WO2018160572A1 - Méthodes de traitement du cancer comprenant des agents se liant au gitr - Google Patents

Méthodes de traitement du cancer comprenant des agents se liant au gitr Download PDF

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Publication number
WO2018160572A1
WO2018160572A1 PCT/US2018/019970 US2018019970W WO2018160572A1 WO 2018160572 A1 WO2018160572 A1 WO 2018160572A1 US 2018019970 W US2018019970 W US 2018019970W WO 2018160572 A1 WO2018160572 A1 WO 2018160572A1
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Prior art keywords
gitr
tumor
cancer
binding agent
subject
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PCT/US2018/019970
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English (en)
Inventor
Ann M. Kapoun
Min Wang
Belinda CANCILLA
Inkyunk PARK
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Oncomed Pharmaceuticals, Inc.
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Publication of WO2018160572A1 publication Critical patent/WO2018160572A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention generally relates to methods for treating cancer using agents that bind human GITR, particularly agents comprising one or more copies of the extracellular domain of GITRL.
  • the basis for immunotherapy is the manipulation and/or modulation of the immune system, including both innate immune responses and adaptive immune responses.
  • the general aim of immunotherapy is to treat diseases by controlling the immune response to a "foreign agent", for example a pathogen or a tumor cell.
  • a "foreign agent” for example a pathogen or a tumor cell.
  • immunotherapy is used to treat autoimmune diseases which may arise from an abnormal immune response against proteins, molecules, and/or tissues normally present in the body.
  • Immunotherapy may include methods to induce or enhance specific immune responses or to inhibit or reduce specific immune responses.
  • the immune system is a highly complex system made up of a great number of cell types, including but not limited to, T-cells, B-cells, natural killer cells, antigen-presenting cells, dendritic cells, monocytes, and macrophages. These cells possess complex and subtle systems for controlling their interactions and responses.
  • the cells utilize both activating and inhibitory mechanisms and feedback loops to keep responses in check and not allow negative consequences of an uncontrolled immune response (e.g., autoimmune diseases or a cytokine storm).
  • cancer immunosurveillance is based on the theory that the immune system can recognize tumor cells, mount an immune response, and suppress the development and/or progression of a tumor.
  • cancerous cells have developed mechanisms to evade the immune system which can allow for uninhibited growth of tumors.
  • Cancer/tumor immunotherapy focuses on the development of new and novel agents that can activate and/or boost the immune system to achieve a more effective attack against tumor cells resulting in increased killing of tumor cells and/or inhibition of tumor growth.
  • the present invention provides methods for treatment of cancer (i.e., inhibiting tumor growth) using agents that bind human glucocorticoid-induced tumor necrosis factor-related protein (GITR; TNFRSF18).
  • GITR human glucocorticoid-induced tumor necrosis factor-related protein
  • the term "agent” includes, but is not limited to, polypeptides, fusion proteins, homodimeric molecules, heterodimeric molecules, and bispecific molecules. Generally, the agents may be referred to as "GITR-binding agents".
  • the GITR-binding agent is a GITR agonist.
  • the GITR-binding agent is an agonist polypeptide.
  • the GITR-binding agent is an agonist antibody.
  • the GITR-binding agent comprises a soluble GITR ligand (GITRL). In some embodiments, the GITR- binding agent comprises at least one copy of the extracellular domain of GITRL. In some embodiments, the GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL. In some embodiments, the GITR-binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of GITRL. In some embodiments, the GITR-binding agent comprises a single chain polypeptide comprising (a) a trimer of the extracellular domain of GITRL and (b) a human Fc region.
  • GITRL soluble GITR ligand
  • the invention provides methods that comprise using the GITR-binding agents for cancer immunotherapy or immuno-oncology.
  • a method comprises using a GITR-binding agent (e.g., wherein the agent comprises an extracellular domain of GITRL or is a GITRL agonist antibody) to induce, activate, promote, increase, enhance, or prolong an immune response.
  • a method comprises using a GITR-binding agent to induce, activate, promote, increase, enhance, or prolong an immune response to cancer, a tumor, and/or tumor cells.
  • a method comprises using a GITR-binding agent to promote the formation of tertiary lymphoid structures within a tumor or tumor microenvironment. In some embodiments, a method comprises using a GITR-binding agent to inhibit the growth of a tumor or tumor cells. In some embodiments, a method comprises using a GITR-binding agent for the treatment of cancer. In some embodiments, a method comprises inhibiting the growth of cancer cells.
  • the invention also provides compositions comprising the GITR-binding agents described herein. In some embodiments, the compositions are pharmaceutical compositions comprising the GITR-binding agents described herein. Polynucleotides encoding the agents and methods of making the agents are also provided.
  • the invention provides methods of inhibiting tumor growth comprising contacting a tumor or tumor cell with an effective amount of a GITR-binding agent described herein.
  • a method of inhibiting growth of a tumor comprises contacting a tumor microenvironment with an effective amount of a GITR-binding agent.
  • a method of inhibiting tumor growth in a subject comprising administering to the subject a
  • GITR-binding agent described herein
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a GITR-binding agent (e.g., wherein the agent comprises an extracellular domain of GITRL or is a GITRL agonist antibody), wherein the tumor is a head and neck cancer, an esophageal cancer, a gastric cancer, a melanoma, a uterine cancer, a non-small cell lung cancer (NSCLC), a NSCLC adenocarcinoma, a NSCLC squamous cell carcinoma, a triple negative breast cancer, a hepatocellular carcinoma (HCC), or a microsatellite instability (MSI)-high tumor.
  • a GITR-binding agent e.g., wherein the agent comprises an extracellular domain of GITRL or is a GITRL agonist antibody
  • the tumor is a head and neck cancer, an esophageal cancer, a gastric cancer, a melanom
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a GITR-binding agent, wherein the tumor is a head and neck cancer, an esophageal cancer, a gastric cancer, a melanoma, a uterine cancer, a non-small cell lung cancer (NSCLC), a NSCLC adenocarcinoma, a NSCLC squamous cell carcinoma, a triple negative breast cancer, a hepatocellular carcinoma (HCC), or a MSI-high tumor; and wherein the GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • the tumor is a head and neck cancer, an esophageal cancer, a gastric cancer, a melanoma, a uterine cancer, a non-small cell lung cancer (NSCLC), a NSCLC adenocarcinoma, a NSCLC squa
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor is a head and neck cancer, an esophageal cancer, a gastric cancer, a melanoma, a uterine cancer, a non-small cell lung cancer (NSCLC), a NSCLC
  • a human GITR-binding agent wherein the tumor is a head and neck cancer, an esophageal cancer, a gastric cancer, a melanoma, a uterine cancer, a non-small cell lung cancer (NSCLC), a NSCLC
  • GITR-binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of human GITRL.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor is a head and neck cancer, an esophageal cancer, a gastric cancer, a melanoma, a uterine cancer, a non-small cell lung cancer (NSCLC), a NSCLC adenocarcinoma, a NSCLC squamous cell carcinoma, a triple negative breast cancer, a hepatocellular carcinoma (HCC), or a MSI-high tumor; and wherein the GITR-binding agent comprises a single chain polypeptide comprising (a) a trimer of the extracellular domain of human GITRL and (b) a human Fc region.
