WO2020146772A1 - Régulation de la neuritine de l'anergie des lymphocytes t et de la fonction des lymphocytes t régulateurs - Google Patents

Régulation de la neuritine de l'anergie des lymphocytes t et de la fonction des lymphocytes t régulateurs Download PDF

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Publication number
WO2020146772A1
WO2020146772A1 PCT/US2020/013151 US2020013151W WO2020146772A1 WO 2020146772 A1 WO2020146772 A1 WO 2020146772A1 US 2020013151 W US2020013151 W US 2020013151W WO 2020146772 A1 WO2020146772 A1 WO 2020146772A1
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cells
inhibitor
cell
neuritin
tumor
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PCT/US2020/013151
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English (en)
Inventor
Drew M. Pardoll
Hong Yu
Joseph Barbi
Fan Pan
Charles G. DRAKE
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The Johns Hopkins University
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Publication of WO2020146772A1 publication Critical patent/WO2020146772A1/fr

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    • 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
    • C07K16/2818—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 against CD28 or CD152
    • 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
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • 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

Definitions

  • compositions for the abrogation of suppression of an antigen specific immune response comprise neuritin inhibitors and checkpoint inhibitors.
  • Methods for treating cancer or other disorders in which an immune response has been suppressed include the administration of these compositions.
  • Immune homeostasis is achieved through both central and peripheral tolerance mechanisms. Thymic central tolerance shapes the peripheral T cell pool by eliminating most high-affinity self-reactive T cells. However, central tolerance is incomplete and thus, a significant number of peripheral CD4 + and CD8 + cells are self-reactive, displaying a broad TCR repertoire h These self-reactive T cells are kept in a quiescent state through peripheral tolerance mechanisms, including“ignorance”, peripheral deletion, induction of T cell anergy and suppression by regulatory T cells (Treg) 2 4 . Understanding these tolerance-promoting mechanisms and their restraint of self-reactive T cells is not only important for autoimmune disease prevention and therapy, it also has increasing implications for cancer immunotherapy.
  • Embodiments of the invention are directed to compositions which abrogate anergic T cell and T regulatory cell functions, thereby allowing for an effective immune response to antigens.
  • Methods of treating cancer are provided that include the use of these compositions.
  • a method of treating cancer comprises administering to a subject, a therapeutically effective amount of a neuritin inhibitor, wherein the neuritin inhibitor abrogates anergic T cell and/or T regulatory cell functions.
  • a method of inducing a tumor specific immune response in a subject comprises administering to a subject, a therapeutically effective amount of a neuritin inhibitor, wherein the neuritin inhibitor abrogates anergic T cell and/or T regulatory cell functions.
  • a method of abrogating T cell anergy comprises administering to a subject in need thereof, a therapeutically effective amount of a neuritin inhibitor and one or more checkpoint inhibitors.
  • a method of modulating T regulatory (Treg) cell function comprises administering to a subject in need thereof, a therapeutically effective amount of a neuritin inhibitor and one or more checkpoint inhibitors.
  • a method of inducing a tumor specific immune response in a subject comprises isolating T cells from a biological sample from the subject, culturing the T cells with an effective amount of a neuritin inhibitor and one or more checkpoint inhibitors, wherein the neuritin inhibitor abrogates anergic T cell and/or T regulatory cell function or activity, adoptively transferring the T cells to the subject thereby, inducing the tumor specific immune response.
  • a method of inducing a tumor specific immune response in a subject comprises administering to a subject, a therapeutically effective amount of a neuritin inhibitor and one or more checkpoint inhibitors, wherein the neuritin inhibitor abrogates anergic T cell and/or T regulatory cell function or activity, thereby, inducing the tumor specific immune response.
  • a tumor specific antigen is co administered to the subject.
  • a method of stimulating an immune response in a subject comprises isolating immune cells, e.g. T cells; contacting the immune cells with a pharmaceutical composition comprising the neuritin inhibitors and/or checkpoint inhibitors embodied herein and/or tumor antigens or tumor cells from the subject; reinfusing the immune cells into the subject; thereby, stimulating the immune response in a subject.
  • the immune cells comprise autologous, haplo-identical, haplotype matched or combinations thereof.
  • the immune cells are derived from autologous or allogeneic stem cells.
  • the immune cells comprise NK cells, T cells, stem cell memory T cells, activated NK (aNK) cells, chimeric antigen receptor- NK (CAR-NK) cells, chimeric antigen receptor-T (CAR-T) cells, or combinations thereof.
  • one or more adjuvants are optionally administered with the soluble fusion protein complexes embodied herein.
  • the adoptively transferred immune cells or pharmaceutical composition comprising the neuritin inhibitors and/or checkpoint inhibitors are administered at least one time per month, e.g., twice per month, once per week, twice per week, once per day, twice per day, every 8 hours, every 4 hours, every 2 hours, or every hour.
  • Suitable modes of administration for the adoptively transferred immune cells include systemic administration, intravenous administration, or local administration. Suitable modes of administration for the
  • compositions include systemic administration, intravenous administration, local administration, subcutaneous administration, intramuscular administration, intratumoral administration, inhalation, and intraperitoneal administration.
  • the effective amounts of the adoptively transferred immune cells are between 1 x 10 4 cells/kg and 1 x 10 10 cells/kg.
  • the patient is pretreated or preconditioned to facilitate engraftment or survival of the adoptively transferred cells.
  • preconditioning include treatment with cyclophosphamide and fludarabine.
  • the patient may be treated with agents that promote activation, survival or persistence of the adoptively transferred cells pre- and/or post-cell transfer. Examples of such treatment include use of IL-2, IL-15 or other immunostimulatory agents.
  • Other therapeutic approaches of known in the field of adoptive cell therapy i.e., including but not limited to allogeneic, autologous, haploidentical, DLI, stem cell, NK92 -based and CAR NK therapies may also be used in the methods herein.
  • the neuritin inhibitor comprises: an anti-neuritin specific antibody or fragments thereof, antisense oligonucleotides, an siRNA, gene editing agents, nucleases, oligonucleotides, polynucleotides, small molecules, peptides, peptidomimetics, natural ligands and derivatives of natural ligands, oligonucleotides or combinations thereof.
  • one or more checkpoint inhibitors are administered, the checkpoint inhibitors comprising an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4, TGFR-b or combinations thereof.
  • the checkpoint inhibitor comprises an inhibitor of PD-1, PD-L1, CTLA4 or combinations thereof.
  • the checkpoint inhibitor comprises: an antibody or fragments thereof, antisense oligonucleotides, an siRNA, gene editing agents, nucleases, oligonucleotides, polynucleotides, small molecules, peptides, peptidomimetics, natural ligands and derivatives of natural ligands, oligonucleotides, organic or inorganic molecules, enzymes, interfering RNAs or
  • the present method further comprise administration of: anti- CD25 antibodies, inhibitors of molecules associated with T cell anergy development, Toll like receptor 2 (TLR2) agonists, 4- IBB agonists, 0X40 agonists, tumor necrosis factor receptor 2 (TNFR2) agonists, cytokines or combinations thereof.
  • TLR2 Toll like receptor 2
  • TNFR2 tumor necrosis factor receptor 2
  • the cytokines comprise: interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL-4), interleukin- 7 (IL-7), interleukin- 15 (IL-15), interleukin-21 (IL- 21), tumor necrosis factor alpha (TNFa) or combinations thereof.
  • IL-2 interleukin-2
  • IL-6 interleukin- 6
  • IL-4 interleukin- 4
  • IL-7 interleukin- 7
  • IL-15 interleukin- 15
  • IL- 21 interleukin-21
  • TNFa tumor necrosis factor alpha
  • molecules associated with T cell anergy development comprise: Cbl-b, TRAF6 or SHP-1.
  • a pharmaceutical composition comprises a therapeutically effective amount of a neuritin inhibitor and one or more checkpoint inhibitors.
  • a pharmaceutical composition comprises an anti-neuritin inhibitor and a first and second checkpoint inhibitor.
  • the first and second checkpoint inhibitors are distinct agents.
  • the checkpoint inhibitors comprise an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4, and/or TGFR-b or combinations thereof.
  • the checkpoint inhibitor comprises an inhibitor of PD-1, PD-L1, CTLA4 or combinations thereof.
  • the first checkpoint inhibitor is an inhibitor of PD-1 and the second checkpoint inhibitor is an inhibitor of CTLA4.
  • the pharmaceutical composition is administered intra muscularly, systemically, intratumorally, intraperitoneally or combinations thereof.
  • the methods described herein inhibit the growth or progression of cancer, e.g., a tumor, or a viral infection in a subject.
  • the methods described herein inhibit the growth of a tumor by at least 1%, e.g., by at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%, relative an untreated control (e.g. tumor cells not treated with a neuritin inhibitor).
  • an untreated control e.g. tumor cells not treated with a neuritin inhibitor.
  • the methods described herein reduce the size of a tumor by at least 1 mm in diameter, e.g., by at least 2 mm in diameter, by at least 3 mm in diameter, by at least 4 mm in diameter, by at least 5 mm in diameter, by at least 6 mm in diameter, by at least 7 mm in diameter, by at least 8 mm in diameter, by at least 9 mm in diameter, by at least 10 mm in diameter, by at least 11 mm in diameter, by at least 12 mm in diameter, by a least 13 mm in diameter, by at least 14 mm in diameter, by at least 15 mm in diameter, by at least 20 mm in diameter, by at least 25 mm in diameter, by at least 30 mm in diameter, by at least 40 mm in diameter, by at least 50 mm in diameter or more, relative to an untreated control (e.g. tumor cells not treated with a neuritin inhibitor).
  • the subject has had the bulk of the tumor resected.
  • the subject is preferably a mammal in need of such treatment, e.g., a subject that has been diagnosed with cancer or a viral infection, or a predisposition thereto, i.e., at risk of developing cancer or a viral infection.
  • the mammal is any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a horse, as well as livestock or animals grown for food consumption, e.g., cattle, sheep, pigs, chickens, and goats.
  • the mammal is a human.
  • Modes of administration include intravenous, systemic, oral, rectal, topical, intraocular, buccal, intravaginal, intracistemal, intracerebroventricular, intratracheal, nasal, transdermal, within/on implants, or parenteral routes.
  • parenteral includes subcutaneous, intrathecal, intravenous, intramuscular, intraperitoneal, or infusion.
  • Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. They could, however, be preferred in emergency situations.
  • Compositions comprising a composition of the invention can be added to a physiological fluid, such as blood. Oral administration can be preferred for prophylactic treatment because of the convenience to the patient as well as the dosing schedule. Parenteral modalities
  • subcutaneous or intravenous may be preferable for more acute illness, or for therapy in patients that are unable to tolerate enteral administration due to gastrointestinal intolerance, ileus, or other concomitants of critical illness. Inhaled therapy is also provided.
  • the composition is administered in a form selected from the group consisting of pills, capsules, tablets, granules, powders, salts, crystals, liquids, serums, syrups, suspensions, gels, creams, pastes, films, patches, and vapors.
  • the methods described herein are used in conjunction with one or more agents or a combination of additional agents, e.g., an anti-cancer agent.
  • Suitable agents include current pharmaceutical and/or surgical therapies for an intended application, such as, for example, cancer.
  • the methods described herein can be used in conjunction with one or more chemotherapeutic or anti-neoplastic agents, e.g., chemotherapy, targeted cancer therapy, cancer vaccine therapy, or immunotherapy.
  • the additional chemotherapeutic agent is radiotherapy.
  • the chemotherapeutic agent is a cell death-inducing agent.
  • Treatment with immunotherapeutic methods or compositions described herein may be a stand-alone treatment, or may be one component or phase of a combination therapy regime, in which one or more additional therapeutic agents are also used to treat the patient.
  • “about” can mean within 1 or more than 1 standard deviation, per the practice in the art.
  • “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value or range.
  • the term can mean within an order of magnitude within 5-fold, and also within 2-fold, of a value.
  • the term“agent” is meant to encompass any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent capable of preventing, ameliorating, or treating a disease or other medical condition.
  • the term includes small molecule compounds, antisense oligonucleotides, siRNA reagents, antibodies, antibody fragments bearing epitope recognition sites, such as Fab, Fab’, F(ab’)2 fragments, Fv fragments, single chain antibodies, antibody mimetics (such as DARPins, affibody molecules, affilins, affitins, anticalins, avimers, fynomers, Kunitz domain peptides and monobodies), peptoids, aptamers; enzymes, peptides organic or inorganic molecules, natural or synthetic compounds and the like.
  • An agent can be assayed in accordance with the methods of the invention at any stage during clinical trials, during pre-trial testing, or following FDA-approval.
  • the term“agonist” refers to an agent that binds to a receptor and activates the receptor to produce a biological response. Whereas an agonist causes an action, an antagonist blocks the action of the agonist, and an inverse agonist causes an action opposite to that of the agonist.
  • ameliorate is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • anti-plastic agent is used herein to refer to agents that have the functional property of inhibiting a development or progression of a neoplasm in a human, particularly a malignant (cancerous) lesion. Inhibition of metastasis is frequently a property of antineoplastic agents.
  • cancer or“tumor” or“hyperproliferative” refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain
  • Such cells exhibit such characteristics in part or in full due to the expression and activity of immune checkpoint inhibitors, such as PD-1, PD-L1, PD-L2, and/or CTLA-4.
  • Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell.
  • the term“cancer” includes premalignant as well as malignant cancers.
  • Cancers include, but are not limited to, B cell cancer, e.g., multiple myeloma, Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematologic tissues, and the like.
  • the heavy chain diseases such as, for
  • leukemias e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non- Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.
  • leukemias e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia);
  • cancers are epithelial in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.
  • the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer.
  • the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma.
  • the epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.
  • checkpoint inhibitor means a group of molecules on the cell surface of CD4 + and/or CD8 + T cells that fine-tune immune responses by down-modulating or inhibiting an anti-tumor immune response.
  • Immune checkpoint proteins are well known in the art and include, without limitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7- H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-
  • Anti-immune checkpoint inhibitor therapy refers to the use of agents that inhibit immune checkpoint inhibitors. Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer.
  • agents useful for inhibiting immune checkpoint inhibitors include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and/or inactivate or inhibit immune checkpoint proteins, or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and/or activity of immune checkpoint inhibitor nucleic acids, or fragments thereof.
