and cultured in a 96-flat bottom well plate for 1 week in complete medium supplemented with IL-2.
  • CTV CellTrace Violet
  • the plate was previously coated with different combination of the following proteins: a-CD3 Ab (50 ng/ml) (HIT3a clone; Biolegend) or its isotype control (MOPC-173; Biolegend), rhPVR (10 ⁇ g/ml) (Life Technologies) or its hlgGl control (Biolegend).
  • a-CD3 Ab 50 ng/ml
  • MOPC-173 isotype control
  • rhPVR 10 ⁇ g/ml
  • hlgGl control Biolegend
  • V51 + cells The initial population of V51 + cells was 1.12% of lymphocytes.
  • Cells were seeded at 2 x 10 5 cells/well into 96 well flat bottom plates (Corning) or at 2 x 106 cells/well into 24 well plates (Corning) for expansion and supplemented with factors at the following concentrations: IL-2 (100 U/ml), IL-15 (10 ng/ml), IL-4 (5 ng/ml) and IL-21 (10 ng/ml).
  • IL-2 100 U/ml
  • IL-15 10 ng/ml
  • IL-4 5 ng/ml
  • IL-21 10 ng/ml
  • Example 1 ⁇ + T cell subpopulations in human PBMC and skin
  • lymphocytes using somatic gene rearrangement to generate diverse surface receptors, B cells, ⁇ T cells and ⁇ T cells respectively. Only ⁇ T cells show compartmentalization, with V52 TCR positive cells dominating the human blood whilst T cells recombining other TCR genes (eg. V51, 53, 54, 55, 56, 57, 58) are predominantly found in human tissues such as the skin, the gut, the lungs, the liver and others ( Figure 1 A).
  • TCR genes eg. V51, 53, 54, 55, 56, 57, 58
  • Peripheral blood mononuclear cells from 11 donors and human skin derived lymphocytes from 27 donors were isolated as described above. Flow cytometry was performed using the following antibody-fluorochrome conjugates: V51-FITC, CD3-BV510 and V52-PE. Samples were also stained for viability using the eFluor 770 NIR live/dead dye. All T lymphocytes present were gated using the marker CD3, and within this population the presence of V52 + ⁇ T cells or V51 + ⁇ T cells made visible using the according antibodies. The results show that V52 TCR positive cells are dominating in the human blood with almost no V51 T cells being present.
  • PB-derived ⁇ + T cells and Skin-derived ⁇ + T cells were tested by FACS analysis for the presence of DNAM1 and TIGIT on their surface.
  • DNAM1 and TIGIT expression was evaluated on a pan ⁇ + T cell population.
  • DNAMl was expressed by both PB- derived ⁇ + T cells and Skin-derived ⁇ + T cells.
  • ⁇ - ⁇ + T cells expressed TIGIT only upon activation, obtained in this case with PHA treatment.
  • TIGIT was already expressed by unstimulated Skin-derived ⁇ + T cells.
  • DNAMl and TIGIT expression was assessed by staining PB-derived and Skin-derived lymphocytes with anti- ⁇ and anti-V02 Abs, in addition to the pan ⁇ Ab.
  • This staining strategy allowed to gate three different populations into the pan ⁇ + gate: ⁇ 1 + , ⁇ 2 + and V61 " V62 " (named here Double Negative, DN).
  • ⁇ - ⁇ + T cells are mostly represented by the ⁇ 2 + population, and in a smaller percentage by ⁇ 1 + or DN.
  • the ⁇ 2 + population largely expressed DNAMl, more than 70% in all donors, while only a small proportion, less than 25%, or none of them expressed TIGIT.
  • ⁇ 1 + and DN populations present in the PB expressed DNAMl, even if to a lower level compare to the ⁇ 2 + population, and shared TIGIT constitutive expression as the Skin-derived ⁇ + T cells.
  • Skin-derived ⁇ + T cells are composed by ⁇ 1 + and DN cells, and even in this second more detailed staining, we confirmed that ⁇ 1 + and DN cells constitutively expressed
  • DNAMl and TIGIT are expressed by more than 70% of ⁇ and DN cells in all donors, and TIGIT is expressed by more than 50%> of ⁇ 1 + cells.
  • DN T cells showed a more variable TIGIT expression because it is a heterogeneous population and it is represented by populations expressing TIGIT and populations not expressing TIGIT, in a different proportion in every donor.
  • Figure 3 shows ⁇ T cells derived either from PBMCs or skin tissue express DNAMl .
  • ⁇ T cells isolated from human PBMCs ( Figure 4) and lymphocytes isolated from human skin tissue ( Figure 5) were stained with anti-TIGIT antibodies and antibodies specific for either the ⁇ chain or ⁇ chain TCR and subjected to FACS analysis.
  • V52 + ⁇ T cells isolated form PBMCs expressed high levels of DNAM1 ( Figure 6) and low levels of TIGIT ( Figure 7) while V51 + ⁇ T cells expressed low levels of DNAMl ( Figure 6) and high levels of TIGIT ( Figure 7).
  • DN cells (V5T V52 " ) expressed moderate levels of DNAMl ( Figure 8) and high levels of TIGIT.
  • ⁇ ⁇ T cells and DN cells isolated from human skin leukocytes expressed high levels of DNAMl and TIGIT ( Figures 8 and 9).
  • Example 3 aCD3-induced cytokine production is inhibited by PVR in V51 + and DN ⁇ T cells.
  • soluble PVR was added to aCD3 -stimulated DN and V51 + ⁇ T cells isolated from either human PBMCs (figure 10 and 11) or human skin lymphocytes ( Figures 11). PB-derived and Skin-derived lymphocytes were stimulated for 6 hours by plate-bound aCD3 Ab in
  • FIG. 11 shows collective plots for IFNy and TNFa production in ⁇ T cells derived from PB and Skin
  • V51 and DN ⁇ T cells The aCD3-induced proliferation of V51 and DN ⁇ T cells was impaired in the presence of PVR compare to its control, further confirming the inhibitory role of the PVR/TIGIT axis on the TCR-induced activation of V51 + and DN T cells.
  • PVR when is not in the context of a TCR stimulation, doesn't work as an inhibitor through TIGIT but works as a costimulator through DNAM1, as demonstrated by the stimulation with PVR only: V51 + and DN T cells are induced to produce, even if in a small amount, IFNy and TNFa and this production is reverted by a blocking aDNAMl mAb ( Figures 15 and 16).
  • Example 6 PVR inhibitory effect is lost on V51 + and V53 + cells not expressing
  • Tissue-derived V51 + and V53 + cells cultured and expanded ex-vivo in the presence of IL-2 and IL-15 (culture method 1) expressed high amounts of TIGIT
  • V51 + and V53 + cells cultured in the presence of IL-2, IL-4, IL-15 and IL-21 (culture method 2), expressed low levels of TIGIT
  • Figure 18 FACS analysis of tissue-derived V51 + and V53 + cells from individual donors cultured in the presence of IL-2, IL-4, IL-15 and IL-21, demonstrated that PVR inhibition of INFy and TNFa production was lost in V51 + and V53 + cells expressing low levels of TIGIT ( Figures 19 and 20).
  • the TCR stimulation is a strong activator of V51+ and DN T cells, in terms of production of inflammatory cytokines like IFNy and TNFa but also in terms of proliferation.
  • the TCR-induced activation of V51 + and DN T cells is normally kept under control by the PVR/TIGIT axis.
  • TIGIT is constitutively expressed by PB- and Skin-derived V51 + and DN T cells.
  • the inhibitory receptor TIGIT like has been extensively demonstrated, has greater affinity for its ligand PVR compare to the activator receptor DNAM1.
  • PVR preferentially binds TIGIT and triggers its inhibitory signaling into the cells dampening the activation of V51 + and DN T cells.
  • PVR is available to bind the costimulatory receptor DNAM1 and help the activation of V51 + and DN T cells.