  • the tumor is a head and neck cancer, an esophageal cancer, a gastric cancer, a melanoma, a uterine cancer, a non-small cell lung cancer (NSCLC), a NSCLC
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor is an esophageal cancer, a non-small cell lung cancer (NSCLC), a NSCLC adenocarcinoma, a NSCLC squamous cell carcinoma, a triple negative breast cancer, or a hepatocellular carcinoma (HCC); and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • NSCLC non-small cell lung cancer
  • HCC hepatocellular carcinoma
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor is a head and neck tumor, a gastric tumor, a melanoma, a uterine tumor, or a MSI-high tumor; and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent (e.g., wherein the agent comprises an extracellular domain of GITRL or is a GITRL agonist antibody), wherein the tumor expresses GITR.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR- binding agent, wherein the tumor expresses GITR; and wherein the GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor expresses GITR; and wherein the GITR-binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a
  • a human GITR-binding agent wherein the tumor expresses GITR; and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent (e.g., wherein the agent comprises an extracellular domain of GITRL or is a GITRL agonist antibody), wherein the tumor is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist.
  • a human GITR-binding agent e.g., wherein the agent comprises an extracellular domain of GITRL or is a GITRL agonist antibody
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist; and wherein the GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist; and wherein the GITR-binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of human GITRL.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist; and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent (e.g., wherein the agent comprises an extracellular domain of GITRL or is a GITRL agonist antibody), wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent.
  • a human GITR-binding agent e.g., wherein the agent comprises an extracellular domain of GITRL or is a GITRL agonist antibody
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent; and wherein the GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent; and wherein the GITR-binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of human GITRL.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent; and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • the invention provides methods of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent (e.g., wherein the agent comprises an extracellular domain of GITRL or is a GITRL agonist antibody), wherein the cancer is head and neck cancer, esophageal cancer, gastric cancer, melanoma, uterine cancer, non-small cell lung cancer (NSCLC), NSCLC adenocarcinoma, NSCLC squamous cell carcinoma, triple negative breast cancer, hepatocellular carcinoma (HCC), or a MSI-high cancer.
  • NSCLC non-small cell lung cancer
  • HCC hepatocellular carcinoma
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is head and neck cancer, esophageal cancer, gastric cancer, melanoma, uterine cancer, non-small cell lung cancer (NSCLC), NSCLC adenocarcinoma, NSCLC squamous cell carcinoma, triple negative breast cancer, hepatocellular carcinoma (HCC), or a MSI-high cancer; and wherein the GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • the cancer is head and neck cancer, esophageal cancer, gastric cancer, melanoma, uterine cancer, non-small cell lung cancer (NSCLC), NSCLC adenocarcinoma, NSCLC squamous cell carcinoma, triple negative breast cancer, hepatocellular carcinoma (HCC), or a MSI-high cancer
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is head and neck cancer, esophageal cancer, gastric cancer, melanoma, uterine cancer, non-small cell lung cancer (NSCLC), NSCLC adenocarcinoma, NSCLC squamous cell carcinoma, triple negative breast cancer, hepatocellular carcinoma (HCC), or a MSI-high cancer; and wherein the GITR-binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of human GITRL.
  • the cancer is head and neck cancer, esophageal cancer, gastric cancer, melanoma, uterine cancer, non-small cell lung cancer (NSCLC), NSCLC adenocarcinoma, NSCLC squamous cell carcinoma, triple negative breast cancer, hepatocellular carcinoma (HCC), or a MSI-high cancer
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is an esophageal cancer, a non-small cell lung cancer (NSCLC), a NSCLC adenocarcinoma, a NSCLC squamous cell carcinoma, a triple negative breast cancer, or a hepatocellular carcinoma (HCC); and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • the cancer is an esophageal cancer, a non-small cell lung cancer (NSCLC), a NSCLC adenocarcinoma, a NSCLC squamous cell carcinoma, a triple negative breast cancer, or
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is head and neck cancer, gastric cancer, melanoma, uterine cancer, or MSI-high cancer; and wherein the
  • GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent (e.g., wherein the agent comprises an extracellular domain of GITRL or is a GITRL agonist antibody), wherein the cancer expresses GITR.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer expresses GITR; and wherein the GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer expresses GITR; and wherein the GITR- binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of human GITRL.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer expresses GITR; and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist; and wherein the GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist; and wherein the GITR-binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of human GITRL.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist; and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent; and wherein the GITR- binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent; and wherein the GITR-binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of human GITRL.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR- binding agent, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent; and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • a method of treating cancer or inhibiting tumor growth in a subject comprises administering to the subject any of the GITR-binding agents described herein, wherein the cancer or tumor is selected from the group consisting of: lung, non-small cell lung cancer (NSCLC), NSCLC adenocarcinoma, NSCLC squamous cell carcinoma, liver, hepatocellular carcinoma, breast, triple negative breast cancer, renal cell carcinoma, kidney, prostate, gastrointestinal, gastric, melanoma, cervical, bladder, glioblastoma, head and neck, pancreatic, ovarian, colorectal, endometrial, and esophageal.
  • the tumor is a head and neck tumor.
  • the tumor is a gastric tumor. In some embodiments, the tumor is an esophageal tumor. In some embodiments, the tumor is a melanoma. In some embodiments, the tumor is a triple negative breast tumor. In some embodiments, the tumor is a hepatoma/hepatocellular carcinoma. In some embodiments, the tumor is a MSI (microsatellite instability) high tumor. In some embodiments, the tumor is a NSCLC tumor. In some embodiments, the tumor is a uterine tumor. In certain embodiments of the methods described herein, the cancer or tumor is resistant or refractory to treatment with an antagonist anti-PD-1 antibody.
  • the cancer or tumor is resistant or refractory to treatment with an antagonist anti-PD-Ll antibody.
  • the subject has previously been treated with an antagonist anti-PD-1 antibody.
  • the subject has previously been treated with an antagonist anti-PD-Ll antibody.
  • the cancer or tumor recurrence is local recurrence, regional recurrence, or distant recurrence.
  • the cancer or tumor comprises tumor-infiltrating lymphocytes (TILs).
  • the cancer or tumor comprises regulatory T- cells (Tregs).
  • the cancer or tumor comprises tertiary lymphoid structures.
  • a method of treating cancer or inhibiting tumor growth in a subject comprises administering to the subject any of the GITR-binding agents described herein, wherein the cancer or tumor is selected from the group consisting of head and neck squamous cell carcinoma, lung squamous cell carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, uterine corpus endometrial carcinoma, lung adenocarcinoma, breast invasive carcinoma, esophageal carcinoma, bladder urothelial carcinoma, uterine carcinosarcoma, testicular germ cell tumor, thymoma, thymic carcinoma, cholangiocarcinoma, mesothelioma, sarcoma, ovarian serous cystadenocarcinoma, skin cutaneous melanoma, thyroid carcinoma, stomach adenocarcinoma, pancreatic adenocarcinoma, prostate adenocarcinoma
  • the GITR-binding agent comprises SEQ ID NO: 3. In some aspects and embodiments of the methods described herein, the GITR-binding agent comprises SEQ ID NO:6. In some aspects and embodiments of the methods described herein, the human Fc region is from an IgGl, IgG2, IgG3, or IgG4 immunoglobulin. In some aspects and embodiments of the methods described herein, the human Fc region comprises SEQ ID NO: 9. In some aspects and embodiments of the methods described herein, the GITR-binding agent comprises SEQ ID NO:8. In some aspects and embodiments of the methods described herein, the stalk region of GITRL comprises LQLETAK (SEQ ID NO:4).
  • the GITR-binding agent comprises a polypeptide encoded by the plasmid deposited with ATCC as Designation No. PTA-122112.
  • the method increases T effector cells within the cancer or tumor. In some aspects and embodiments of the methods described herein, the method increases T effector cells within the microenvironment of the cancer or tumor. In some aspects and embodiments of the methods described herein, the method increases T effector cell activity within the cancer or tumor. In some aspects and embodiments of the methods described herein, the method increases T effector cell activity within the microenvironment of the cancer or tumor.