  • Exemplary agents for upregulating an immune response include antibodies against one or more immune checkpoint inhibitor proteins block the interaction between the proteins and its natural receptor(s); a non activating form of one or more immune checkpoint inhibitor proteins (e.g., a dominant negative polypeptide); small molecules or peptides that block the interaction between one or more immune checkpoint inhibitor proteins and its natural receptor(s); fusion proteins (e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fe portion of an antibody or immunoglobulin) that bind to its natural receptor(s); nucleic acid molecules that block immune checkpoint inhibitor nucleic acid transcription or translation; and the like.
  • a non activating form of one or more immune checkpoint inhibitor proteins e.g., a dominant negative polypeptide
  • small molecules or peptides that block the interaction between one or more immune checkpoint inhibitor proteins and its natural receptor(s)
  • fusion proteins e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fe portion of an antibody or immunoglobulin
  • agents can directly block the interaction between the one or more immune checkpoint inhibitors and its natural receptor(s) (e.g., antibodies) to prevent inhibitory signaling and upregulate an immune response.
  • agents can indirectly block the interaction between one or more immune checkpoint proteins and its natural receptor(s) to prevent inhibitory signaling and upregulate an immune response.
  • a soluble version of an immune checkpoint protein ligand such as a stabilized extracellular domain can binding to its receptor to indirectly reduce the effective concentration of the receptor to bind to an appropriate ligand.
  • anti-PD-1 antibodies, anti-PD-Ll antibodies, and anti-CTLA-4 antibodies either alone or used in combination.
  • cancer therapy refers to a therapy useful in treating cancer.
  • anti-cancer therapeutic agents include, but are not limited to, surgery, chemotherapeutic agents, immunotherapy, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti- angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, such as anti-HER-2 antibodies (e.g., HERCEPTINTM), anti-CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (TARCEVATM)), platelet derived growth factor inhibitors (e.g., GLEEVECTM (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the HER-2 antibodies (e.g.
  • A“chemotherapeutic agent” is a chemical compound useful in the treatment of cancer.
  • co-administer refers to the simultaneous presence of two active agents in the blood of an individual. Active agents that are co- administered can be concurrently or sequentially delivered.
  • the terms“comprising,”“comprise” or“comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements— or, as appropriate, equivalents thereof— and that other elements can be included and still fall within the scope/definition of the defined item, composition, apparatus, method, process, system, etc.
  • the phrase“consisting essentially of’ refers to the genera or species of active pharmaceutical agents included in a method or composition, as well as any inactive carrier or excipients for the intended purpose of the methods or compositions.
  • control refers to any reference standard suitable to provide a comparison to the expression products in the test sample.
  • the control comprises obtaining a“control sample” from which expression product levels are detected and compared to the expression product levels from the test sample.
  • a control sample may comprise any suitable sample, including but not limited to a sample from a control cancer patient (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the cancer patient, cultured primary cells/tissues isolated from a subject such as a normal subject or the cancer patient, adjacent normal cells/tissues obtained from the same organ or body location of the cancer patient, a tissue or cell sample isolated from a normal subject, or a primary cells/tissues obtained from a depository.
  • control may comprise a reference standard expression product level from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, survival for one, two, three, four years, etc.) or receiving a certain treatment (for example, standard of care cancer therapy).
  • a certain outcome for example, survival for one, two, three, four years, etc.
  • a certain treatment for example, standard of care cancer therapy
  • control samples and reference standard expression product levels can be used in combination as controls in the methods of the present invention.
  • control may comprise normal or non-cancerous cell/tissue sample.
  • control may comprise an expression level for a set of patients, such as a set of cancer patients, or for a set of cancer patients receiving a certain treatment, or for a set of patients with one outcome versus another outcome.
  • the specific expression product level of each patient can be assigned to a percentile level of expression, or expressed as either higher or lower than the mean or average of the reference standard expression level.
  • control may comprise normal cells, cells from patients treated with combination chemotherapy, and cells from patients having benign cancer.
  • control may also comprise a measured value for example, average level of expression of a particular gene in a population compared to the level of expression of a housekeeping gene in the same population.
  • control comprises a ratio transformation of expression product levels, including but not limited to determining a ratio of expression product levels of two genes in the test sample and comparing it to any suitable ratio of the same two genes in a reference standard; determining expression product levels of the two or more genes in the test sample and determining a difference in expression product levels in any suitable control; and determining expression product levels of the two or more genes in the test sample, normalizing their expression to expression of housekeeping genes in the test sample, and comparing to any suitable control.
  • control comprises a control sample which is of the same lineage and/or type as the test sample.
  • control may comprise expression product levels grouped as percentiles within or based on a set of patient samples, such as all patients with cancer.
  • a control expression product level is established wherein higher or lower levels of expression product relative to, for instance, a particular percentile, are used as the basis for predicting outcome.
  • a control expression product level is established using expression product levels from cancer control patients with a known outcome, and the expression product levels from the test sample are compared to the control expression product level as the basis for predicting outcome.
  • the methods of the invention are not limited to use of a specific cut-point in comparing the level of expression product in the test sample to the control.
  • an“effective amount,”“therapeutically effective amount” or “effective dose” is an amount of a composition (e.g., a therapeutic composition or agent) that produces at least one desired therapeutic effect in a subject, such as preventing or treating a target condition or beneficially alleviating a symptom associated with the condition.
  • the term“immune cells” generally includes white blood cells
  • leukocytes which are derived from hematopoietic stem cells (HSC) produced in the bone marrow“Immune cells” includes, e.g., lymphocytes (T cells, B cells, natural killer (NK) cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells).
  • HSC hematopoietic stem cells
  • immune response includes T cell mediated and/or B cell mediated immune responses.
  • exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity.
  • immune response includes immune responses that are indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages.
  • the term“in combination” in the context of the administration of a therapy to a subject refers to the use of more than one therapy for therapeutic benefit.
  • the term“in combination” in the context of the administration can also refer to the prophylactic use of a therapy to a subject when used with at least one additional therapy.
  • the use of the term“in combination” does not restrict the order in which the therapies (e.g., a first and second therapy) are administered to a subject.
  • a therapy can be administered prior to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject which had, has, or is susceptible to cancer.
  • the therapies are administered to a subject in a sequence and within a time interval such that the therapies can act together.
  • the therapies are administered to a subject in a sequence and within a time interval such that they provide an increased benefit than if they were administered otherwise. Any additional therapy can be administered in any order with the other additional therapy.
  • “Mammal” covers warm blooded mammals that are typically under medical care (e.g., humans and domesticated animals). Examples include feline, canine, equine, bovine, and human, as well as just human.
  • modulating refers to an increase or decrease in an adaptive immune system response. In a preferred embodiment, this relates to an increased, up-regulated or enhanced adaptive immune system response.
  • An effective amount of an immunomodulatory agent is an amount that when applied or administered in accordance to the techniques herein is sufficient to modulate, preferably up-regulate, an adaptive immune system response.
  • “patient,”“subject,” and“individual” may be used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice and rats).
  • “Pharmaceutical agent,” also referred to as a“drug,” or“therapeutic agent” is used herein to refer to an agent that is administered to a subject to treat a disease, disorder, or other clinically recognized condition that is harmful to the subject, or for prophylactic purposes, and has a clinically significant effect on the body to treat or prevent the disease, disorder, or condition.
  • Therapeutic agents include, without limitation, agents listed in the United States Pharmacopeia (USP), Goodman and Gilman's The Pharmacological Basis of Therapeutics, 12 th Ed., McGraw Hill, 2001; Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw- Hill/ Appleton & Lange; 8 th edition (Sep. 21, 2000); Physician's Desk Reference (Thomson Publishing), and/or The Merck Manual of Diagnosis and Therapy, 18 th ed. (2006), or the 19 th ed (2011), Robert S. Porter, MD., Editor-in-chief and Justin L.
  • the terms“preventing” and grammatical variants thereof refer to an approach for preventing the development of, or altering the pathology of, a condition, disease or disorder. Accordingly,“prevention” may refer to prophylactic or preventive measures.
  • beneficial or desired clinical results include, but are not limited to, prevention or slowing of symptoms, progression or development of a disease, whether detectable or undetectable.
  • a subject e.g., a human
  • the term“prevention” includes slowing the onset of disease relative to the absence of treatment, and is not necessarily meant to imply permanent prevention of the relevant disease, disorder or condition.
  • “preventing” or“prevention” of a condition may in certain contexts refer to reducing the risk of developing the condition, or preventing or delaying the development of symptoms associated with the condition.
  • response refers to an anti-cancer response, e.g. in the sense of reduction of tumor size or inhibiting tumor growth.
  • the terms can also refer to an improved prognosis, for example, as reflected by an increased time to recurrence, which is the period to first recurrence censoring for second primary cancer as a first event or death without evidence of recurrence, or an increased overall survival, which is the period from treatment to death from any cause.
  • To respond or to have a response means there is a beneficial endpoint attained when exposed to a stimulus. Alternatively, a negative or detrimental symptom is minimized, mitigated or attenuated on exposure to a stimulus. It will be appreciated that evaluating the likelihood that a tumor or subject will exhibit a favorable response is equivalent to evaluating the likelihood that the tumor or subject will not exhibit favorable response (i.e., will exhibit a lack of response or be non-responsive).
  • RNA interfering agent is defined as any agent which interferes with or inhibits expression of a target gene by RNA interference (RNAi).
  • RNA interfering agents include, but are not limited to, nucleic acid molecules including RNA molecules which are homologous to the target gene of the invention, or a fragment thereof, short interfering RNA (siRNA), and small molecules which interfere with or inhibit expression of a target nucleic acid by RNA interference (RNAi).
  • RNA interference (RNAi)” is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target nucleic acid results in the sequence specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene (see Cobum, G.
  • PTGS sequence specific degradation or specific post-transcriptional gene silencing
  • RNA is double stranded RNA (dsRNA).
  • dsRNA double stranded RNA
  • RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes processive cleavage of long dsRNA into double- stranded fragments termed siRNAs.
  • siRNAs are incorporated into a protein complex that recognizes and cleaves target mRNAs.
  • RNAi can also be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silence the expression of target nucleic acids.
  • nucleic acid molecules e.g., synthetic siRNAs or RNA interfering agents
  • “inhibition of target nucleic acid expression” or“inhibition of marker gene expression” includes any decrease in expression or protein activity or level of the target nucleic acid or protein encoded by the target nucleic acid. The decrease may be of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the expression of a target nucleic acid or the activity or level of the protein encoded by a target nucleic acid which has not been targeted by an RNA interfering agent.
  • targeted therapy refers to administration of agents that selectively interact with a chosen biomolecule to thereby treat cancer, e.g. immunotherapy targeting tumor antigens.
  • the term“therapeutically effective regimen” refers to a regimen for dosing, timing, frequency, and duration of the administration of one or more therapies for the treatment and/or management of cancer or a symptom thereof.
  • the regimen achieves one, two, three, or more of the following results: (1) a stabilization, reduction or elimination in the cancer cell population; (2) a stabilization or reduction in the growth of a tumor or neoplasm; (3) an impairment in the formation of a tumor; (4) eradication, removal, or control of primary, regional and/or metastatic cancer; (5) a reduction in mortality; (6) an increase in disease-free, relapse-free, progression-free, and/or overall survival, duration, or rate; (7) an increase in the response rate, the durability of response, or number of patients who respond or are in remission; (8) a decrease in hospitalization rate, (9) a decrease in hospitalization lengths, (10) the size of the tumor is maintained and does not increase or increases by less than 10%, preferably less than
  • “treating” or“treatment” and grammatical variants thereof refer to an approach for obtaining beneficial or desired clinical results.
  • the term may refer to slowing the onset or rate of development of a condition, disorder or disease, reducing or alleviating symptoms associated with it, generating a complete or partial regression of the condition, or some combination of any of the above.
  • beneficial or desired clinical results include, but are not limited to, reduction or alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.“Treatment” can also mean prolonging survival relative to expected survival time if not receiving treatment.
  • a subject e.g., a human
  • treatment includes inhibition or reduction of an increase in severity of a pathological state or symptoms relative to the absence of treatment, and is not necessarily meant to imply complete cessation of the relevant disease, disorder or condition.
  • untargeted therapy refers to administration of agents that do not selectively interact with a chosen biomolecule yet treat cancer.
  • Representative examples of untargeted therapies include, without limitation, chemotherapy, gene therapy, and radiation therapy.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • FIGS. 1A-1H are a series of graphs and plots demonstrating Nrnl expression in anergic T cells and Treg.
  • FIGS. 1A-1D Nml expression in anergic CD4 cells.
  • Nml mRNA measurement by qRT-PCR FIG. 1A among 6.5 T cells recovered from C3HA host and wt host infected with VacHA virus (n>3 per group of mice) at various times after transfer;
  • FIG. 1A among 6.5 T cells recovered from C3HA host and wt host infected with VacHA virus (n>3 per group of mice) at various times after transfer
  • FIG. IB among AE7 cells stimulated with plate bound anti-CD3 in the presence (activation) or absence (anergy) of anti-CD28 for the indicated times (O/
  • FIG. ID Detection of Nml expression by anti-Nml antibody staining among CD4 + CD44 + Foxp3- FR4 hi CD73 hi cells from wt Nml +/+ and NrnT /_ splenocytes.
  • FIGS. 1E-1H Nml expression by Treg.
  • FIG. IF Nml expression in CD4 + CD44 low /CD62L high cTreg and CD4 + CD44 hlgh /CD62L low eTreg and upon activation respectively.
  • FIG. 1H Expression of neuritin message by cTreg and eTreg isolated from the peripheral blood of healthy human donors. Data are presented as mean +/- SEM. Data are representative of 2-3 independent experiments.
  • FIGS. 2A-2K are a series of graphs demonstrating that Nrnl deficiency affects CD4 T cells anergy development and pTreg cell conversion in vivo.
  • FIGS. 2A-2C Susceptibility of Nml 7- and Nml +/_ CD4 cells to suppression by Treg and anergy development.
  • FIG. 2A is a schematic representation showing an experimental outline: 2xl0 6 Thyl.l + Nml 7- or Nml +/_ CD4 OTII T cells were co-transferred with 4-5xl0 5 Thyl.2 + Thyl.l- wt Treg cells into TCRa /_ mice. Cells were recovered on day 13 post transfer.