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Abstract

L'invention concerne des méthodes d'activation de lymphocytes T Vδ2- gamma-delta (lymphocytes T γδ) chez un sujet mammifère, comprenant l'antagonisme de l'activité TIGIT de lymphocytes T Vδ2- γδ. L'invention concerne également des méthodes de traitement de cancers à l'aide de lymphocytes T γδ infiltrés dans un tissu et des méthodes de traitement de cancers consistant à administrer des compositions pharmaceutiques comprenant des lymphocytes T Vδ2- γδ.
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WO2022221506A1 (fr) * 2021-04-16 2022-10-20 Acepodia Biotechnologies Ltd. Nouvelles compositions enrichies en lymphocytes t gamma delta, procédés de préparation et utilisations associées
US11643447B2 (en) 2019-01-07 2023-05-09 Shattuck Labs, Inc. Heterodimeric proteins for modulating gamma delta T cells
US11718669B2 (en) 2021-05-04 2023-08-08 Agenus Inc. Anti-TIGIT and anti-CD96 antibodies
US12077586B2 (en) 2018-09-19 2024-09-03 LAVA Therapeutics N.V. Bispecific antibodies for use in the treatment of hematological malignancies
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US12110328B2 (en) 2015-01-27 2024-10-08 LAVA Therapeutics N.V. Single domain antibodies targeting CD1d
US11021537B2 (en) 2017-05-01 2021-06-01 Agenus Inc. Anti-TIGIT antibodies and methods of use thereof
US12077586B2 (en) 2018-09-19 2024-09-03 LAVA Therapeutics N.V. Bispecific antibodies for use in the treatment of hematological malignancies
WO2020095058A1 (fr) * 2018-11-08 2020-05-14 GammaDelta Therapeutics Limited Procédés d'isolement et de multiplication de cellules
US10988517B2 (en) 2019-01-07 2021-04-27 Shattuck Labs, Inc. Heterodimeric proteins for modulating gamma delta T cells
US11098093B2 (en) 2019-01-07 2021-08-24 Shattuck Labs, Inc. Heterodimeric proteins for modulating gamma delta T cells
US11643447B2 (en) 2019-01-07 2023-05-09 Shattuck Labs, Inc. Heterodimeric proteins for modulating gamma delta T cells
CN110157686A (zh) * 2019-05-22 2019-08-23 南京大学 一种新型的免疫检查点激活免疫共刺激的复制型溶瘤腺病毒及其构建方法和应用
CN110157686B (zh) * 2019-05-22 2022-06-21 南京惟亚德生物医药有限公司 一种免疫检查点激活免疫共刺激的复制型溶瘤腺病毒及其构建方法和应用
WO2022221506A1 (fr) * 2021-04-16 2022-10-20 Acepodia Biotechnologies Ltd. Nouvelles compositions enrichies en lymphocytes t gamma delta, procédés de préparation et utilisations associées
US11718669B2 (en) 2021-05-04 2023-08-08 Agenus Inc. Anti-TIGIT and anti-CD96 antibodies

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