  • the method depletes regulatory T-cells within the cancer or tumor. In some aspects and embodiments of the methods described herein, the method depletes regulatory T-cells within the microenvironment of the cancer or tumor. In some aspects and embodiments of the methods described herein, the method inhibits and/or decreases the suppressive activity of Tregs within the cancer or tumor. In some aspects and embodiments of the methods described herein, the method inhibits and/or decreases the suppressive activity of Tregs within the microenvironment of the cancer or tumor. In some aspects and embodiments of the methods described herein, the method promotes the formation of tertiary lymphoid structures within the cancer or tumor or within the microenvironment of the cancer or tumor.
  • the invention provides methods of modulating the immune response of a subject.
  • the invention provides a method of inducing an immune response in a subject comprising administering a GITR-binding agent described herein.
  • the invention provides a method of activating an immune response in a subject comprising administering a GITR-binding agent described herein.
  • the invention provides a method of promoting an immune response in a subject comprising administering a GITR-binding agent described herein.
  • the invention provides a method of increasing an immune response in a subject comprising administering a GITR-binding agent described herein.
  • the invention provides a method of enhancing an immune response in a subject comprising administering a GITR-binding agent described herein. In some embodiments, the invention provides a method of prolonging an immune response in a subject comprising administering a GITR-binding agent described herein.
  • the immune response is to an antigenic stimulation. In some embodiments, the antigenic stimulation is a tumor or a tumor cell.
  • the invention provides a method of increasing the activity of immune cells.
  • the invention provides a method of increasing the activity of immune cells comprising contacting the cells with an effective amount of a GITR-binding agent described herein.
  • the immune cells are T-cells, NK cells, monocytes, macrophages, antigen-presenting cells (APCs), and/or B-cells.
  • the invention provides a method of increasing the activity of NK cells in a subject comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein.
  • the invention provides a method of increasing the activity of T-cells in a subject comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein. In some embodiments, the invention provides a method of increasing the activity of CD4+ and/or CD8+ T-cells in a subject comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein. In some embodiments, the invention provides a method of increasing the activity of CTLs in a subject comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein.
  • the invention provides a method of increasing the activation of T-cells, CTLs, and/or NK cells in a subject comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein. In some embodiments, the invention provides a method of increasing the T- cell response in a subject comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein. In some embodiments, the invention provides a method of inhibiting the activity of Tregs in a subject comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein.
  • the invention provides a method of inhibiting the suppressive activity of Tregs in a subject comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein. In some embodiments, the invention provides a method of inhibiting the activity of MDSCs in a subject comprising administering to the subject a therapeutically effective amount of a GITR- binding agent described herein. In some embodiments, the invention provides a method of inhibiting the suppressive activity of MDSCs in a subject comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein.
  • the invention provides a method of inducing an immune response in a subject without causing substantial side effects and/or immune-based toxicities comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein. In some embodiments, the invention provides a method of inducing an immune response in a subject without causing cytokine release syndrome or a cytokine storm comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein. In some embodiments, increasing the activity of immune cells comprises formation of tertiary lymphoid structures in a tumor or tumor microenvironment, e.g., as determined in an immunohistochemistry assay.
  • the invention provides methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject.
  • the invention provides methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein.
  • the invention provides methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a GITR- binding agent that binds human GITR.
  • the invention provides methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a GITR-binding agent that activates or enhances GITR signaling.
  • the invention provides a method of increasing T-cell activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof capable of binding human GITR.
  • the invention provides a method of increasing CTL activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof capable of binding human GITR.
  • the invention provides a method of increasing NK activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof capable of binding human GITR.
  • the invention provides a method of decreasing or inhibiting Treg activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof capable of binding human GITR.
  • the invention provides a method of decreasing or inhibiting MDSC activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof capable of binding human GITR.
  • the invention provides a method of increasing T-cell activity in a subject, comprising administering to the subject a therapeutically effective amount of any of the GITR-binding agents described herein. In some embodiments, the invention provides a method of increasing CTL activity in a subject, comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein. In some embodiments, the invention provides a method of increasing NK activity in a subject, comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein.
  • the invention provides a method of decreasing or inhibiting Treg activity in a subject, comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein. In some embodiments, the invention provides a method of decreasing or inhibiting MDSC activity in a subject, comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein. In some embodiments of the methods described herein, the subject has cancer. In some embodiments, the invention provides a method of promoting the formation of tertiary lymphoid structures in a tumor or tumor microenvironment in a subject, comprising administering to the subject a therapeutically effective amount of any of the GITR-binding agents described herein.
  • the invention provides a method of enhancing the antigen-specific memory response to a tumor.
  • a method of enhancing the antigen-specific memory response to a tumor comprises administering to a subject a therapeutically effective amount of a GITR-binding agent described herein.
  • a method of enhancing the antigen- specific memory response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof capable of binding human GITR.
  • the invention provides a method of activating or enhancing a persistent or long-term immune response to a tumor.
  • a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a GITR-binding agent described herein.
  • a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof capable of binding human GITR.
  • the invention provides a method of inducing a persistent or long-term immunity which inhibits tumor relapse or tumor regrowth.
  • a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a GITR-binding agent described herein.
  • a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof capable of binding human GITR.
  • the invention provides methods of stimulating a protective response in a subject comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein in combination with an antigen of interest.
  • the antigen of interest is a tumor antigen or tumor-associated antigen (TAA).
  • TAA tumor-associated antigen
  • the antigen of interest is a cancer cell biomarker.
  • the antigen of interest is a cancer stem cell marker.
  • the invention provides methods of promoting the formation of tertiary lymphoid structures within a tumor or tumor microenvironment in a subject, comprising administering to the subject a therapeutically effective amount of any of the GITR-binding agents described herein, wherein the tumor is resistant or refractory to the treatment with a PD-1 antagonist or a PD-L1 antagonist.
  • the tertiary lymphoid structures comprise tumor infiltrating lymphocytes (TILs).
  • the tertiary lymphoid structures comprise immune cells selected from the group consisting of CD8+ T cells, CD4+ T cells, and B-cells.
  • the tumor is or tumor microenvironment is selected from the group consisting of lung, non-small cell lung cancer (NSCLC), NSCLC adenocarcinoma, NSCLC squamous cell carcinoma, liver, hepatocellular carcinoma, breast, triple negative breast cancer, renal cell carcinoma, kidney, prostate, gastrointestinal, gastric, melanoma, cervical, bladder, glioblastoma, head and neck, pancreatic, ovarian, colorectal, endometrial, esophageal, uterine, and MSI-high.
  • NSCLC non-small cell lung cancer
  • NSCLC adenocarcinoma NSCLC squamous cell carcinoma
  • liver hepatocellular carcinoma
  • breast triple negative breast cancer
  • renal cell carcinoma kidney, prostate, gastrointestinal, gastric, melanoma, cervical, bladder, glioblastoma, head and neck
  • pancreatic, ovarian colorectal, endometrial, esophageal, uterine
  • the invention provides methods of promoting the formation of tertiary lymphoid structures within a tumor or tumor microenvironment in a subject, comprising administering to the subject a therapeutically effective amount of any of the GITR-binding agents described herein, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist.
  • the tertiary lymphoid structures comprise tumor infiltrating lymphocytes (TILs).
  • TILs tumor infiltrating lymphocytes
  • the tertiary lymphoid structures comprise immune cells selected from the group consisting of CD8+ T cells, CD4+ T cells, and B-cells.