  • FIG. 2B Proportions of CD4 cells
  • FIG. 2C OTII cell numbers recovered from recipient lymph node and spleen
  • FIG. 2D IF2 secretion from OTII cells upon ex vivo stimulation with OVA peptide.
  • FIG. 2E Foxp3 + cell percentage among Thyl.l + Nrnl 7- or Nrnl +/ CD4 cells.
  • FIGGS.2F-2K Comparison of Nml 7- and Nrnl +/ CD45.2 + T cell response to self-antigen in the absence of Treg cells in CD45.1 wt host.
  • FIGS. 2F Experimental outline: 6xl0 6 CD45.2 + T cells from Nml 7- or Nrnl +/ FDG mice was transferred into CD45.1 congenic wt FDG mice on day 0. Host mice were treated with DT at lpg/mouse i.p. for 3 days to delete all Treg cells both from wt host and from transferred Nml 7- and Nml +/ T cells. Cells were analyzed on dayl l after transfer.
  • FIG. 2G Foxp3 percentage
  • FIG. 2H CD4 + CD44 + FR4 hl CD73 hl cell proportion among adoptive transferred CD45.2 + Nrn l 7 - or Nml +/ CD4 cells and wt host CD45.1+ CD4 cells.
  • FIGS 3A-3J are a series of plots, graphs and photographs of histological stains, demonstrating that loss of Nml affect Treg cell suppression function.
  • FIG. 3 A Acquisition of eTreg traits by in vitro stimulation of Nml +/_ and Nml 7- nTregs.
  • Representative flow plots of post-activation CD62F and CD44 staining are shown from 3 separate experiment.
  • FIGS. 3B-3H Examination of Nrnl 7- Treg cell suppression function in vivo in a colitis model.
  • FIGS. 3B Mean percentage weight changes (+/-SEM) over time of experimental mice.
  • FIGS. 3C and 3D Representative H/E stained colon tissue sections and the averaged pathology scores from recipient mice in FIG. 3B are shown.
  • FIG. 3E The frequencies of cytokine-producing lymphocytes infiltrating the mesenteric lymph node (mLN) and lamina intestinal (FP) were determined by intracellular cytokine staining (ICS) and FACS.
  • FIG. 3 F Representative absolute numbers of FP- infiltrating IENg and IL-17 producing leukocytes from the indicated recipient mice are shown.
  • FIG. 3G Representative flow cytometry analysis of injected Foxp3 + /CD45.2 + Tregs frequency among CD4 + LP cells and FIG.
  • FIG. 31 and 3J The development and resolution of EAE symptoms in Nml _/ mice or in their Nrnl +/ littermates.
  • FIG. 31 Ascending paralysis was scored daily and mean scores (+/-SEM) over time are shown.
  • FIG. 3J Proinflammatory cytokine and Foxp3 expression by the spinal cord-infiltrating CD4 + lymphocytes of Nrnl +/ and Nml _/ mice afflicted with EAE were determined by flow cytometry. Shown are representative findings from at least 3 independent experiments.
  • FIGS. 4A-4G are a series of graphs and heat maps demonstrating that Nml deficiency disrupts anergy and Treg cell signature gene expression patterns.
  • FIGS. 4A-4E Whole transcriptome (RNAseq) analysis of genes expressed by Nml _/ vs Nml +/ OTII cells recovered from the peptide induced anergy model outlined in FIG. 2A.
  • FIG. 4A
  • FIG. 4B Results of a Gene Set Enrichment Analysis (GSEA) comparing the gene expression signature previously reported for anergized Thl cells (GEO:GSE46243) to those of Nrnl +/_ and Nrnl _/ T cells in the aforementioned anergy-inducing model.
  • FIG. 4C GSEA depicting a Treg cell gene profile (GEO: GSE42021) by Nrn 1 +A and Nml 7 T cells from FIG. 2A.
  • FIG. 4D (Left) Heatmap of DEGs enriched in Nrn +/ T cells under anergy-inducing conditions that overlap with Thl anergy gene signature. (Right) Examples of anergy specific gene expression (mean FPKM +/- SEM) are displayed in bar graphs.
  • FIG. 4E GSEA of NFAT dependent transcription gene set (PID_NFAT_TFPATHWAY) expressed by Nrnl +/ and Nrnl _/ T cells from FIG. 2A.
  • FIGS. 4F, 4G RNAseq analysis of the gene expression patterns of Tregs isolated from Nrnl _/ or Nml +/ mice.
  • FIG. 4F GSEA of Treg cell gene signature
  • FIG. 4G an NFAT dependent transcription gene set
  • FIGS.5A-5J are a series of graphs and plots demonstrating that Nrnl deficiency affects adaptive anti-tumor response and tumor progression.
  • FIGS. 5A, 5B Comparison of tumor growth in NmT /_ and Nml +/+ mice.
  • FIG. 5A EL4 tumor growth
  • FIGS. 5C-5F Analysis of TILs in tumor from B 16F10- implanted in NrnL /_ or Nml +/+ mice.
  • FIG. 5C Frequencies of Foxp3 + CD4 + T cells; FIG.
  • FIG. 5D FR4 hi CD73 hi CD4 + T cells
  • FIG. 5C IFNy producing CD4 + cells
  • FIG. 5F IFNy producing CD 8 cells among the B16F10 TILs from NrnL /_ or Nrnl +/+ mice were determined by flow cytometry.
  • FIG. 5G Expression of Nrnl mRNA by Foxp3-expressing Tregs and non-Treg CD4 + cells in s.c. B 16 melanoma.
  • CD4 + T cells were recovered from the tumors and peripheral blood of FDG reporter mice bearing subcutaneous B16 melanomas. Nml expression was assessed by qRT-PCR analysis.
  • FIG. 5G Expression of Nrnl mRNA by Foxp3-expressing Tregs and non-Treg CD4 + cells in s.c. B 16 melanoma.
  • CD4 + T cells were recovered from the tumors and peripheral blood of FDG reporter mice bearing subcutaneous B16 melanomas. Nml expression was assessed by q
  • FIGS. 51, 5J Impact of Nml deficiency in Treg on tumor growth and anti-tumor immunity.
  • Rag2 /_ mice were reconstituted with Treg-depleted CD45.1 + spleen and lymph node cells (2 xlO 6 ) and Tregs from either NmL /_ or Nrnl +/_ mice (CD45.2VCD45.1-, 0.5 x 10 6 ).
  • Rag2 /_ mice were subsequently challenged with s.c. B16F10 cells.
  • FIG. 51 Tumor growth as measured in FIG. 5B.
  • FIG. 5J the frequencies of transferred Tregs (CD45.L) and non-Tregs (CD45.1 + ) in tissues of tumor-bearing recipients were found by flow cytometry. Results from at least 2 trials are presented as mean +/- SEM.
  • FIGS. 6A-6I are a series of graphs and a scan of a photograph demonstrating that the combination of Nml, CTLA4 and PD1 blockade synergistically inhibits tumor growth.
  • FIGS. 6A-6D Effect of Nrnl blockade on the growth of multiple tumors.
  • B16F10, MC38, CT26 or 4T1 average tumor growth curve in mice treated with anti-Nrnl Ab or an inert isotype control Ab (mIgG2b) on day 5-6 after tumor implantation.
  • Mean tumor volume (+/- SEM) over time was plotted (n>8 per group, data represent the results of at least two experiments).
  • FIG. 6E- 6G Flow cytometric analysis of TILs recovered from anti-Nrnl or isotype control treated B16F10 bearing mice 20 days after tumor inoculation.
  • FIG. 6E The CD8/CD4Foxp3 + ratio as well as the FIGS. 6F, 6G, frequencies of Tbet expressing and IFNy expressing among CD8 cells among the TILs of anti-Nrnl or control Ab treated mice.
  • FIGS. 6H, 61 Impact of combining anti-Nrnl, anti-CTLA4 and anti-PDl antibody treatments on B16F10 tumor growth. Anti-Nrnl and anti-CTLA4 antibody treatments were initiated 5-6 days after tumor injection. Anti-PDl blockade was administered starting on day 8 after tumor injection.
  • FIG. 6H Mean tumor volumes (+/- SEM) over time and FIG. 6J, representative tumor sizes 18 days after tumor inoculation. Data representative of at least 3 separate experiments (n>7 per experimental group/trial).
  • FIG. 7A is a graph and a Western blot and FIG. 7B a plot demonstrating that Nrnl is expressed in Treg cells.
  • FIG. 7A Nml message and protein levels were measured across immune cell types isolated from Foxp3DTRgfp reporter mice by qRT-PCR and western blot.
  • FIG. 7B Flow cytometry analysis of surface Nml expression by CD4 + Foxp3 + CD25 + cells.
  • FIGS. 8A-8F are a series of graphs and blots demonstrating that Nrnl affects anergy development and pTreg cell conversion.
  • FIGS. 8A, 8B Induction of anergy in wt B6 host mice. 5xl0 6 congenically marked Nml _/ or Nml +/_ Thyl.l+ OTII cells were transferred into wt Thyl.2 + B6 mice and subjected to 3 treatment of OVAII peptide (100pg i.v) every three days. Thyl.l + cells were recovered 13 days after cell transfer.
  • FIG. 8A, 8B Induction of anergy in wt B6 host mice. 5xl0 6 congenically marked Nml _/ or Nml +/_ Thyl.l+ OTII cells were transferred into wt Thyl.2 + B6 mice and subjected to 3 treatment of OVAII peptide (100pg i.v) every
  • FIG. 8 A IL2 production by recovered OTII cells upon ex vivo restimulation with OVAII peptide measured by ELISA.
  • FIG. 8B the frequencies of Foxp3 + cells among recovered Thyl.l+OTII cells determined by flow cytometry.
  • FIG. 8C Schematic of CD2N Nrnl transgenic mouse design. A transgenic construct expressing Nml under the CD2 promoter was used for pronuclear injection and transgene positive mice were maintained on a C57BL/6 background (referred to as CD2N mice).
  • FIG. 8D Detection of cell surface Nml expression among CD3 + T cells from CD2N and littermate wt Nml +/+ mice by flow cytometry.
  • FIGS. 8E, 8F Anergy development and pTreg conversion among CD2N T cells. 6xl0 6 total T cells from CD45.2 + CD2N + or wt littermate control mice on FDG background were transferred into CD45.1 + FDG hosts and administered DT lug/mouse as indicated in Fig.
  • FIGS 8A-8C, 8E, and 8F depict mean values +/- S EM .
  • FIGS. 9A-9F are a series of graphs, blots and histological stain demonstrating that transgenic Nrnl overexpression and monoclonal antibody-mediated blockade of Nml have divergent effects on Treg function in vivo.
  • FIG. 9A Colitis was induced in Rag2 /_ mice by i.p. injection of 4xl0 5 naive CD4 + T cells. Weight loss in recipient mice was monitored weekly. After the development of colitis symptoms (2-3 weeks later), 3-5xl0 5 wt Tregs, CD2N + Tregs, or no Tregs were transferred to the colitis mice.
  • FIG. 9F Representative H&E staining of colon sections from the mice represented in FIG. 9A are shown.
  • FIG. 9C The absolute numbers of inflammatory cytokine producing leukocyte recovered from the lamina propria (LP) of the indicated recipient mice were found after intracellular cytokine staining. Shown are representative results of 3 experiments.
  • FIG. 9D Percentages of Foxp3-expressing cells in the original transferred Treg population (CD45.1 + ) recovered from the LP of recipient mice.
  • FIGS. 9E, 9F Nml functional blockade by antibody results in exacerbated EAE. Disease was induced in a cohort of age/sex matched wt C57BL/6 mice as in FIG. 31.
  • FIG. 9E Clinical scoring of EAE disease severity in mice given either an anti- Nmlantibody or an isotype control at the time of disease induction.
  • FIG. 9F Production of proinflammatory cytokines by CNS-infiltrating T cells recovered from mice treated with anti- Nml or isotype control Ab. Shown are the representative findings of at least 3 independent experiments.
  • FIGS. 10A-10D are a series of graphs and heatmaps demonstrating that Nrnl deficiency impacts gene expression by CD4 cells activated under anergy-inducing condition in vivo.
  • Analysis of RNAseq data from Nrnl _/ or Nml +/ OTII cells recovered from an i.v. peptide driven model of anergy (outlined in FIG. 2A).
  • FIG. 10A molecular function GO term analysis was carried out using the Molecular Signature Database (MSigDB) based on the DEGs that were enriched in Nml +/ T cells relative to Nrnl _/ T cells recovered from the anergy model. The top 5 terms are shown.
  • MSigDB Molecular Signature Database
  • FIG. 10B Top 5 terms identified by GO term analysis with KEGG pathway using DEGs enriched in Nrnl +/_ T cells.
  • FIG. IOC GSEA of DEGs from Nml +/_ and NrnT /_ cells recovered from anergy condition (FIG. 2A) to a previously reported anergic CD4+ T cell gene set (GSE5960) and a AE7 gene set.
  • FIG. 10D Representative expression of cation transport and Ca 2+ binding related genes by T cells recovered from in vivo anergy-inducing condition (mean FPKM +/- SEM).
  • FIGS. 11A-11C are a series of graphs and a scan of a photograph demonstrating the effect of genetic modulation of Nml expression on B16F10 tumor progression.
  • FIG. 11A, B16F10 mean tumor volumes per group (+/-SEM) in NmT /_ , Nml +/_ and Nml +/+ host mice.
  • FIGS. 11B, llC, B16F10 mean tumor volumes per group (+/- SEM) and representative tumor sizes at 20 days after tumor injection in CD2N mice, wt CD2N littermate controls and NrnT /_ mice. (n>4 per group; data represent of at least 2 trials).
  • FIGS. 12A-12D are a series of graphs demonstrating the impact of Nml blockade on tumor progression in multiple murine cancer models.
  • FIGS. 4A-4D The indicated tumor cell lines were implanted s.c. into age-/sex-matched wt C57BL/6 mice. 5 days post implantation. Mice were treated with anti- Nml or an isotype control. Changes in the tumor volumes of individual mice in treatment and control cohorts are shown for FIG. 12A, B16F10; FIG. 12B, MC38; FIG. 12C, CT26 and FIG. 12D, 4T1 tumors. (N>7 mice/group/trial). Data shown are representative at least two independent experiments).