  • the tumor is or tumor microenvironment is selected from the group consisting of lung, non-small cell lung cancer (NSCLC), NSCLC adenocarcinoma, NSCLC squamous cell carcinoma, liver, hepatocellular carcinoma, breast, triple negative breast cancer, renal cell carcinoma, kidney, prostate,
  • NSCLC non-small cell lung cancer
  • NSCLC adenocarcinoma NSCLC adenocarcinoma
  • NSCLC squamous cell carcinoma liver, hepatocellular carcinoma, breast, triple negative breast cancer
  • renal cell carcinoma kidney, prostate
  • tertiary lymphoid structures formation of the tertiary lymphoid structures is detected by an immunohistochemistry assay.
  • the methods further comprise administering to the subject at least one additional therapeutic agent.
  • the at least one additional therapeutic agent is a chemotherapeutic agent.
  • the at least one additional therapeutic agent is a second immunotherapeutic agent.
  • the at least one additional therapeutic agent is a checkpoint inhibitor.
  • the second immunotherapeutic agent is a compound that has a therapeutically active property.
  • the second immunotherapeutic agent is a compound that has a therapeutically active property.
  • the immunotherapeutic agent is selected from the group consisting of GM-CSF, M-CSF, G-CSF, IL-3, IL-12, IL-1, IL-2, B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-Ll antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody.
  • the additional therapeutic agent is an anti-PD-1 antibody.
  • the additional therapeutic agent is an anti-PD-Ll antibody.
  • the invention provides pharmaceutical compositions comprising a GITR- binding agent described herein and a pharmaceutically acceptable carrier.
  • a subject e.g., a human
  • Methods of treating cancer and/or inhibiting tumor growth in a subject comprising administering to the subject an effective amount of a composition comprising a GITR-binding agent described herein are also provided.
  • the agent is isolated. In certain embodiments, the agent is substantially pure.
  • the invention provides polynucleotides comprising a polynucleotide that encodes a GITR-binding agent described herein.
  • the polynucleotide is isolated.
  • the invention provides vectors that comprise the polynucleotides, as well as cells that comprise the vectors and/or the polynucleotides.
  • the invention also provides cells comprising or producing a GITR-binding agent described herein.
  • the cell is a monoclonal cell line.
  • the present invention encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members.
  • the present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.
  • Figure 1 Prevalence of GITR gene expression in tumors from TCGA database.
  • Figure 2 Analysis of GITR gene expression in tumor samples from patient-derived xenograft models.
  • FIG. 3 Inhibition of tumor growth by a mGITRL-mFc fusion protein (336B3).
  • the murine breast carcinoma line EMT6 was implanted subcutaneously into Balb/c mice. Mice were injected on days 8, 12, and 15 with 200 ⁇ g per mouse of 336B3 or control antibody, or on days 8, 12, 15, 20, 23, and 27 with 200 ⁇ g per mouse of anti-PD-1 antibody. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points. Data is shown as tumor volume (mm 3 ) over days post cell injection. Left panel: The mean values ⁇ SEM for each group. Upper right panel: Tumor volumes of each individual mouse from the group treated with control antibody. Middle right panel: Tumor volumes of each individual mouse from the group treated with anti-PDl antibody. Bottom right panel: Tumor volumes of each individual mouse from the group treated with 336B3.
  • Figures 4A-4B Induction of tertiary lymphoid structures induced by a mGITRL-mFc fusion protein (336B3) in PD-1 resistant EMT6 tumors.
  • Figure 4A Immunohistochemistry of EMT6 tumors from control antibody treated mice.
  • Figure 4B Immunohistochemistry of EMT6 tumors from 336B3 treated mice.
  • the present invention provides methods of using novel agents that specifically bind human GITR, wherein the agents include, but not limited to, polypeptides, soluble proteins, fusion proteins, homodimeric bispecific molecules, and heterodimeric bispecific molecules that modulate the immune response. Methods of using the novel agents for treatment of cancer and/or for inhibiting tumor growth are provided.
  • Methods of using the novel agents for activating an immune response, for stimulating an immune response, for promoting an immune response, for increasing an immune response, for activating NK cells, for activating T-cells, including CTLs, for increasing the activity of NK cells, for increasing the activity of T-cells, including CTLs, for promoting the activity of NK cells, for promoting the activity of T-cells, including CTLs, for inhibiting the activity of Tregs, for inhibiting the activity of MDSCs, and for promoting the formation of tertiary lymphoid structures in a tumor or tumor microenvironment are further provided.
  • agonist and “agonistic” as used herein refer to or describe an agent that is capable of, directly or indirectly, substantially inducing, activating, promoting, increasing, or enhancing the biological activity of a target and/or a pathway.
  • agonist is used herein to include any agent that partially or fully induces, activates, promotes, increases, or enhances the activity of a protein or other target of interest.
  • antagonists and antagonistic refer to or describe an agent that is capable of, directly or indirectly, partially or fully blocking, inhibiting, reducing, or neutralizing a biological activity of a target and/or pathway.
  • antagonist is used herein to include any agent that partially or fully blocks, inhibits, reduces, or neutralizes the activity of a protein or other target of interest.
  • modulation and “modulate” as used herein refer to a change or an alteration in a biological activity. Modulation includes, but is not limited to, stimulating an activity or inhibiting an activity. Modulation may be an increase in activity or a decrease in activity, a change in binding characteristics, or any other change in the biological, functional, or immunological properties associated with the activity of a protein, a pathway, a system, or other biological targets of interest.
  • soluble protein refers to a protein or a fragment thereof that can be secreted from a cell in soluble form.
  • fusion protein or "fusion polypeptide” as used herein refers to a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes.
  • linker refers to a linker inserted between a first polypeptide (e.g., copies of a GITRL extracellular domain or fragments thereof) and a second polypeptide (e.g., a Fc region).
  • the linker is a peptide linker.
  • Linkers should not adversely affect the expression, secretion, or bioactivity of the polypeptides. Preferably, linkers are not antigenic and do not elicit an immune response.
  • antibody refers to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing, through at least one antigen-binding site wherein the antigen-binding site is usually within the variable region of the immunoglobulin molecule.
  • the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) antibodies, multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen- binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen- binding site as long as the antibodies exhibit the desired biological activity.
  • antibody fragments such as Fab, Fab', F(ab')2, and Fv fragments
  • scFv single chain Fv
  • An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy -chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • the different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations.
  • Antibodies can be naked or conjugated to other molecules, including but not limited to, toxins and radioisotopes.
  • antibody fragment refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments. "Antibody fragment” as used herein comprises an antigen-binding site or epitope-binding site. [050] The term “monoclonal antibody” as used herein refers to a homogenous antibody population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies that typically include a mixture of different antibodies directed against different antigenic determinants.
  • monoclonal antibody encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single chain (scFv) antibodies, fusion proteins comprising an antibody fragment, and any other modified immunoglobulin molecule comprising an antigen-binding site.
  • monoclonal antibody refers to such antibodies made by any number of techniques, including but not limited to, hybridoma production, phage selection, recombinant expression, and transgenic animals.
  • epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing.
  • An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.
  • the terms “selectively binds” or “specifically binds” mean that an agent interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including related and unrelated proteins.
  • “specifically binds” means, for instance, that an agent binds a protein or target with a K D of about 0. ImM or less, but more usually less than about 1 ⁇ .
  • “specifically binds” means that an agent binds a target with a K D of at least about 0.1 ⁇ or less, at least about 0.01 ⁇ or less, or at least about InM or less.
  • specific binding can include an agent that recognizes a protein or target in more than one species.
  • specific binding can include an agent that recognizes more than one protein or target.
  • an agent that specifically binds a first target may or may not specifically bind a second target.