  • FIGS. 13A-13C are a series of graphs showing results obtained from the treatment of B16F10 tumor-bearing mice with combinedNml blockade and checkpoint inhibition therapy.
  • FIG. 13A Anti-Nrnl combination therapy with anti-CTLA4.
  • the following experimental groups are included for assessing the efficacy of anti-Nrnl and anti-CTLA4 combination therapy: isotype control (mIgG2b), anti-Nrnl, anti- CTLA4, anti-PDl, anti-Nrnl with anti-CTLA4, and anti-CTLA4 with anti-PDl.
  • FIG. 13B Anti-Nml combination therapy with anti-PDl. Anti- PDl was administered 3 days after the initiation of Nml blockade.
  • FIG. 13C Individual tumor growth curve for tumor bearing mice subjected to triple combination therapy with anti- Nml, anti-CTLA4 mAh and anti-PDl blockade. Anti-Nml and anti-CTLA4 were administered on day5 post tumor establishment, anti-PDl was administered 3 days after the initiation of anti-Nrnl and anti-CTLA4 treatment. Mean tumor volume (bolded line) and individual tumor volume were plotted over time. Data represents 2-3 independent experiments (n>7 per group/trial).
  • FIG. 14 shows representative flow cytometry gating strategy for analyzing anergic CD4 + CD44 + FR4 hl CD73 hl cells, anergic cells, cTreg and eTreg cells.
  • Cancer immunotherapy strategies rely on activating (or reactivating) T cell populations specific for either tumor specific, mutation associated neoantigens; viral antigens (for vims-associated cancers); de -repressed endogenous retroviral antigens; and tumor- associated self-antigens. It has been shown that tumor antigen specific CD4 T cells can become anergized and convert to Treg rapidly after tumor emergence 5 . These cells and pre existing Treg cells can accumulate in the tumor microenvironment and are capable of preventing effector T cell infiltration and obstmcting cytotoxic activity at the tumor site 5 6 . Understanding the precise mechanisms and molecules involved in inducing and maintaining the immune-tolerant state has already and will continue to underpin the success of immunotherapy for autoimmune disease and cancer. Accordingly, embodiments of the invention are directed to compositions which target molecules involved in inducing and maintaining the immune-tolerant state thereby abrogating the suppression of the immune response to a particular tumor.
  • T cell anergy and Treg cell mediated suppression are two complementary peripheral tolerance mechanisms.
  • T cell anergy is historically defined as a state of functional inactivation wherein T cells lose the capacity to respond and produce cytokine upon re encountering cognate antigen 7 .
  • T cell anergy can be induced in vitro by providing TCR signal in the absence of costimulation 1 .
  • anergy is observed in CD4 + T cells after systemic, repeated exposure to a soluble antigen (Ag) or superantigen in the absence of infection or adjuvant 8 .
  • Ag soluble antigen
  • Treg autoreactive T cell responses against self-antigen and modulating immune activation throughout the life span of normal animals.
  • Most Treg differentiate in the thymus and are referred to as thymic Treg or natural Treg (tTreg or nTreg, respectively), whereas others originate in the periphery from conventional Foxp3(-) CD4 + T cells and are referred to as peripherally induced Treg (pTreg, or iTreg if generated in vitro).
  • pTreg peripherally induced Treg
  • Both nTreg and pTreg subsets are marked by the canonical Treg transcription factor Foxp3 and are required for optimal immune homeostasis 2 15 16 .
  • Anergic T cells and Treg are functionally related. Although anergic T cells are generally non-proliferative, they are not functionally inert. These cells may function as suppressor cells and anergy development in vivo is often associated with the emergence of pTreg 17-2a Further linking these cell types are observations that deletion of pTreg within the anergic T cell pool largely abrogates its suppressive function 18 19 ⁇ Thus, anergic T cells may contribute to the establishment of peripheral tolerance partly through induction of or differentiation into pTreg 3 . On the other hand, target cells suppressed by Treg exhibit anergic cell characteristics, including defective cytokine production.
  • Nml neuritin
  • Treg T regulatory cells
  • Nml is important in anergy development and Treg mediated suppressive function. Accordingly, growth of multiple tumors was significantly inhibited in Nml deficient mice. Treatment with antibodies to Nml alone and in combination with anti-CTLA4 and anti-PDl significantly inhibited growth of established B16F10 melanoma, a poorly immunogenic tumor.
  • a method of treating cancer comprises administering to a subject, a therapeutically effective amount of a neuritin inhibitor, wherein the neuritin inhibitor abrogates anergic T cell and T regulatory cell functions.
  • a method of inducing a tumor specific immune response in a subject comprises administering to a subject, a therapeutically effective amount of a neuritin inhibitor, wherein the neuritin inhibitor abrogates anergic T cell and T regulatory cell functions.
  • a method of abrogating T cell anergy comprises
  • a pharmaceutical composition comprising a therapeutically effective amount of a neuritin inhibitor and one or more checkpoint inhibitors.
  • a method of modulating T regulatory (Treg) cell function comprises administering to a subject in need thereof, a pharmaceutical composition comprising a therapeutically effective amount of a neuritin inhibitor and one or more checkpoint inhibitors.
  • Checkpoint Inhibitors In certain embodiments, a method of treating cancer, administering one or more checkpoint inhibitors.
  • the one or more checkpoint inhibitors comprises an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG- 3, CEACAM-1, CEAC AM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4, TGFR-b or combinations thereof.
  • the checkpoint inhibitor comprises an inhibitor of PD-1, PD-L1, CTLA4 or combinations thereof.
  • the at least one or more checkpoint inhibitors are administered to the patient prior to, during and/or after administration of the neuritin inhibitors. Examples of checkpoint inhibitors include without limitation, an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3,
  • CEACAM-1 CEAC AM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-b.
  • the duration and/or dose of treatment with checkpoint inhibitor therapies may vary according to the particular anti-immune checkpoint inhibitor agent or combination thereof (e.g., anti-ARGl agents like small molecule inhibitors in combination with inhibitors of PD- 1, PD-L1, PD-L2, CTLA4, and the like).
  • An appropriate treatment time for a particular cancer therapeutic agent will be appreciated by the skilled artisan.
  • dosage concentrations and dosing regimens are configured based upon one or more cancer related factors such as tumor size, tumor volume, cancer stage of a cancer patient or group of cancer patients (such as pre or post metastatic cancer).
  • the invention contemplates the continued assessment of optimal treatment schedules for each cancer therapeutic agent, where the phenotype of the cancer of the subject as determined by the methods of the invention is a factor in determining optimal treatment doses and schedules.
  • the Programmed Death 1 (PD-1) protein is an inhibitory member of the extended CD28/CTLA-4 family of T cell regulators (Okazaki et al. (2002) Curr Opin Immunol 14: 391779-82; Bennett et al. (2003) J. Immunol. 170:711-8).
  • Other members of the CD28 family include CD28, CTLA-4, ICOS and BTLA.
  • Two cell surface glycoprotein ligands for PD-1 have been identified, Program Death Ligand 1 (PD-L1) and Program Death Ligand 2 (PD- L2).
  • PD-L1 and PD-L2 have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al.
  • PD-L1 (also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7- Hl)) is a 40 kDa type 1 transmembrane protein. PD-L1 binds to its receptor, PD-1, found on activated T cells, B cells, and myeloid cells, to modulate activation or inhibition. Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1, but do not bind to CD28 or CTLA-4 (Blank et al. (2005) Cancer Immunol Immunother. 54:307-14).
  • Binding of PD-L1 with its receptor PD- 1 on T cells delivers a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation.
  • the mechanism involves inhibition of ZAP70 phosphorylation and its association with CD3z (Sheppard et al. (2004) FEBS Lett. 574:37-41).
  • PD-1 signaling attenuates PKC-0 activation loop phosphorylation resulting from TCR signaling, necessary for the activation of transcription factors NF-KB and AP-1, and for production of IL-2.
  • PD-L1 also binds to the costimulatory molecule CD80 (B7-1), but not CD86 (B7-2) (Butte et al. (2008) Mol Immunol. 45:3567-72).
  • PD-L1 has been shown to be upregulated through IFN-g stimulation.
  • PD-L1 expression has been found in many cancers, including human lung, ovarian and colon carcinoma and various myelomas, and is often associated with poor prognosis (Iwai et al. (2002) PNAS 99:12293-7; Ohigashi et al. (2005) Clin Cancer Res 11:2947-53; Okazaki et al. (2007) Intern. Immun. 19:813-24; Thompson et al. (2006) Cancer Res. 66:3381-5).
  • PD-L1 has been suggested to play a role in tumor immunity by increasing apoptosis of antigen-specific T-cell clones (Dong et al.
  • checkpoint inhibitor antibodies for cancer therapy have generated unprecedented response rates in cancers previously thought to be resistant to cancer treatment (see, e.g., Ott & Bhardwaj, 2013, Frontiers in Immunology 4:346; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85; Pardoll, 2012, Nature Reviews 12:252-264).
  • Therapy with antagonistic checkpoint blocking antibodies against CTLA-4, PD-1 and PD-L1 are one of the most promising new avenues of immunotherapy for cancer and other diseases.
  • checkpoint inhibitor do not target tumor cells directly, but rather target lymphocyte receptors or their ligands in order to enhance the endogenous antitumor activity of the immune system.
  • ADCs antibody-drug conjugates
  • PD-1 Programmed cell death protein 1
  • CD279 encodes a cell surface membrane protein of the immunoglobulin superfamily, which is expressed in B cells and NK cells (Shinohara et al., 1995, Genomics 23:704-6; Blank et al., 2007, Cancer Immunol Immunother 56:739-45; Finger et al., 1997, Gene 197:177-87; Pardoll, 2012, Nature Reviews 12:252-264).
  • Anti-PDl antibodies have been used for treatment of melanoma, non- small-cell lung cancer, bladder cancer, prostate cancer, colorectal cancer, head and neck cancer, triple negative breast cancer, leukemia, lymphoma and renal cell cancer (Topalian et al., 2012, N Engl J Med 366:2443-54; Lipson et al., 2013, Clin Cancer Res 19:462-8; Berger et al., 2008, Clin Cancer Res 14:3044-51; Gildener-Leapman el al., 2013, Oral Oncol 49:1089-96;
  • anti-PDl antibodies include pembrolizumab (MK-3475, Merck), nivolumab (BMS-936558, Bristol-Myers Squibb), and pidilizumab (CT-011, Curetech LTD.).
  • Anti-PDl antibodies are commercially available, for example from ABCAMTM
  • BIOLEGENDTM EH12.2H7, RMP1-14
  • Affymetrix Ebioscience J105, J116, MIH4.
  • Programmed cell death 1 ligand 1 (PD-L1, also known as CD274) is a ligand for PD- 1, found on activated T cells, B cells, myeloid cells and macrophages.
  • the complex of PD-1 and PD-L1 inhibits proliferation of CD8 + T cells and reduces the immune response (Topalian et al., 2012, N Engl J Med 366:2443-54; Brahmer et al., 2012, N Eng J Med 366:2455-65).
  • Anti-PDLl antibodies have been used for treatment of non-small cell lung cancer, melanoma, colorectal cancer, renal-cell cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, and hematologic malignancies (Brahmer et al., 2012, N Eng J Med 366:2455-65; Ott et al., 2013, Clin Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res 19:5541; Menzies & Long, 2013, TherAdv Med Oncol 5:278-85; Berger et al., 2008, Clin Cancer Res 14:13044-51).
  • anti-PDLl antibodies include MDX-1105 (MEDAREX), durvalumab (MEDI4736, MEDIMMUNE) atezolizumab (TECENTRIQTM, MPDL3280A,
  • Anti-PDLl antibodies are also commercially available, for example from AFFYMETRIX EBIOSCIENCE (MIH1).
  • CTLA-4 (CD 152) is a B7/CD28 family member that inhibits T cell functions. It is constitutively expressed by Tregs but can also be upregulated by other T cell subsets, especially CD4 + T cells, upon activation (Chan DV, et al. Genes Immun. 2014 Jan; 15(1):25- 32). Exhausted T cells are also often characterized by the expression of CTLA-4 among other inhibitory receptors. CTLA-4 is mostly located in intracellular vesicles and is only transiently expressed upon activation in the immunological synapse before being rapidly endocytosed (Leung HT, et al. J Biol Chem. 1995 Oct 20; 270(42):25107-14).
  • CTLA-4 mediates immunosuppression by indirectly diminishing signaling through the co-stimulatory receptor CD28. Although both receptors bind CD80 and CD86, CTLA-4 does so with much higher affinity, effectively outcompeting CD28 (Rudd CE, Taylor A, Schneider H Immunol Rev. 2009 May; 229(1): 12-26). CTLA-4 may also remove CD80 and CD86 (including their cytoplasmic domains) from the cell surfaces of antigen-presenting cells via trans-endocytosis (Qureshi OS, et al. Science. 2011 Apr 29; 332(6029):600-3), therefore reducing the availability of these stimulatory receptors to other CD28-expressing T cells. Indeed, this process is one mechanism by which Tregs mediate immune suppression on bystander cells (Wing K, et al. Science. 2008 Oct 10; 322(5899):271-5).
  • CTLA-4 increases the activation threshold of T cells, reducing immune responses to weak antigens such as self- and tumor antigens.
  • the central role that CTLA-4 plays in immunological tolerance is exemplified by experiments in mice that lack the CTLA-4 gene globally or specifically in the Forkhead box P3 (FoxP3) + Treg compartment. These animals develop lymphoproliferative disorders and die at a young age (Wing K, et al. Science. 2008 Oct 10; 322(5899):271-5).
  • polymorphisms within the CTLA-4 gene are associated with autoimmune diseases in humans (Gough SC, et al. Immunol Rev. 2005 Apr; 204(): 102-15).
  • CTLA-4 signaling has been shown to dampen immune responses against infections and tumor cells (Nakamoto N, et al. PLoS Pathog. 2009 Feb; Curran MA, et al. Proc Natl Acad Sci U S A. 2010 Mar 2;
  • An exemplary anti-CTLA-4 antibody is IPILIMUMAB (trade name Yervoy).
  • a checkpoint inhibitor is an RNA interfering agent
  • Tregs also employ a diverse repertoire of suppressive mechanisms, including secretion of suppressive cytokines, cytotoxicity, metabolic disruption, and modulation of antigen-presenting cell (APC) function (Sakaguchi S, Wing K, Miyara M. Regulatory T cells - a brief history and perspective. Eur J Immunol. 2007 Nov; 37 Suppl 1:S116-23).