  • “specific binding” does not necessarily require (although it can include) exclusive binding, i.e. binding to a single target.
  • an agent may, in certain embodiments, specifically bind more than one target.
  • multiple targets may be bound by the same antigen-binding site on the agent.
  • an antibody may, in certain instances, comprise two identical antigen- binding sites, each of which specifically binds the same epitope on two or more proteins.
  • an antibody may be bispecific and comprise at least two antigen-binding sites with differing specificities.
  • binding means "specific binding".
  • polypeptide and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids
  • the polypeptides of this invention may be based upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, the polypeptides can occur as single chains or as associated chains.
  • polynucleotide and “nucleic acid” and “nucleic acid molecule” are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and
  • RNA RNA
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • the percent identity may be measured using sequence comparison software or algorithms or by visual inspection.
  • Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof.
  • two nucleic acids or polypeptides of the invention are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
  • identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between.
  • identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between.
  • identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.
  • a "conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains
  • substitution of a phenylalanine for a tyrosine is a conservative substitution.
  • conservative substitutions in the sequences of the polypeptides, soluble proteins, and/or antibodies of the invention do not abrogate the binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence, to the target binding site.
  • Methods of identifying amino acid conservative substitutions which do not eliminate binding are well-known in the art.
  • vector means a construct, which is capable of delivering, and usually expressing, one or more gene(s) or sequence(s) of interest in a host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.
  • a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is "isolated” is a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature.
  • Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature.
  • a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.
  • substantially pure refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
  • immune response includes responses from both the innate immune system and the adaptive immune system. It includes both cell-mediated and/or humoral immune responses. It includes both T-cell and B-cell responses, as well as responses from other cells of the immune system such as natural killer (NK) cells, monocytes, macrophages, etc.
  • NK natural killer
  • subject refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment.
  • subject and patient are used interchangeably herein in reference to a human subject.
  • pharmaceutically acceptable refers to a substance approved or approvable by a regulatory agency of the Federal government or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.
  • pharmaceutically acceptable excipient, carrier or adjuvant or “acceptable pharmaceutical carrier” refer to an excipient, carrier or adjuvant that can be administered to a subject, together with at least one agent of the present disclosure, and which does not destroy the
  • an agent described herein e.g., a fusion protein, a soluble receptor, an antibody, a polypeptide, a polynucleotide, a small organic molecule, or other drug
  • an agent described herein e.g., a fusion protein, a soluble receptor, an antibody, a polypeptide, a polynucleotide, a small organic molecule, or other drug
  • the therapeutically effective amount of an agent e.g., polypeptide, soluble protein, or antibody
  • an agent e.g., polypeptide, soluble protein, or antibody
  • the therapeutically effective amount of an agent has a therapeutic effect and as such can boost the immune response, boost the anti-tumor response, increase cytolytic activity of immune cells, increase killing of tumor cells by immune cells, reduce the number of tumor cells; decrease tumorigenicity, tumorigenic frequency or tumorigenic capacity; reduce the number or frequency of cancer stem cells; reduce the tumor size; reduce the cancer cell population; inhibit or stop cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibit and stop tumor or cancer cell metastasis; inhibit and stop tumor or cancer cell growth; relieve to some extent one or more of the symptoms associated with the cancer; reduce morbidity and mortality; improve quality of life; or a combination of such effects.
  • an agent e.g., polypeptide, soluble protein, or antibody
  • treating or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that may cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and/or prophylactic or preventative measures that may prevent or slow the development of a targeted pathologic condition or disorder.
  • those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.
  • the terms may refer to administration to a subject or patient of an agent described herein.
  • a subject is "treated" according to the methods of the present invention if the patient shows one or more of the following: an increased immune response, an increased anti-tumor response, increased cytolytic activity of immune cells, increased killing of tumor cells by immune cells, a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer cells into soft tissue and bone; inhibition of or an absence of tumor or cancer cell metastasis; inhibition or an absence of cancer growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity; reduction in the number or frequency of cancer stem cells; or some combination of these effects.
  • tertiary lymphoid structures refers to lymphoid aggregates that can form in non-lymphoid tissues, for example, in the context of chronic inflammation or cancer.
  • Tertiary lymphoid structures have been detected in tumors or tumor microenvironments, where they are believed to orchestrate local and systemic anti-tumor responses.
  • tertiary lymphoid structures have been described as important sites for the initiation and/or maintenance of local and systemic T- and B-cell responses against NSCLC tumors.
  • Tertiary lymphoid structure densities in tumors or tumor microenvironments have been found to favorably correlate with clinical outcome in a variety of patients having different cancers.
  • Tertiary lymphoid structures have been described, for example, by Sautes-Fridman et al. (2016) Front Immunol. Vol 7 Article 407.
  • the GITR-binding agents of the invention are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as immunotherapy for cancer.
  • a GITR-binding agent described herein is useful for activating, promoting, increasing, and/or enhancing an immune response, inhibiting tumor growth, reducing tumor volume, inducing tumor regression, increasing tumor cell apoptosis, and/or reducing the tumorigenicity of a tumor.
  • the methods of use may be in vitro, ex vivo, or in vivo methods.
  • the present invention provides methods for activating an immune response in a subject using a GITR-binding agent described herein.
  • a method comprises promoting an immune response in a subject using a GITR-binding agent described herein.
  • a method comprises increasing an immune response in a subject using a GITR-binding agent described herein.
  • a method comprises enhancing an immune response in a subject using a GITR-binding agent described herein.
  • the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing cell-mediated immunity.
  • the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing Thl-type responses.
  • the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CD4+ T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CD8+ T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CTL activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T-cell activity and increasing NK cell activity.
  • the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CTL activity and increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of Treg cells. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of MDSCs. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing the number of the percentage of memory T-cells. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing long-term immune memory function.
  • the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing long-term memory. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises no evidence of substantial side effects and/or immune-based toxicities. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises no evidence of cytokine release syndrome (CRS) or a cytokine storm.
  • the immune response is a result of antigenic stimulation. In some embodiments, the antigenic stimulation is a tumor cell. In some embodiments, the antigenic stimulation is cancer. In some embodiments, activating the immune response comprises promoting the formation of a tertiary lymphoid structure in a tumor or tumor microenvironment, e.g., as detected in an
  • a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent binds human GITR. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent is an agonist antibody that specifically binds GITR. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent is a fusion protein that specifically binds GITR.
  • a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a single chain fusion polypeptide that specifically binds GITR.
  • a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises an extracellular domain of GITRL or a GITR-binding fragment thereof.
  • a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a single chain GITRL trimer.
  • the GITR-binding agent comprises a single chain GITRL trimer and a Fc region.
  • the GITR-binding agent is 336B3.
  • the GITR-binding agent is OMP-336B11.
  • a method of activating or enhancing a persistent or long-term immune response to a tumor comprises administering to a subject a therapeutically effective amount of a GITR-binding agent which binds human GITR.
  • a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent is an agonist antibody that specifically binds GITR.
  • a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a fusion protein that specifically binds GITR. In some embodiments, a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a single chain fusion polypeptide that specifically binds GITR.
  • a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises an extracellular domain of GITRL or a GITR-binding fragment thereof.
  • a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a single chain GITRL trimer.
  • the GITR- binding agent comprises a single chain GITRL trimer and a Fc region.
  • the GITR-binding agent is 336B3.
  • the GITR-binding agent is OMP-336B11.
  • a method of inducing a persistent or long-term immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a GITR-binding agent which binds human GITR.
  • a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a GITR- binding agent described herein, wherein the agent is an agonist antibody that specifically binds GITR.
  • a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a GITR- binding agent described herein, wherein the agent comprises a fusion protein that specifically binds GITR.
  • a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a single chain fusion polypeptide that specifically binds GITR.
  • a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises an extracellular domain of GITRL or a GITR-binding fragment thereof.
  • a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a single chain GITRL trimer.
  • the GITR- binding agent comprises a single chain GITRL trimer and a Fc region.
  • the GITR-binding agent is 336B3.
  • the GITR-binding agent is OMP-336B11.
  • a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a GITR- binding agent which binds human GITR.
  • a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a GITR- binding agent described herein, wherein the agent is an agonist antibody that specifically binds GITR.
  • a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a fusion protein that specifically binds to GITR.
  • a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a single chain fusion polypeptide that specifically binds to GITR.
  • a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises an extracellular domain of GITRL or a GITR-binding fragment thereof.
  • a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a single chain GITRL trimer.
  • the GITR-binding agent comprises a single chain GITRL trimer and a Fc region.
  • the GITR-binding agent is 336B3.
  • the GITR-binding agent is OMP-336B11.
  • the present invention also provides methods for inhibiting growth of a tumor using a GITR- binding agent described herein.
  • the method of inhibiting growth of a tumor comprises contacting a cell mixture with a GITR-binding agent in vitro.
  • a GITR-binding agent for example, an immortalized cell line or a cancer cell line mixed with immune cells (e.g., T-cells, cytolytic T-cells, or NK cells) is cultured in medium to which is added a test agent.
  • tumor cells are isolated from a patient sample such as, for example, a tissue biopsy, pleural effusion, or blood sample, mixed with immune cells (e.g., T-cells, cytolytic T-cell, and/or NK cells), and cultured in medium to which is added a test agent.
  • immune cells e.g., T-cells, cytolytic T-cell, and/or NK cells
  • the GITR-binding agent increases, promotes, and/or enhances the activity of the immune cells.
  • the GITR-binding agent inhibits tumor cell growth.
  • the method of inhibiting growth of a tumor comprises contacting the tumor or tumor cells with a GITR-binding agent in vivo.
  • contacting a tumor or tumor cell with an agent is undertaken in an animal model.
  • a test agent may be administered to mice which have tumors.
  • the agent increases, promotes, and/or enhances the activity of immune cells in the mice.
  • the agent inhibits tumor growth.
  • the agent is administered at the same time or shortly after introduction of tumor cells into the animal to prevent tumor growth ("preventative model").
  • the agent is administered as a therapeutic after tumors have grown to a specified size ("therapeutic model").
  • a method of inhibiting growth of a tumor in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein. In certain embodiments, a method of inhibiting growth of a tumor in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent is an agonist antibody that specifically binds GITR. In certain embodiments,
  • a method of inhibiting growth of a tumor in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a fusion protein.
  • a method of inhibiting growth of a tumor in a subject comprises administering to the subject a therapeutically effective amount of a GITR- binding agent described herein, wherein the agent comprises a single chain fusion polypeptide that specifically binds GITR.
  • a method of inhibiting growth of a tumor in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises an extracellular domain of GITRL or a GITR- binding fragment thereof.
  • a method of inhibiting growth of a tumor in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a single chain GITRL trimer.
  • a method of inhibiting growth of a tumor in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a single chain GITRL trimer and a Fc region.
  • the GITR- binding agent is 336B3.
  • the GITR-binding agent is OMP-336B11.
  • the subject is a human.
  • the subject has a tumor or the subject had a tumor which was removed.
  • a method of inhibiting growth of a tumor in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein.
  • the tumor comprises cancer stem cells.
  • the frequency of cancer stem cells in the tumor is reduced by administration of the agent.
  • a method of reducing the frequency of cancer stem cells in a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a GITR-binding agent is provided.
  • the GITR-binding agent is 336B3.
  • the GITR-binding agent is OMP-336B11.
  • the invention provides a method of reducing the tumorigenicity of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein.
  • the tumor comprises cancer stem cells.
  • the tumorigenicity of a tumor is reduced by reducing the frequency of cancer stem cells in the tumor.
  • the methods comprise using a GITR-binding agent described herein.
  • the frequency of cancer stem cells in the tumor is reduced by administration of a GITR-binding agent.
  • the tumor is a solid tumor.
  • the tumor is a tumor selected from the group consisting of: colorectal tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, neuroendocrine tumor, gastrointestinal tumor, gastric tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor.
  • the tumor is a colorectal tumor.
  • the tumor is an ovarian tumor.
  • the tumor is a lung tumor.
  • the tumor is a pancreatic tumor.
  • the tumor is a head and neck tumor.
  • the tumor is a melanoma tumor. In some embodiments, the tumor is a bladder tumor. In some embodiments, the tumor is an esophageal tumor. In certain embodiments, the tumor is a gastric tumor. In some embodiments, the tumor is a non-small cell lung cancer (NSCLC). In some embodiments, the tumor is a NSCLC adenocarcinoma. In some embodiments, the tumor is a NSCLC squamous cell carcinoma. In some embodiments, the tumor is a triple negative breast cancer. In some embodiments, the tumor is a hepatocellular carcinoma (HCC). In certain embodiments, the tumor is a hepatoma.
  • NSCLC non-small cell lung cancer
  • the tumor is a microsatellite instability -high tumor.
  • the tumor is selected from the group consisting of: head and neck squamous cell carcinoma, lung squamous cell carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, uterine corpus endometrial carcinoma, lung
  • adenocarcinoma breast invasive carcinoma, esophageal carcinoma, bladder urothelial carcinoma, uterine carcinosarcoma, testicular germ cell tumor, thymoma, thymic carcinoma, cholangiocarcinoma, mesothelioma, sarcoma, ovarian serous cystadenocarcinoma, skin cutaneous melanoma, thyroid carcinoma, stomach adenocarcinoma, pancreatic adenocarcinoma, prostate adenocarcinoma, kidney carcinoma, kidney renal papillary cell carcinoma, colon adenocarcinoma, liver hepatocellular carcinoma, rectum adenocarcinoma, kidney renal clear cell carcinoma, adrenocortical carcinoma, glioblastoma multiforme, pheochromocytoma, paraganglioma, kidney chromophobe renal cell carcinoma, lower grade glioma, and uveal mela
  • the tumor expresses or overexpresses a tumor antigen targeted by the agent, such as a bispecific agent which comprises an antigen-binding site that specifically binds the tumor antigen.
  • a tumor antigen targeted by the agent such as a bispecific agent which comprises an antigen-binding site that specifically binds the tumor antigen.
  • the present invention further provides methods for treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein.
  • the agent binds GITR and inhibits or reduces growth of the cancer.
  • a method for treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent is an agonist antibody.
  • a method for treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a fusion protein.
  • a method for treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a single chain fusion polypeptide that specifically binds GITR.
  • a method for treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises an extracellular domain of GITRL or a GITR-binding fragment thereof.
  • a method for treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a single chain GITRL trimer.
  • a method for treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein, wherein the agent comprises a single chain GITRL trimer and a Fc region.
  • the GITR-binding agent is 336B3.
  • the GITR-binding agent is OMP-336B11.
  • the present invention provides for methods of treating cancer comprising administering to a subject (e.g., a subject in need of treatment) a therapeutically effective amount of a GITR-binding agent described herein.
  • a subject e.g., a subject in need of treatment
  • a therapeutically effective amount of a GITR-binding agent described herein in certain embodiments, the subject is a human.
  • the subject has a cancerous tumor.
  • the subject has had a tumor removed.