  • APC antigen-presenting cell
  • IL-6 Trinschek B, et al. Kinetics of IL-6 production defines T effector cell responsiveness to regulatory T cells in multiple sclerosis. PLoS One (2013) 8:e77634; Schneider A, et al. In active relapsing- remitting multiple sclerosis, effector T cell resistance to adaptive T(regs) involves IL-6- mediated signaling. Sci Transl Med (2013) 5:170), TNFa (Wehrens EJ, et al.
  • Anti-tumor necrosis factor a targets protein kinase B/c- Akt-induced resistance of effector cells to suppression in juvenile idiopathic arthritis.
  • Arthritis Rheum (2013) 65:3279—84 IL-15 (Proinflammatory mediator-induced reversal of CD4 + ,CD25 + regulatory T cell-mediated suppression in rheumatoid arthritis van Amelsfort JM, el al. Arthritis Rheum. 2007 Mar; 56(3):732-42), IL-21 (IL-21 counteracts the regulatory T cell-mediated suppression of human CD4 + T lymphocytes.
  • Peluso I el al. J Immunol. 2007 Jan 15; 178(2):732-9
  • IL-Ib Protein kinase B/c- Akt-induced resistance of effector cells to suppression in juvenile idiopathic arthritis.
  • IL-15 Proinflammatory mediator-induced reversal of CD4 + ,CD25 + regulatory T cell-mediated suppression in rheumatoi
  • IL-2 has also long been known to overrule Treg suppression in vitro (Takahashi T, et al., Int Immunol. 1998 Dec; 10(12): 1969-80).
  • a method of treating cancer or abrogating anergic T cell and/or Treg-mediated suppression comprises administration of cytokines to a subject in need thereof.
  • the cytokines comprise: interleukin-2 (IL-2), interleukin- 6 (IL-6), interleukin- 4 (IL-4), interleukin- 7 (IL-7), interleukin- 15 (IL-15), interleukin-21 (IL- 21), tumor necrosis factor alpha (TNFa) or combinations thereof.
  • TNF receptors 4- IBB, 0X40, GITR, and TNFR2 can induce T cell resistance to Treg suppression, as they provide costimulatory signals similar to CD28 ligation.
  • 4- IBB, 0X40, and TNFR2 signaling induce PI3K/Akt activation via TRAF adaptor proteins.
  • Toll-like receptors 1, 2, 4, 8, and 9, as well as IL-1R, also a member of the TLR family have been shown to induce Treg resistance. Of these, only signaling through TLR2 and TLR9 has been shown to activate the PI3K/Akt pathway via recruitment of adaptor protein MyD88, which in turn recruits and activates PI3K via its Toll/interleukin- 1 receptor domain.
  • Intracellular signaling molecules Cbl-b and SHP-1 act as negative regulators downstream of TCR signaling, and genetic deficiency in either induces Treg resistance.
  • Cbl-b enforces the requirement for CD28 costimulatory signaling by inhibiting the recruitment of PI3K to CD28.
  • TRAF6 also negatively regulates activation of PI3K downstream of CD28 costimulation by an as yet undefined mechanism (Mercadante, E. R., & Lorenz, U. M.
  • the method further comprises administration of: anti-CD25 antibodies, inhibitors of molecules associated with T cell anergy development, Toll-like receptor 2 (TLR2) agonists, 4-1BB agonists, 0X40 agonists, tumor necrosis factor receptor 2 (TNFR2) agonists, cytokines or combinations thereof.
  • TLR2 Toll-like receptor 2
  • 4-1BB 4-1BB
  • 0X40 0X40
  • TNFR2 tumor necrosis factor receptor 2
  • cytokines cytokines
  • the method further comprises administering an agonist of a co stimulatory receptor.
  • exemplary agonists include an anti-glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR) antibody, a Toll-like receptor 2 (TLR2) agonists, antibodies to molecules associated with T cell anergy development, an anti- CD27 antibody, an anti-4-lBB antibody, an anti-OX40 antibody, an anti-inducible T-cell co stimulator (ICOS) antibody, and an anti-CD40 antibody, an anti-Toll-like receptor 2 (TLR2) antibody, or any combination thereof.
  • TNFR tumor necrosis factor receptor
  • TLR2 Toll-like receptor 2
  • Toll-like Receptors are an essential line of defense against microbial and viral pathogens.
  • Various pathogen-derived ligands signal through TLRs, which recruit adaptor molecules such as MyD88 to trigger the production of pro-inflammatory mediators (Cohen P. J Cell ScL 2014 Jun 1 : 127(Pt 11)i2383-9Q).
  • the goal of TLR signaling is to sense a pathogenic threat and mount innate and adaptive immune responses.
  • TLR ligands can influence conventional T (Tcon) cells i.e.
  • T lymphocytes that express an ab T cell receptor (TCR), as well as a co-receptor CD4 or CD8, responses via direct receptor activation or indirectly, by inducing antigen presenting cells (APCs) to produce cytokines that affect T cells (Sutmuller RP, el al. Trends Immunol. 2006 Aug; 27(8):387-93).
  • APCs antigen presenting cells
  • DCs mouse dendritic cells
  • CpG TLR4 and 9 agonists, respectively
  • TLR4 and 9 agonists, respectively induced their production of IL-6, contributing to Tcon cell resistance to Treg suppression (Pasare C, Medzhitov R. Science. 2003 Feb 14; 299(5609): 1033-6). While TLR signaling directly affects Tregs there is also evidence that TLR signaling can directly induce Tcon cell resistance to suppression (Sutmuller RP, et al., Trends Immunol. 2006 Aug;
  • TLR engagement acts as a costimulatory signal to T cells and subsequently activates the PI3K/Akt pathway, consistent with a role in inducing Tcon cells to resist Treg suppression (Rahman AH, Taylor DK, Turka LA. Immunol Res. 2009; 45(l):25-36).
  • TNF Receptors Engagement of certain tumor necrosis factor receptors (TNFRs) on T cells provides costimulatory signals that lead to activation, proliferation, differentiation, and survival (Watts THT. NF/TNFR family members in costimulation of T cell responses. Annu Rev Immunol. 2005; 23:23-68.).
  • TNFRs tumor necrosis factor receptors
  • the four TRAF-binding TNFRs have been found to render Tcon cells resistant to Treg suppression (Mercadante, E. R., et al. (2016). Frontiers in immunology, 7, 193).
  • Evidence supports a role, in particular for TRAF2, in activating PI3K/Akt downstream of TNFRs (So T, et al. Front Immunol (2013)
  • TNFRs do not contain PI3K-binding motifs, they utilize TRAF adaptor proteins to activate the PI3K pathway. These TNFRs are constitutively expressed on Tregs and become upregulated on activated Tcon cells. The ligands for these TNFRs are generally expressed on APCs, but can also be induced on other cell types during infection (Stephens GL, et al. J Immunol (2004) 173:5008-20). TNFRs, like TLRs, play an important role during an infectious threat by allowing Tcon cells to become efficiently activated in order to mount a response, unrestrained by Tregs.
  • TNFR ligand expression becomes upregulated during inflammatory conditions and provides costimulatory signals to both Tregs and Tcon cells, with Tcon cells becoming activated, producing IL-2, and resisting Treg suppression.
  • Tregs can assume control of the immune response (Stephens GL, et al. J Immunol (2004) 173:5008-20.10.4049/jimmunol.l73.8.5008).
  • GITR GITR signaling in murine Tcon cells enhanced their proliferation and allowthem to resist Treg-mediated suppression.
  • 4- IBB Signaling through 4- IBB in murine Tcon cells has been shown to induce proliferation and enhance survival, especially in CD8 + T cells (Wang C, et al. Immunol Rev (2009) 229:192-21). Treatment with agonistic 4- IBB antibodies has beneficial effects on CD8 + T cell-mediated viral clearance and antitumor immunity.
  • 4- IBB signaling In vitro studies of 4- IBB signaling have shown a clear role for its CD28-independent costimulation of Tcon cells as well as its ability to induce resistance to Treg-mediated suppression (Choi BK, et al. J Leukoc Biol (2004) 75:785-91; Elpek KG, et al. J Immunol (2007) 179:7295-304; Robertson SJ, et al.
  • 0X40 0X40 signaling provides costimulation for Tcon cells, promoting their survival and development into memory cells (Ishii N, et al. Adv Immunol (2010) 105:63-98).
  • Several studies are in agreement that 0X40 signaling in murine Tcon cells induces resistance to Treg- mediated suppression (Takeda I, et al. J Immunol (2004) 172:3580-9; Piconese S, et al. J Exp Med (2008) 205:825-39; Voo KS, et al. J Immunol (2013) 191:3641-50),
  • TNFR2 TNFR2 Originally characterized by its expression on activated/memory Treg cells, TNFR2 marks potently suppressive Tregs present in peripheral lymphoid tissues as well as in tumors, but can also be induced upon T cell receptor (TCR) activation on Tcon cells (Chen X, et al. J Immunol (2008) 180:6467-71).
  • the method further comprises administering antagonists or inhibitors of molecules associated with T cell anergy development.
  • molecules associated with T cell anergy development comprise: Cbl-b, TRAF6 or SHP-1.
  • Cbl-b is an E3 ubiquitin ligase that catalyzes the ubiquitylation of target proteins, which can result in their degradation by the proteasome, translocation inside the cell, or alteration in function (Paolino M, Penninger JM. Semin Immunopathol. 2010 Jun; 32(2): 137-48).
  • Cbl-b sets the threshold for weak antigen stimulation (Chiang YJ, et al. Nature. 2000 Jan 13; 403(6766):216-20) and enforces the need for costimulation, or “signal 2,” by regulating CD28 signaling (Li D, et al. J Immunol. 2004 Dec 15;
  • Cbl-b negatively regulates the recruitment of the p85 subunit of PI3K to CD28, thereby enforcing T cell anergy and tolerance when signal 2 is lacking (Fang D, Liu YC. Nat Immunol. 2001 Sep; 2(9):870-5).
  • CD28 signaling Cbl-b itself becomes ubiquitylated and degraded, allowing PI3K recruitment and other downstream signaling required for full T cell activation (Gruber T, et al. Sci Signal. 2009 Jun 23; 2(76):ra30).
  • Cbl-b knockout mice develop systemic autoimmunity due to hyper-proliferation and increased activation of lymphocytes, with T cells that can be activated in the absence of CD28 costimulation (Bachmaier K, et al. Nature. 2000 Jan 13; 403(6766):211-6).
  • TRAF6 belongs to the E3 ubiquitin ligase family and transduces signals downstream of members of the TNFR superfamily, including IL-lR/TLRs (Wu H, Arron JR. Bioessays. 2003 Nov; 25(11): 1096- 105), thereby activating NFKB, NFAT, MAP kinases, and Akt signaling pathways (Yang WL, et al. Science. 2009 Aug 28; 325(5944): 1134-8).
  • a 2006 study demonstrated that TRAF6 KO mice developed multi-organ inflammatory disease characterized by hyper-activated T cells (King CG, et al. Nat Med. 2006 Sep; 12(9): 1088- 92).
  • TRAF6 KO Tregs were normal, the Tcon cells resisted Treg suppression both in vitro and in vivo.
  • Re-expression of TRAF6 via retroviral transduction restored susceptibility of Tcon cells to Treg-mediated suppression (King CG, et al. Nat Med. 2006 Sep; 12(9): 1088- 92).
  • TRAF6 KO T cells could also be activated independently of CD28 costimulation, and showed enhanced Akt activation upon TCR signaling.
  • sensitivity to Tregs could by restored by overexpression of PTEN, an inhibitor of PI3K/Akt.
  • SHP-1 a protein tyrosine phosphatase, negatively regulates TCR signaling by dephosphorylating signaling mediators such as Zap70, Vav, Lck, and SLP76 (Lorenz U. Immunol Rev. 2009 Mar; 228(l):342-59).
  • SHP-1 KO mice develop inflammation in skin and lungs due to myeloid hyper-proliferation (Abram CL, et al. Immunity. 2013 Mar 21;
  • mice 38(3):489-501). These mice also accumulate memory T cells, and T cells are hyper- responsive to TCR stimulation. SHP-1 KO Tregs have an increased suppressive capacity (Iype T, et al. J Immunol. 2010 Nov 15; 185(10):6115-27). Tcon cells deficient in SHP-1 via genetic deletion or pharmacological inhibition can resist Treg suppression in vitro
  • SHP-1 regulates conventional T cell resistance to suppression by regulatory T cells. AAAS Annu. Meet. 2016 Washington, DC (2016)). SHP-1 also negatively regulates activation of STAT3 in response to IL-6 signaling, with SHP-1 -deficient cells being hyper- sensitive to IL-6 (Mauldin IS, Tung KS, Lorenz UM. Blood. 2012 May 10;
  • the methods of treatment inhibit the growth or progression of cancer, e.g., a tumor or a viral-induced tumor, in a subject.
  • the methods described herein inhibit the growth of a tumor by at least 1%, e.g., by at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%.
  • the methods described herein reduce the size of a tumor by at least 1 mm in diameter, e.g., by at least 2 mm in diameter, by at least 3 mm in diameter, by at least 4 mm in diameter, by at least 5 mm in diameter, by at least 6 mm in diameter, by at least 7 mm in diameter, by at least 8 mm in diameter, by at least 9 mm in diameter, by at least 10 mm in diameter, by at least 11 mm in diameter, by at least 12 mm in diameter, by a least 13 mm in diameter, by at least 14 mm in diameter, by at least 15 mm in diameter, by at least 20 mm in diameter, by at least 25 mm in diameter, by at least 30 mm in diameter, by at least 40 mm in diameter, by at least 50 mm in diameter or more.
  • the subject has had the bulk of the tumor resected.
  • methods of treating cancer can include one or more cancer therapies as part of the treatment regimen.
  • the compositions of the invention embodied herein can be administered with one or more alternative treatment regimens, such as targeted and/or untargeted anti-cancer therapies can be administered.
  • Combination therapies are also contemplated and can comprise, for example, one or more chemotherapeutic agents and radiation, one or more chemotherapeutic agents and immunotherapy, or one or more chemotherapeutic agents, radiation and chemotherapy.
  • cancer therapy refers to a therapy useful in treating cancer.