  • the cancer is a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, neuroendocrine cancer, bladder cancer, brain cancer, glioblastoma, and head and neck cancer.
  • the cancer is selected from the group consisting of: head and neck squamous cell carcinoma, lung squamous cell carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, lung adenocarcinoma, breast invasive carcinoma, esophageal carcinoma, bladder urothelial carcinoma, uterine carcinosarcoma, testicular germ cell tumor, thymoma, thymic carcinoma, cholangiocarcinoma, mesothelioma, sarcoma, ovarian serous cystadenocarcinoma, skin cutaneous melanoma, thyroid carcinoma, stomach adenocarcinoma, pancreatic adenocarcinoma, prostate adenocarcinoma, kidney carcinoma, kidney renal papillary cell carcinoma, colon adenocarcinoma, liver hepatocellular carcinoma, rectum adeno
  • the cancer is pancreatic cancer. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is prostate cancer. In some embodiments, the cancer is uterine cancer. In certain embodiments, the cancer is lung cancer. In certain embodiments, the cancer is melanoma. In some embodiments, the cancer is a head and neck cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • the cancer is NSCLC adenocarcinoma. In some embodiments, the cancer is NSCLC squamous cell carcinoma. In some embodiments, the cancer is triple negative breast cancer. In some embodiments, the cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is a microsatellite instability-high cancer.
  • HCC hepatocellular carcinoma
  • the cancer is a hematological cancer.
  • the cancer is lymphoma.
  • the cancer is lymphoid neoplasm diffuse large B-cell lymphoma.
  • the cancer is a T-cell lymphoma.
  • the cancer is leukemia.
  • the cancer is acute myeloid leukemia.
  • a method of inhibiting tumor growth comprises contacting a tumor or tumor cell with an effective amount of a GITR-binding agent described herein.
  • a method of inhibiting growth of a tumor comprises contacting a tumor
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a
  • a GITR-binding agent wherein the tumor is a head and neck cancer, an esophageal cancer, a gastric cancer, a melanoma, a uterine cancer, a non-small cell lung cancer (NSCLC), a NSCLC adenocarcinoma, a NSCLC squamous cell carcinoma, a triple negative breast cancer, a hepatocellular carcinoma (HCC), or a MSI-high tumor.
  • the tumor is a head and neck cancer, an esophageal cancer, a gastric cancer, a melanoma, a uterine cancer, a non-small cell lung cancer (NSCLC), a NSCLC adenocarcinoma, a NSCLC squamous cell carcinoma, a triple negative breast cancer, a hepatocellular carcinoma (HCC), or a MSI-high tumor.
  • NSCLC non-small cell lung cancer
  • HCC hepatocellular carcinoma
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a GITR-binding agent, wherein the tumor is a head and neck cancer, an esophageal cancer, a gastric cancer, a melanoma, a uterine cancer, a non-small cell lung cancer (NSCLC), a NSCLC adenocarcinoma, a NSCLC squamous cell carcinoma, a triple negative breast cancer, a hepatocellular carcinoma (HCC), or a MSI-high tumor; and wherein the GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a
  • a human GITR-binding agent wherein the tumor is a head and neck cancer, an esophageal cancer, a gastric cancer, a melanoma, a uterine cancer, a non-small cell lung cancer (NSCLC), a NSCLC adenocarcinoma, a NSCLC squamous cell carcinoma, a triple negative breast cancer, a hepatocellular carcinoma (HCC), or a MSI-high tumor; and wherein the GITR- binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of human GITRL.
  • NSCLC non-small cell lung cancer
  • HCC hepatocellular carcinoma
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor is an esophageal cancer, a non-small cell lung cancer (NSCLC), a NSCLC adenocarcinoma, a NSCLC squamous cell carcinoma, a triple negative breast cancer, or a hepatocellular carcinoma
  • NSCLC non-small cell lung cancer
  • NSCLC adenocarcinoma adenocarcinoma
  • NSCLC squamous cell carcinoma a triple negative breast cancer
  • a hepatocellular carcinoma hepatocellular carcinoma
  • the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor is a head and neck tumor, a gastric tumor, a melanoma, a uterine tumor, or a MSI-high tumor; and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor expresses GITR.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor expresses GITR; and wherein the GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a
  • a human GITR-binding agent wherein the tumor expresses GITR; and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist; and wherein the GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist; and wherein the GITR-binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of human GITRL.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount a human GITR-binding agent, wherein the tumor is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist; and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent; and wherein the GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent; and wherein the GITR-binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of human GITRL.
  • a method of inhibiting tumor growth in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent; and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • the invention provides methods of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a GITR-binding agent described herein.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is head and neck cancer, esophageal cancer, gastric cancer, melanoma, uterine cancer, non-small cell lung cancer (NSCLC), NSCLC adenocarcinoma, NSCLC squamous cell carcinoma, triple negative breast cancer, hepatocellular carcinoma (HCC), or a MSI-high cancer.
  • NSCLC non-small cell lung cancer
  • HCC hepatocellular carcinoma
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is head and neck cancer, esophageal cancer, gastric cancer, melanoma, uterine cancer, non-small cell lung cancer (NSCLC), NSCLC adenocarcinoma, NSCLC squamous cell carcinoma, triple negative breast cancer, hepatocellular carcinoma (HCC), or a MSI-high cancer; and wherein the GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • the cancer is head and neck cancer, esophageal cancer, gastric cancer, melanoma, uterine cancer, non-small cell lung cancer (NSCLC), NSCLC adenocarcinoma, NSCLC squamous cell carcinoma, triple negative breast cancer, hepatocellular carcinoma (HCC), or a MSI-high cancer
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is head and neck cancer, esophageal cancer, gastric cancer, melanoma, uterine cancer, non-small cell lung cancer (NSCLC), NSCLC adenocarcinoma, NSCLC squamous cell carcinoma, triple negative breast cancer, hepatocellular carcinoma (HCC), or a MSI-high cancer; and wherein the GITR-binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of human
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is esophageal cancer, non-small cell lung cancer (NSCLC), NSCLC adenocarcinoma, NSCLC squamous cell carcinoma, triple negative breast cancer, or hepatocellular carcinoma (HCC); and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • a human GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is head and neck cancer, gastric cancer, melanoma, uterine cancer, or MSI-high cancer; and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer expresses GITR.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer expresses GITR; and wherein the GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer expresses GITR; and wherein the GITR- binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of human GITRL.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer expresses GITR; and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist; and wherein the GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist; and wherein the GITR-binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of human GITRL.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the cancer is resistant or refractory to treatment with a PD-1 antagonist or a PD-L1 antagonist; and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent; and wherein the GITR- binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR-binding agent, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent; and wherein the GITR-binding agent comprises a single chain polypeptide comprising a trimer of the extracellular domain of human GITRL.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a human GITR- binding agent, wherein the subject has previously been treated with a PD-1 antagonist or a PD-L1 antagonist as a single agent; and wherein the GITR-binding agent comprises a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITRL, wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • the cancer or tumor is selected from the group consisting of: lung, non-small cell lung cancer (NSCLC), NSCLC adenocarcinoma, NSCLC squamous cell carcinoma, liver, hepatocellular carcinoma, breast, triple negative breast cancer, renal cell carcinoma, kidney, prostate, gastrointestinal, gastric, melanoma, cervical, bladder, glioblastoma, head and neck, pancreatic, ovarian, colorectal, endometrial, and esophageal.
  • the cancer or tumor is resistant or refractory to treatment with an antagonist anti-PD-1 antibody.
  • the cancer or tumor is resistant or refractory to treatment with an antagonist anti-PD-Ll antibody.
  • the subject has previously been treated with an antagonist anti-PD-1 antibody.