  • anti-cancer therapeutic agents include, but are not limited to, surgery, chemotherapeutic agents, immunotherapy, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti- angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, such as anti-HER-2 antibodies (e.g., HERCEPTINTM), anti-CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (TARCEVATM)), platelet derived growth factor inhibitors (e.g., GLEEVECTM (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the HER-2 antibodies (e.g.
  • the methods of treatment include the administration of one or more chemotherapeutic agents.
  • chemotherapeutic agents include Erlotinib (TARCEVATM, Genentech/OSI Pharm.), Bortezomib (VELCADETM, Millennium Pharm.), Fulvestrant (FASLODEXTM, Astrazeneca), Sutent (SU11248, Pfizer), Letrozole
  • Astrazeneca AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as Thiotepa and CYTOXANTM cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa;
  • ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine;
  • acetogenins especially bullatacin and bullatacinone
  • a camptothecin including the synthetic analogue topotecan
  • bryostatin callystatin
  • CC-1065 including its adozcicsin, carzcicsin and bizcicsin synthetic analogues
  • cryptophycins particularly cryptophycin 1 and cryptophycin 8
  • dolastatin duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide,
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, ADRIAMYCINTM doxorubicin (including morpholino-doxorubicin,
  • vindesine dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosinc; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOLTM paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANETM Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERETM doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZARTM gemcitabine; 6-thioguanine; mercaptopurine;
  • taxoids e.g., TAXOLTM paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.),
  • methotrexate platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; NAVELBINETM vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • platinum analogs such as cisplatin and carboplatin
  • vinblastine platinum
  • ifosfamide mitoxantrone
  • vincristine platinum
  • NAVELBINETM vinorelbine novantrone
  • teniposide edatre
  • chemotherapeutic agent also included in this definition of“chemotherapeutic agent” are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen
  • aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASETM (megestrol acetate), AROMASINTM (exemestane), formestanie, fadrozole, RIVISORTM (vorozole), FEMARATM (letrozole), and ARIMIDEXTM (anastrozole);
  • anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as
  • troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (viii) ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYMETM (ribozyme)) and a HER2 expression inhibitor; (ix) vaccines such as gene therapy vaccines, for example,
  • PROLEUKINTM rIL-2 PROLEUKINTM rIL-2; LURTOTECANTM topoisomerase 1 inhibitor; ABARELIXTM rmRH; (x) anti- angiogenic agents such as bevacizumab (AVASTINTM, Genentech); and (xi) pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • chemotherapeutic agents are illustrative, and are not intended to be limiting.
  • radiation therapy is used.
  • the radiation used in radiation therapy can be ionizing radiation.
  • Radiation therapy can also be gamma rays, X-rays, or proton beams.
  • Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125 palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and/or total abdominal and pelvic radiation therapy.
  • the radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source.
  • the radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass.
  • photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy- hypocrellin A; and 2BA-2-DMHA.
  • hormone therapy is used.
  • Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).
  • hormonal antagonists e.g., flutamide, bicalu
  • Adoptive cell therapy (including allogeneic and autologous hematopoietic stem cell transplantation (HSCT) and recombinant cell (i.e., CAR T) therapies) is the treatment of choice for many malignant disorders (for reviews of HSCT and adoptive cell therapy approaches, see, Rager & Porter, Ther Adv Hematol (2011) 2(6) 409-428; Roddie & Peggs, Expert Opin. Biol. Ther. (2011) ll(4):473-487; Wang et al. Int. J. Cancer: (2015)136, 1751-1768; and Chang, Y.J. and X.J. Huang, Blood Rev, 2013. 27(1): 55- 62).
  • ACT Adoptive cell therapy
  • Such adoptive cell therapies include, but are not limited to, allogeneic and autologous hematopoietic stem cell transplantation, donor leukocyte (or lymphocyte) infusion (DLI), adoptive transfer of tumor infiltrating lymphocytes, or adoptive transfer of T cells or NK cells (including recombinant cells, i.e., CAR T, CAR NK).
  • a method of stimulating an immune response in a subject comprises isolating immune cells, e.g. T cells; contacting the immune cells with a pharmaceutical composition comprising the neuritin inhibitors and/or checkpoint inhibitors embodied herein and/or tumor antigens or tumor cells from the subject; reinfusing the immune cells into the subject; thereby, stimulating the immune response in a subject.
  • the immune cells comprise autologous, haplo-identical, haplotype matched or combinations thereof.
  • the immune cells are derived from autologous or allogeneic stem cells.
  • the immune cells comprise NK cells, T cells, stem cell memory T cells, activated NK (aNK) cells, chimeric antigen receptor- NK (CAR-NK) cells, chimeric antigen receptor-T (CAR-T) cells, or combinations thereof.
  • aNK activated NK
  • CAR-NK chimeric antigen receptor- NK
  • CAR-T chimeric antigen receptor-T cells
  • one or more adjuvants are optionally administered with the soluble fusion protein complexes embodied herein.
  • the adoptively transferred immune cells or pharmaceutical composition comprising the neuritin inhibitors and/or checkpoint inhibitors are administered at least one time per month, e.g., twice per month, once per week, twice per week, once per day, twice per day, every 8 hours, every 4 hours, every 2 hours, or every hour.
  • Suitable modes of administration for the adoptively transferred immune cells include systemic administration, intravenous administration, or local administration. Suitable modes of administration for the
  • compositions include systemic administration, intravenous administration, local administration, subcutaneous administration, intramuscular administration, intratumoral administration, inhalation, and intraperitoneal administration.
  • compositions of the invention can be administered as pharmaceutical
  • a pharmaceutical composition comprises a therapeutically effective amount of a neuritin inhibitor and one or more checkpoint inhibitors.
  • a pharmaceutical composition comprises an anti-neuritin inhibitor and a first and second checkpoint inhibitor.
  • the checkpoint inhibitors comprise an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1,
  • the checkpoint inhibitor comprises an inhibitor of PD-1, PD-L1, CTLA4 or combinations thereof.
  • the first checkpoint inhibitor is an inhibitor of PD- 1 and the second checkpoint inhibitor is an inhibitor of CTLA4.
  • the neuritin inhibitor comprises: an anti-neuritin specific antibody or fragments thereof, antisense oligonucleotides, an siRNA, gene editing agents, nucleases, oligonucleotides, polynucleotides, small molecules, peptides, peptidomimetics, natural ligands and derivatives of natural ligands, oligonucleotides or combinations thereof.
  • the checkpoint inhibitor comprises: an antibody or fragments thereof, antisense oligonucleotides, an siRNA, gene editing agents, nucleases,
  • oligonucleotides polynucleotides, small molecules, peptides, peptidomimetics, natural ligands and derivatives of natural ligands, oligonucleotides, organic or inorganic molecules, enzymes, interfering RNAs or combinations thereof.
  • compositions of the present invention may be specially formulated, in pharmaceutically acceptable carriers or salts, for parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; intravaginally or intrarectally, for example, as a pessary, cream or foam; or aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.
  • phrases“pharmaceutically acceptable” is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • phrases“pharmaceutically-acceptable carrier” as used herein means a
  • composition or vehicle such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body.
  • a liquid or solid filler such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be“acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose, (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar, (14) buffering agents, such as magnesium hydroxide and aluminum hydro
  • pharmaceutically-acceptable salts refers to the relatively non-toxic, inorganic and organic acid addition salts of the agents that modulates (e.g., inhibits) biomarker expression and/or activity, or expression and/or activity of the complex encompassed by the invention. These salts can be prepared in situ during the final isolation and purification of the agents, or by separately reacting a purified agent in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like.
  • the agents useful in the methods of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically- acceptable salts with pharmaceutically-acceptable bases.
  • pharmaceutically- acceptable salts refers to the relatively non-toxic, inorganic and organic base addition salts of agents that modulates (e.g., inhibits) biomarker expression and/or activity, or expression and/or activity of the complex.
  • salts can likewise be prepared in situ during the final isolation and purification of the agents, or by separately reacting the purified agent in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine.
  • a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine.
  • Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.
  • Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.
  • kits for the treatment or prevention of a cancer includes a therapeutic or prophylactic composition containing an effective amount of an agent described herein.
  • the kit comprises a sterile container that contains a therapeutic or prophylactic composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • an agent of the invention is provided together with instructions for administering the agent to a subject having or at risk of developing a cancer.
  • the instructions will generally include information about the use of the composition for the treatment or prevention of a cancer.
  • the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of a cancer or symptoms thereof; precautions; warnings; indications; counter- indications; overdosage information; adverse reactions; animal pharmacology;
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container
  • Example 1 The neurotrophic factor Neuritin regulates T cell anergy and T regulatory cell function and is a target for cancer immunotherapy
  • Nml also known as CPG15
  • GPI glycosylphosphatidylinositol
  • Nml plays multiple roles in the processes of neural development, synaptic plasticity, and synaptic maturation 26 29 ⁇ It is also highly conserved across species, with 100% sequence homology in the extracellular region and 98% overall homology between the murine and human protein. While Nrnl has been found in gene expression profile lists of murine Foxp3 + Treg 30 , anergized CD8 cells 31 tumor infiltrating lymphocytes in mouse tumor models and in human Treg infiltrating breast cancer tumor tissue 6 ’ 32 35 , its role in immune system regulation has never been studied.
  • Nml is highly expressed in both anergic T cells and Treg. Deletion of Nml resulted in defective T cell anergy induction, reduced pTreg cell generation and compromised Treg cell suppressive function in vivo. The growth of multiple tumors is highly inhibited in NmT /_ mice coincident with diminished suppression of anti-tumor T cell function in the tumor microenvironment. Furthermore, antibody blockade of Nrnl alone or in combination with anti-CTLA4 and anti-PD-1 significantly reduced tumor progression. These results implicate neuritin as a hitherto unappreciated promoter of immune tolerance and a yet untapped immunotherapy target.
  • mice 61 The Nml _/ mice 61 , Foxp3-DTR-GFP (FDG) 45 mice and TCRD _/ mice were obtained from Jackson laboratory. OTII mice on Thyl.l + background was kindly provided by Dr. Jonathan Powell. Rag2 _/ mice were maintained in our mouse facility. 6.5 TCR transgenic mice specific for HA antigen on B10.D2 background and C3HA mice on B10.D2 background has been described previously 24 .
  • FDG Foxp3-DTR-GFP
  • CD2N neuritin transgenic mice were generated as follows: an EcoRI/Smal fragment containing the Nrnl cDNA (PCR product from Nrnl cDNA clone NM_153529) was cloned into the EcoKUSmal sites between the human CD2 promoter and the CD2 locus control region of the pBS CD2 construct. The construct was then digested with KpnVNotl to remove plasmid backbone. Purified DNA fragment was injected into fertilized C57BL/6 Jax eggs and transgenic mice were generated using standard microinjection techniques at the Johns Hopkins Transgenic Mouse Core Facility. Nml expression driven by the CD2 promoter was confirmed by qRT-PCR and cell surface staining.
  • Nml _/ mice were crossed with FDG mice to generate Nrnl /_ FDG and Nrnl +/ FDG mice. Nml _/ mice were also crossed with OTII mice to generate Nrnl _/ Thyl.l + OTII + mice, Nrnl +/ Thyl.l + OTII mice.
  • mice colonies were maintained in accordance with the guidelines of Johns Hopkins University and the institutional animal care and use committee. Antibodies and Reagents.
  • Single-cell suspension of lymph node (LN) and spleen (Sp) cells were obtained by grinding the tissue through a IOOmM nylon mesh.
  • Purified CD4 + or total CD3 + T cell population was obtained by magnetic bead-based negative selection.
  • cells were incubated with anti-CD45R (B220, clone RA3-6B2), anti-I-A/I-E (Clone M5/114. 15.2), anti- CDl lb(clone Ml/70) and anti-Ly-6G (Clone RB6-8C5) with or without anti-CD8 (clone 53- 6.72).
  • Treg were isolated by sorting from the FDG CD4 + fraction based on the following profile: CD4 + CD25 + GFP + (total Treg) or CD4 + CD25 + GFP + CD62L hlgh (cTreg) and CD4 + CD25 + GFP + CD62L low (eTreg) expression.
  • Treg were enriched from CD4 cells by positive selection. Specifically, enriched CD4 cells from negative selection were incubated with anti-CD25- biotin Ab. Cells were washed and incubated with streptavidin microbeads (Miltenyi), washed and passed over a magnetic column (Miltenyi), and the positive fraction was collected.
  • lxlO 6 HA specific Thyl.U 6.5 CD4 cells from donor mice on a B10.D2 background were transferred into C3HA recipient mice, where HA is expressed as self antigen in the lung; or into wt B10.D2 mice followed by infection with Vac-HA virus (lxlO 6 pfu).
  • HA- reactive T cells were recovered from the lung draining lymph node of C3HA host mice or from wt B10.D2 Vac-HA infected mice at indicated time points by cell sorting. RNA from sorted cells was used for qRT-PCR assay examining Nml expression.
  • Nrnl _/ or Nml +/ OTII mice were transferred into wt C57BL/6 mice and subjected to the same treatment and analysis as in TCRoU hosts.
  • CD45.2 + Nml +/ or Nrnl _/ mice bred to FDG background were transferred into congenic marked CD45.U FDG mice.
  • host mice were treated with DT at lpg/mouse for 3 days. Spleen and lymph nodes were processed on Dayl l.
  • Adoptively transferred CD45.2 + cells were enriched by staining with anti-CD45.2- FITC followed by selection with anti-FITC beads and LS columns (Miltenyi Biotec). Enriched cells were further subject to cell surface staining and activation by PMA and ionomycin in the presence of brefeldin A for 4 hrs at 37°C followed by intracellular cytokine staining.
  • Naive CD4 + T cells from C57BL/6 mice were isolated from pooled lymph nodes and spleens by FACS sorting. 4xl0 5 naive T cells were injected intraperitoneally (i.p.) into lymphopenic Rag2 /_ mice. On the same day or 14-21 days post-naive T cell injection (as indicated), upon the appearance of colitis symptoms, 3-4xl0 5 congenically distinct, Treg freshly isolated from Nrnl +/+ , Nml _/ , or CD2N mice were also injected into separate cohorts of Rag2 _/ recipients. Changes in body weight were assessed weekly, and upon conclusion of the experiment, colons were removed and fixed in 10% formalin.
  • grade 1 minimal scattered mucosal inflammatory cell infiltrates, with or without minimal epithelial hyperplasia
  • grade 2 mild scattered to diffuse inflammatory cell infiltrates, sometimes extending into the submusoca and associated with erosions, with mild to moderate epithelial hyperplasia and mild to moderate mucin depletion from goblet cells
  • grade 3 moderate inflammatory cell infiltrates that were sometimes transmural, with moderate to severe epithelial hyperplasia and mucin depletion
  • grade 4 marked inflammatory cell infiltrates that were often transmural and associated with crypt abscesses and occasional ulceration, with marked epithelial hyperplasia, mucin depletion
  • grade 5 marked transmural inflammation with severe ulceration and loss of intestinal glands.
  • Transferred naive and Treg cell populations were characterized by surface marker and intracellular staining for cytokines and Foxp3.
  • Eeukocytes recovered from recipient lymph node, spleen and gut lamina propria were re-stimulated before surface staining for CD4, CD45.1 and CD45.2.
  • EAE was induced in Nrnl _/ and Nrnl +/ mice or in wt C57BE/6 mice by s.c. injection of 200pg MOG35-55 peptide in an emulsion of complete Freund Adjuvant oil (500 pg M.
  • mice received 400 ng pertussis toxin i.p. on days 0 and 2 after immunization. Clinical signs of EAE were assessed daily according to the standard 5 point scale 63 . Anti-Nrnl Ab or isotype control
  • Primary murine cells were cultured in 10%RPMI and were stimulated by indicated dose of plate bound CD3 and CD28 in a 96-well round bottom plate at lxl0 5 cells /well.
  • 5x10 s to 2xl0 6 cells/ml were activated in 24 well plate with 5pg/ml plate bound anti-CD3, 10011 I/ml IL2 and 4pg/ml anti-CD28 in the presence of 5 pg/mL of exogenous TGF .
  • gcggtgcaaatagcttacctg forward, SEQ ID NO: 1
  • cggtcttgatgttcgtcttgtc reverse, SEQ ID NO:
  • RNAseq analysis At least lOOng total RNA was extracted from sorted cells using an Qiagen RNEASY Kit (QIAGEN) according to the manufacturer’s instructions. Samples were randomly primed and prepared based on the manufacturer’ s recommendation (NEBNext Ultra RNA Library Prep Kit for Illumina). Samples were pooled and sequenced on a HiSeq with a read length configuration of 150 PE. Samples were sequenced and analyzed by Admera Health (South Plainfield, NJ). Sample quality was checked by FastQC vO.10.1. The reads were mapped to the GRCM38 and reported as fragments per kilobase of transcript per million mapped reads units.
  • the heatmap was generated using the Morpheus software from the Broad Institute, Cambridge, MA (broadinstitute.org/morpheus/). Differentially expressed transcripts were identified using the sleuth R package. In addition to a sleuth p- value ⁇ 0.05, expression > 1.0 FPKM was required on average across the samples. GSEA was employed in the context of gene ontology. Enrichment analysis with MSigDB gene sets was computed as the
  • Thl anergy gene set was also generated from upregulated genes in anergized Thl cells comparing to the control activated Thl cells based on the GSE46243 data sets using NCBI GE02R 47 ⁇ Genes overexpressed in anergized Thl cells with Benjamini & Hochberg false discover rate P ⁇ 0.001 were selected into Thl anergy gene set.
  • B16F10 melanoma, EL4 lymphoma, CT26 colon carcinoma and 4T1 breast cancer cell lines were purchased from ATCC and were culture according to ATCC instructions.
  • MC38 was cultured in 10% DMEM.
  • cells were trypsinized, washed and resuspended in PBS at an indicated concentration. While maintaining
  • mice were injected s.c. into the shaved right flanks of age-, sex- matched, female mice of the indicated strains (B16F10 and EL4). 3-4xl0 5 cells were inoculated in CT26, MC38 and 4T1 experiment. Tumor volume was calculated every 2-3 days by the following formula, 0.5xLxW 2 . Length (L) and width (W) measurements were taken perpendicular to one another, with length describing the longer measurement. Measurements were made using a digital caliper. At indicated time points post injection, cohorts of mice were euthanized. For the antibody study, the mice received an i.p.
  • Nrnl is highly expressed in anergic T cells and Treg.
  • a system was used to identify tolerance- associated genes. The gene expression patterns associated with either a T effector/memory response or tolerance induction both triggered by the same antigen but under divergent in vivo conditions were compared 24 .
  • the T effector/memory response or tolerance induction both triggered by the same antigen but under divergent in vivo conditions were compared 24 .
  • effector/memory response was induced by influenza hemagglutinin (HA) antigen-specific TCR transgenic CD4 T cells transferred into WT recipients and activated by HA-expressing vaccinia-HA (Vac-HA) virus.
  • Tolerance induction was achieved by the same TCR transgenic T cells when adoptively transferred into hosts transgenically expressing HA as self antigen (C3-HA mice) 24 .
  • One of the most highly differentially expressed genes upregulated under the in vivo anergy inducing conditions was Nml 26 29 ⁇ Nml expression by in vivo tolerized HA-specific T cells recovered at different time points after transfer was confirmed by qRT-PCR (FIG. 1A). While Nrnl was highly expressed in all time points by cells recovered from C3-HA hosts, it was minimally expressed in T cells recovered from VacHA infected mice (FIG. 1A).
  • Nrnl expression was examined in the classic AE7 in vitro T cell clonal anergy model 36 .
  • stimulation of a Thl cell line with TCR signal alone results in the development of anergy, whereas stimulation with TCR signal in the presence of
  • anti-CD28 results in cell activation and expansion. Nrnl expression was induced under anergy-inducing conditions (stimulation with anti-CD3 alone) within 9 hours with levels peaking after 24 hours. In contrast, in vitro activation (treatment with both anti-CD3 and anti-CD28) failed to significantly enhance Nml expression (FIG. IB).
  • CD4 + CD44 + FR4 CD73- cells recovered from mice under steady state conditions (FIG. 1C). Nml protein expression was also detected by cell surface staining with an anti-Nrnl antibody among CD44 + FR4 hi CD73 hi CD4 cells (FIG. ID).
  • Nrnl is expressed by Foxp3 + Treg.
  • FACS-isolated from murine lymphoid tissues revealed Foxp3 + /CD25 + Tregs indeed express Nrnl, an observation confirmed by flow cytometric staining of surface Nml protein (FIGS. 7A, 7B). Foxp3 + Treg are known to be heterogeneous in vivo.
  • Treg Resting or central Treg
  • cTreg are characterized by expression of high levels of CD62L and lower expression of CD44, primarily localized in secondary lymphoid tissues 37 .
  • Tregs acquire an effector-like phenotype (eTregs), characterized by heightened suppressive potency, high expression of CD44, GITR, ICOS, chemokine receptors, and preferential accumulation at peripheral tissue sites, such as tumor tissues 38 39,4041 .
  • eTregs effector-like phenotype
  • Nml message was highly expressed by induced Treg (iTreg) generated in vitro (FIG. IE). Nml RNA levels are much higher in CD62L low eTreg than CD62L hlgh cTreg under steady state (FIG. IF). Consistent with a link between Nrnl expression and the process of Treg activation, in vitro activation with TCR signal and IL2 resulted in considerable Nml upregulation by purified CD44 low /CD62L hlgh Tregs. Activation of CD44 hlgh /CD62L low Tregs resulted in similarly enhanced Nml levels (FIG. IF).
  • Nrnl is important for the induction of T cell anergy and pTreg development in vivo.
  • T cell anergy refers to a hyporesponsive state induced in Tconv as a result of certain conditions 7 including suppression by Treg during their activation 11 13 .
  • Tconv that are suppressed by Treg can develop anergy and convert to pTreg (a process associated with Foxp3 induction), thereby amplifying“infectious tolerance” 10 14 ’ 42 .
  • anergy among Tconv cells can also be induced upon encounter persistent self antigen in the absence of Treg cells 9 10 ’ 43 .
  • the high expression of Nml in anergic T cells (FIGS.
  • Nrnl _/ mice were obtained. Nml deficiency does not interfere with thymocytes development nor bone marrow B cell development; similar proportions of T and B cells were observed in wild type (WT, Nml +/+ ) and Nrnl _/ mice (data not shown). OVA antigen specific TCR transgenic OTII mice were crossed onto the Nml _/ background to facilitate the observation of antigen specific CD4 cell response in the absence of Nml.
  • OTII TCR transgenic mice Upon crossing with OTII TCR transgenic mice, there was comparable development of OTII CD4 T cells, and comparable reduction of the Foxp3 + Treg population due to TCR transgenic expression (2-4% in OTII transgenic mice vs 10-15% in wt C57BL/6 mice) in both Nml _/ or Nml +/_ offspring.
  • Nrnl The first potential role of Nrnl in CD4 cell anergy development that was analyzed herein, was the susceptibility of CD4 Tconv cells to Treg mediated suppression during antigen specific anergy induction in vivo.
  • the classic soluble intravenous (i.v.) peptide injection anergy-induction model was used 14 .
  • OTII+Nrnl 7 or control OTII+Nml +/_ CD4 Tconv cells, depleted of CD25 hl Treg were cotransferred with wt Treg into TCRa knockout mice (TCRoU) (FIG. 2A).
  • Transferred OTII Tconv cells could be distinguished from co-transferred Treg via distinct congenic markers (OTII Thyl.U; Treg Thyl.2 + ).
  • Reconstituted mice were injected i.v. with soluble OVA peptide every three days for three treatments - a standard protocol to induce anergy 7 14 .
  • a component of anergy induction by soluble i.v. antigen involves Treg mediated suppression operating upon antigen- specific Tconv cells 7 ’ 22 ’ 23 .
  • Mice were harvested on day 13 after adoptive transfer (ADT) and OTII T cell expansion, cytokine production and conversion to pTreg (Foxp3 induction) were examined.
  • Nrnl _/ T cells showed increased expansion evidenced by increased percentage of OTII+Nml 7 cells in the CD4 + population both in lymph node and spleen (FIG. 2B). The total number of OTIRNml 7 cells was also increased compared to OTII Nml +/_ T cells (FIG. 2C). Upon OVA peptide restimulation in vitro, OTII Nml _/ cells produced significantly more IL2 than control OTII Nml +/ cells (FIG. 2D). Moreover, OTII Nml _/ T cell demonstrated significantly reduced conversion to Foxp3 + Treg relative to control (FIG. 2E).
  • a second tolerance system was used to study the role of Nml in anergy induction and the generation of pTreg among polyclonal T cells (to complement the findings made in the monoclonal OTII system described above) in the absence of Treg suppression.
  • Persistent self antigen has been shown to induce tolerance among naive self-Ag specific T cells and to mediate their conversion into pTreg 43 .
  • Transient depletion of Treg in adult mice can result in self-reactive CD4 + T cell responses, followed by recovery of self-tolerance accompanied by reemergence of Treg and reestablishment of anergy among peripheral Tconv cells in 10- 14 days 44 .
  • Nrnl regulates Treg reemergence and the restoration of self-tolerance was used to determine whether Nrnl regulates Treg reemergence and the restoration of self-tolerance.
  • Nml _/ mice were crossed onto a Foxp3DTRgfp (FDG) background where Treg can be deleted by administration of diphtheria toxin (DT) due to the expression of diphtheria toxin receptor (DTR) 45
  • T cells from Nml _/ or Nml +/_ control mice on the FDG background were co-transferred into congenic CD45.1expressing FDG wt hosts as outlined in FIG. 2F. Because all Treg (host and adoptively transferred) in this system express DTR, DT administration after T cell transfer completely eliminates all Treg from the adoptively transferred mice. Because adoptively transferred T cells are congenically distinguished from endogenous T cells, this enabled the evaluation of both cell-intrinsic and cell-extrinsic effects of Nrnl deficiency on anergy induction as well as conversion of Tconv to pTreg.
  • DT was administered the day after T cell transfer to delete Treg from both transferred T cells and endogenous T cells. Mice were harvested 11 days after DT treatment, at the recovery phase from the autoimmune response induced by Treg cell depletion 44 . De novo Foxp3 + Treg generation was consistently reduced among Nrnl 7 cells compared to Nml +/ cells (FIG. 2G). Anergic T cell populations (FR4 hl CD73 hl ) were also reduced (FIG. 2H).
  • Nml _/ T cells have an intrinsic defect in generating Foxp3 + Treg and FR4 hl CD73 hl anergic cells under this self-reactivity model, it was also found that they negatively impacted the generation of endogenous Treg and anergic cells.
  • T cells in the host mice (CD45.1 + ) receiving adoptive transfer of Nml _/ T cells consistently showed reduced generation of Foxp3 + and FR4 hl CD73 hl cells among CD4 cells relative to those receiving Nrnl +/ control T cells (FIGS. 2G, 2H). They also showed higher levels of proliferation as indicated by Ki67 staining and higher levels of Thl generation evidenced by IFNy and Tbet staining (FIGS.
  • Nrnl deficiency not only caused a cell-intrinsic defect in generation of Foxp3 + and FR4 hl CD73 hl anergic T cells, it also resulted in cell extrinsic effects impacting the proinflammatory capacity of wt T cells.
  • CD2-Nml CD2N mice
  • FIG. 8C CD2N mice
  • an elevated level of Nrnl expression was detected by mRNA and surface staining among CD3 + cells from CD2N mice
  • FIG. 8D CD2N mice were also crossed to the FDG background to generate CD2N-FDG mice.
  • CD2N-FDG T cell responses to self-antigen upon transfer into FDG mice were tested as outlined in FIG. 2F.
  • Nrnl expression by Treg supports their in vivo suppressive function. Having demonstrated a role for Nrnl in anergy induction and pTreg conversion, the importance of this molecule in the suppressive function of Tregs, was further explored. Nrnl was found to be highly expressed by Treg and in particular by activated eTreg (FIGS. IE, IF, 1H).
  • Nrnl may also participate in the acquisition of the eTreg phenotype and consequent upregulation of suppressive potency. It was first investigated whether eTreg can be effectively activated and converted to eTreg in the absence of Nml in vitro.
  • CD44 Low /CD62L Hlgh cTreg marked with GFP were sorted from Nml _/ or Nml +/_ mice on the FDG background and activated by anti- CD3 and CD28 in vitro. Indeed, activation of purified Nml _/ cTreg resulted in less efficient generation of CD44 Hlgh /CD62L Low cells compared to the activation of Nml +/ cTreg (FIG.
  • Treg T cell- induced colitis model was used to test the importance of Nml for the control of inflammation by Treg.
  • naive CD62L H 7CD25
  • CD4 + T cells lymphopenic recipient mice
  • Treg isolated from Nml _/ and their littermates Nrnl +/ were transferred into recipient mice cohorts, and their ability to ameliorate colitis was determined.
  • Nrnl +/ Treg prevented further development of colitis, which progressed unabated in control mice not receiving Treg.
  • Treg isolated from Nrnl _/ mice failed to rescue recipients from severe colitis, evidenced by wasting and colon pathology (FIGS. 3B-3D). Additionally, proinflammatory cytokine-producing T cells (IRNg or IL-17) were more prevalent in the gut-draining lymph node and lamina propia of mice receiving Nrnl _/ Treg compared to Nml +/_ Treg recipients (FIGS. 3E, 3F). Tracking the fate of injected Treg revealed that Nrnl deficiency significantly reduced the number of Treg present in these tissues as well as the expression of Foxp3 (FIGS. 3G, 3H).
  • IRNg or IL-17 proinflammatory cytokine-producing T cells
  • Nrnl _/ mice unlike their wt counterparts, failed to control inflammation in this widely used model of multiple sclerosis. Specifically, Nml _/ mice showed no partial recovery during the late stages of disease, when Treg normally accumulate in affected tissues and mediate their suppressive activity 46 (FIG. 31).
  • Nrnl affects anergy and Treg cell signature gene expression.
  • gene expression profiles were compared between Nml _/ and Nrnl +/ T cells under different conditions.
  • an RNAseq analysis of soluble peptide anergized OTII+Nrnl 7 vs OTII+Nml +/ T cells that were cotransferred with wt Treg into TCRa _/ host mice was performed as outlined in FIG. 2A. 868 genes were revealed gene categories related to receptor binding as the most affected category upon loss of Nrnl.
  • Kegg pathway analysis also provided evidence that defects in the T cell receptor signaling pathway and the cytokine- receptor interaction pathways were among the top effects of Nml deletion in anergized T cells (FIGS. 10A, 10B).
  • GSEA Gene set enrichment analysis
  • Nrnl +/_ cells While the gene set enriched in Treg was prominent in Nrnl +/_ cells, it was not in Nrnl _/ cells, consistent with a defect in pTreg cell generation by Nml _/ cells (FIG. 4C).
  • Genes upregulated in Nml +/_ T cells overlapping with the Thl anergy gene set are shown in the heatmap in FIG. 4D.
  • PD-1, Lag3, ICOS and CD73 are all down regulated in Nrnl 7 T cells.
  • SLC9b2, SLC4al, SlOOal and NSMf are also reduced in Nrnl 7 T cells (FIG. 10D).
  • GSEA analysis against NFAT target genes reveal reduced NFAT target gene expression among Nrnl 7 OTII cells (FIG. 4E), providing evidence of reduced or unbalanced NFAT signaling.
  • Treg were also sorted from Nrnl 7 or Nml +/ mice under steady state and subjected them to RNAseq gene expression profile comparison analysis. Consistent with reduced Treg cell suppression function in vivo, GSEA analysis showed reduced expression of Treg cell signature genes (FIG. 4F). NFAT signaling plays a significant role in Treg cell suppression function 50 . Similar to the finding of Nrnl 7 OTII cell gene expression under the anergy setting (FIG. 4E), Nrnl 7 eTreg showed reduced expression of genes in the NFAT target gene set (FIG. 4G).
  • Nrnl 7 vs Nml +/ cells The reduced expression of anergy related signature genes in Nrnl 7 vs Nml +/ cells are consistent with the phenotype of compromised T cell anergy induction.
  • Treg cell expression profile analysis between Nrnl 7 and Nml +/ cells also reveals reduced Treg signature gene expression in the absence of Nrnl expression, explaining the defective suppressive function of Nrnl 7 Tregs in vivo.
  • Nrnl has been shown to modulate NFAT down- regulated and 432 genes were up-regulated in Nrnl 7 OTII cells compared to Nml +/ OTII cells (FIG. 4A).
  • Nrnl 7 down-regulated genes signaling pathway in neurons and NFAT signaling is important for both anergy development and Treg function 50 52 .
  • GSEA analysis reduction in the NFAT target signature genes was found in Nrnl 7 T cells recovered from anergic conditions and in eTreg recovered from Nrnl 7_ mice under steady state.
  • Nrnl deficiency or blockade alone or in combination with CTLA4 and PD1 blockade inhibits tumor growth.
  • Tumor antigen specific T cells can quickly become anergized or convert to a pTreg fate in the cancer setting 5 53 ⁇
  • Treg facilitate the induction of anergy and pTreg cell differentiation within the pool of potential anti-tumor effector T cells during tumor emergence and progression 5 .
  • Nrnl 7 mice and their Nml +/ littermates were challenged with the moderately immunogenic EL4 thymoma and the aggressive, poorly immunogenic B16 melanoma subcutaneously (s.c).
  • Nml The important role for Nml in regulating the anti-cancer immune response was further evidenced by its expression in T cells in the tumor bearing mice.
  • TIFs B16F10 tumor infiltrating lymphocytes
  • Non-Treg CD4 (Foxp3 , gfp ) cells among the TIFs from these mice also show higher Nrnl expression compared to peripheral blood CD4 cells (FIG. 5G), an observation in line with Nrnl expression by multiple suppressor T cell populations.
  • 6.5 T cells specific for HA antigen were adoptively transferred into A20HA B cell lymphoma bearing mice or into wt mice followed by infection with VacHA virus. Nrnl is highly expressed in tumor antigen specific 6.5 T cells isolated from tumor bearing host compared to T cells activated by VacHA infection (FIG. 5H).
  • Treg cell suppression of adaptive immunity can contribute to tumor progression.
  • Rag2 /_ mice were reconstituted with Nml +/+ , CD45.1 + spleen and lymph node cells depleted of CD25 + Treg and co- transferred congenically marked Treg from Nml +/ or Nrnl _/ mice (CD45.1 ) before B16 tumor challenge. Tumors grew much slower in Nml _/ Treg recipients compared to those reconstituted with Nml +/_ controls (FIG. 51).
  • Nrnl _/ Treg effector cells
  • Nml is a promising target for tumor immunotherapy.
  • anti-Nml monoclonal antibody (mAh) treatment of B16F10 tumor bearing mice 5-6 days after implantation results in marked tumor growth inhibition relative to isotype control treatment (FIG. 6A and FIG. 12A). Similar trends were obtained in the MC38 (colon), CT26 (colon) and 4T1 (breast) tumor models (FIGS. 6B-6D and FIGS.
  • TILs from B16F10 tumors revealed an increased CD8/Treg ratio and increased expression of Tbet and effector cytokines production among CD 8 cells in mice treated with anti-Nrnl mAh (FIGS. 6E-6G).
  • triple combination treatment of anti-Nrnl with anti-CTLA4 and anti-PDl may allow comprehensive blockade against the development of anergy and exhaustion among tumor specific T cells, thus achieve further improvement in treatment efficacy.
  • administration of a triple combination of anti-Nml, anti-CTLA4 and anti-PDl mAh to mice with established B16F10 tumors resulted in a significant inhibition of tumor growth (FIGS. 6H, 61 and 13C).
  • Nml as a novel tolerance-promoting factor with a role in the generation and biology of two major suppressor T cell populations. They also clearly implicate Nml to be a high-value target for anti-tumor immunotherapy. Nml blockade results in enhanced CD8 cell tumor infiltration and effector function. Furthermore, combination of anti-Nml with checkpoint inhibition regimens can significantly improve tumor treatment efficacy.
  • Nml was discovered in neurons as an activity induced GPI-anchored membrane protein. It has been shown to promote synapse maturation in neurons 29,54 ⁇ No functional role of Nml in the immune system has been previously reported. Its expression in Treg and anergized Tconv cells, its role in maintaining Treg and anergy- dependent tolerance and the anti-tumor effects resulting from its deletion or functional blockade is characterized here. Indeed, increased Nml expression has been observed on various expression profiling lists, not only in CD4 + anergic and Treg 30 ⁇ 34 ⁇ 55 ⁇ but also in anergic CD 8 cells 31 34 . Any potential role for Nml in the biology of these immunoregulatory cells prior to the findings herein, have gone unexplored.
  • Nrnl in conventional CD4 cells results in compromised anergy induction and pTreg development in a number of different in vivo systems including a model of immune tolerance induced by soluble i.v. antigen and an autoimmune disease model triggered by transient Treg deletion (FIGS. 2A-2K).
  • the defective anergy induction among Nrnl _/ cells was accompanied with reduced expression of anergy and Treg related genes as revealed by GSEA analysis (FIGS. 4B, 4C).
  • loss of Nml in Treg resulted in reduced Treg cell signature gene expression and compromised suppression function in vivo (FIGS. 3A-3J; FIG. 4F).
  • Treg facilitate the induction of anergy, and anergic T cells are prone to develop into pTreg and spread self-antigen-specific suppression 3 .
  • Nml contributes to the maintenance of peripheral tolerance through both T cell anergy and Treg cell suppression.
  • Nrnl may affect T cell function intrinsically or it may act extrinsically affecting neighboring cell function, impacting the establishment of tolerance environment.
  • FIGS. 2E, 2G reduced pTreg generation in the absence of Nml
  • Nrnl may also have a cell-extrinsic effect influencing the establishment of tolerance environment.
  • Nml has been shown to function in the CNS both when it is attached on the cell surface and as a soluble protein 26,29 .
  • Nrnl-Fc fusion protein binding of Nrnl to various cell types including dendritic cells was observed (data not shown).
  • Nml has been shown to increase calcium concentration and NFAT activation in the granule neurons of the cerebellum 51 .
  • Reduced NFAT target gene expression was found in Nml 7 Tconv cells and NrnT /_ Foxp3+ effector Treg (FIGS. 4E, 4G).
  • NFAT also contributes to the activation of the anergic gene expression program in Tconv cells and NFAT1 deletion results in defective anergy induction 52 .
  • NFAT can interact with Foxp3 and facilitate Treg cell suppression function 50 .
  • Dysregulation of calcium mobilization can contribute to reduced NFAT activation and target gene transcription, thus compromising T cell anergy development and Treg cell suppression.
  • Nrnl has been identified in the AMPA-type ionotropic glutamate receptor (AMPAR) complex 54,57 .
  • AMPAR ionotropic glutamate receptor
  • the ionotropic AMPAR can permeate calcium directly or indirectly 58 .
  • Nml may affect calcium mobilization through AMPAR. It remains to be determined how the function of Nrnl is related to AMPAR in the immune system.
  • CTLA-4 and PD- 1 are both immune checkpoint inhibitors that have shown clear clinical efficacy but have different functional mechanisms 59 .
  • CTLA-4 primarily attenuates T cell activation in the priming phase through cell intrinsic and extrinsic mechanisms.
  • PD-1 appears on exhausted T cells and primarily attenuates T cell activity in peripheral tissues 60 .
  • Nrnl as a novel target and our strategy of combination therapy targeting different aspect of tumor specific T cell dysfunction can significantly improve the outcome of tumor therapy.
  • Nml was identified as a novel cell surface molecule with preferential expression by suppressive T cell populations and clear function in T cell mediated immune tolerance in vivo.
  • Nrnl is highly expressed in anergic T cells and Treg, deletion of which affect CD4 T cell anergy development and Treg cell suppression function.
  • Nml blockade can enhance anti-tumor adaptive T cell responses and combination therapy with anti-CTLA4 and anti-PDl greatly enhances treatment efficacy to an aggressive, non-immunogenic murine tumor model.
  • Egr2 early growth response gene 2

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Abstract

La présente invention se rapporte à la neuritine (Nml), une molécule de surface d'ancrage GPI conservée, importante pour l'excroissance des neurites et la maturation des synapses dans les neurones, qui est fortement exprimée dans les lymphocytes T anergiques et les Treg, dans la périphérie et le microenvironnement tumoral. L'invention concerne des compositions pour l'annulation de la suppression d'une réponse immunitaire spécifique d'un antigène comprennent des inhibiteurs de neuritine et des inhibiteurs de point de contrôle.
PCT/US2020/013151 2019-01-11 2020-01-10 Régulation de la neuritine de l'anergie des lymphocytes t et de la fonction des lymphocytes t régulateurs WO2020146772A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100040636A1 (en) * 2005-09-09 2010-02-18 The Johns Hopkins University Manipulation of Regulatory T Cell and Dc Function By Targeting Neuritin Gene Using Antibodies, Agonists and Antagonists
WO2018186924A1 (fr) * 2017-01-17 2018-10-11 The University Of Chicago Cellules t cd8+ spécifiques d'un antigène dysfonctionnelles dans le microenvironnement tumoral
WO2018191676A1 (fr) * 2017-04-13 2018-10-18 Galera Labs, Llc Poly-immunothérapie anticancéreuse basée sur un complexe de type cycle macrocyclique pentaaza
US20180312929A1 (en) * 2013-03-15 2018-11-01 The University Of Chicago Methods and compositions related to t-cell activity
US20180312809A1 (en) * 2006-09-13 2018-11-01 The Trustees Of Columbia University In The City Of New York Agents and methods to elicit anti-tumor immune response

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100040636A1 (en) * 2005-09-09 2010-02-18 The Johns Hopkins University Manipulation of Regulatory T Cell and Dc Function By Targeting Neuritin Gene Using Antibodies, Agonists and Antagonists
US20180312809A1 (en) * 2006-09-13 2018-11-01 The Trustees Of Columbia University In The City Of New York Agents and methods to elicit anti-tumor immune response
US20180312929A1 (en) * 2013-03-15 2018-11-01 The University Of Chicago Methods and compositions related to t-cell activity
WO2018186924A1 (fr) * 2017-01-17 2018-10-11 The University Of Chicago Cellules t cd8+ spécifiques d'un antigène dysfonctionnelles dans le microenvironnement tumoral
WO2018191676A1 (fr) * 2017-04-13 2018-10-18 Galera Labs, Llc Poly-immunothérapie anticancéreuse basée sur un complexe de type cycle macrocyclique pentaaza

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