  • the subject has previously been treated with an antagonist anti-PD-Ll antibody.
  • the cancer or tumor recurrence is local recurrence, regional recurrence, or distant recurrence.
  • the cancer or tumor comprises immune cells.
  • the cancer or tumor comprises tumor-infiltrating lymphocytes (TILs).
  • the cancer or tumor comprises regulatory T- cells (Tregs).
  • the cancer or tumor comprises tertiary lymphoid structures. In certain embodiments of the methods described herein, the method promotes the formation of tertiary lymphoid structures in the cancer, tumor, or tumor
  • microenvironment e.g., PD-1 antagonist or PD-L1 antagonist resistant or refractory.
  • the invention provides methods of promoting the formation of tertiary lymphoid structures within a tumor or tumor microenvironment in a subject.
  • the methods of promoting the formation of tertiary lymphoid structures comprise administering to the subject a therapeutically effective amount of any of the human glucocorticoid-induced tumor necrosis factor receptor (GITR)-binding agent described herein.
  • the tumor is resistant or refractory to the treatment with a PD-1 antagonist or a PD-L1 antagonist.
  • the subject has previously been treated with a PD-1 antagonist (e.g., anti-PD-1 antibody) or a PD-L1 antagonist (e.g., anti-PD-Ll antibody) as a single agent.
  • a PD-1 antagonist e.g., anti-PD-1 antibody
  • a PD-L1 antagonist e.g., anti-PD-Ll antibody
  • the GITR-binding agent is a single chain polypeptide comprising: (a) a trimer of the extracellular domain of human GITR ligand (GITRL), wherein each copy of the extracellular domain comprises the stalk region of GITRL; and the trimer does not comprise any peptide linkers; and (b) a human Fc region.
  • the tertiary lymphoid structures comprise tumor- infiltrating lymphocytes (TILs).
  • the tertiary lymphoid structures comprise immune cells selected from the group consisting of CD8+ T cells, CD4+ T cells, and B-cells.
  • the tumor or tumor microenvironment is selected from the group consisting of: lung, non-small cell lung cancer (NSCLC), NSCLC adenocarcinoma, NSCLC squamous cell carcinoma, liver, hepatocellular carcinoma, breast, triple negative breast cancer, renal cell carcinoma, kidney, prostate, gastrointestinal, gastric, melanoma, cervical, bladder, glioblastoma, head and neck, pancreatic, ovarian, colorectal, endometrial, esophageal, uterine and microsatellite instability-high (MSI-high).
  • the formation of the tertiary lymphoid structures is detected by an immunohistochemistry assay.
  • the GITR-binding agent comprises SEQ ID NO:3. In some aspects and embodiments of the methods described herein, the GITR-binding agent comprises SEQ ID NO:6. In some aspects and embodiments of the methods described herein, the human Fc region is from an IgGl, IgG2, IgG3, or IgG4 immunoglobulin. In some aspects and embodiments of the methods described herein, the human Fc region comprises SEQ ID NO:9. In some aspects and embodiments of the methods described herein, the GITR-binding agent comprises SEQ ID NO:8. In some aspects and embodiments of the methods described herein, the stalk region of GITRL comprises LQLETAK (SEQ ID NO:4).
  • the GITR-binding agent comprises a polypeptide encoded by the plasmid deposited with ATCC as Designation No. PTA-122112. In some aspects and embodiments of the methods described herein, the GITR-binding agent OMP-336B11.
  • the GITR-binding agent can be MEDI1873 or any of those disclosed in PCT Publication WO 2017/025610; any of those disclosed in U.S. Patent No. 8,450,460; or any of those disclosed in WO 2005/103077.
  • the GITR-binding agent is an antibody, particularly an agonist antibody.
  • the methods can employ, e.g., any agonist antibody known in the art.
  • the antibody can be selected from the group consisting of INCAGN01876 or any of the antibodies described in U.S. Patent Publication No. 2015/0368349; MK-4166, MK-1248, or any of those described in U.S. Patent Publication No. 2014/0348841; AMG228 or US Patent No.
  • the method increases T effector cells within the cancer or tumor. In some aspects and embodiments of the methods described herein, the method increases T effector cells within the microenvironment of the cancer or tumor. In some aspects and embodiments of the methods described herein, the method increases T effector cell activity within the cancer or tumor. In some aspects and embodiments of the methods described herein, the method increases T effector cell activity within the microenvironment of the cancer or tumor. In some aspects and embodiments of the methods described herein, the method depletes regulatory T-cells within the cancer or tumor. In some aspects and embodiments of the methods described herein, the method depletes regulatory T-cells within the microenvironment of the cancer or tumor.
  • the method inhibits and/or decreases the suppressive activity of Tregs within the cancer or tumor. In some aspects and embodiments of the methods described herein, the method inhibits and/or decreases the suppressive activity of Tregs within the microenvironment of the cancer or tumor. In some aspects and embodiments of the methods described herein, the method promotes the formation of tertiary lymphoid structures within the microenvironment of the cancer or tumor.
  • the invention also provides a method of activating or enhancing GITR signaling in a cell comprising contacting the cell with an effective amount of a GITR-binding agent described herein.
  • a method of activating or enhancing GITR signaling in a cell comprises contacting the cell with an effective amount of a GITR-binding agent described herein.
  • the agent is 336B3.
  • the agent is OMP-336B11.
  • the cell is a T-cell.
  • the cell is a cytolytic cell.
  • the cell is a CTL.
  • the cell is a NK cell.
  • the method is an in vivo method wherein the step of contacting the cell with the agent comprises administering a therapeutically effective amount of the agent to the subject.
  • the method is an in vitro or ex vivo method.
  • the present invention provides methods of determining the level of expression of GITR and/or GITRL.
  • the level of expression of GITR is determined.
  • the level of expression of GITR in a sample e.g., a tissue, a cell, etc.
  • the level of expression of GITRL is determined.
  • the level of expression of GITRL in a sample e.g., a tissue, a cell, etc.
  • Methods for determining the level of nucleic acid expression in a cell, tumor, or cancer are known by those of skill in the art.
  • Methods for determining the level of protein expression in a cell, tumor, or cancer include, but are not limited to, Western blot analyses, protein arrays, ELISAs, immunohistochemistry (IHC), and FACS.
  • the expression level of GITR and/or GITRL in a tumor sample is determined to be at a high level.
  • the expression level of GITR in a sample is compared to a pre-determined expression level of GITR.
  • the expression level of GITRL in a sample is compared to a pre-determined expression level of GITRL.
  • the pre-determined expression level of GITR expression is an expression level of GITR in a reference tumor sample, a reference normal tissue sample, a series of reference tumor samples, or a series of reference normal tissue samples.
  • the pre-determined expression level of GITRL expression is an expression level of GITRL in a reference tumor sample, a reference normal tissue sample, a series of reference tumor samples, or a series of reference normal tissue samples.
  • the expression level of GITR and/or GITRL is determined using PCR-based methods, such as but not limited to, reverse transcription PCR (RT-PCR), quantitative RT-PCR (qPCR), TaqManTM, or TaqManTM low density array (TLDA).
  • RT-PCR reverse transcription PCR
  • qPCR quantitative RT-PCR
  • TLDA TaqManTM low density array
  • the expression level of a biomarker is determined using a microarray.
  • the expression level GITR and/or GITRL is determined using protein-based methods.
  • the expression level of a biomarker is measured or determined by multi-analyte profile testing, radioimmunoassay (RIA), Western blot assay, immunofluorescent assay, enzyme immunoassay, enzyme linked immunosorbent assay (ELISA), immunoprecipitation assay, chemiluminescent assay, immunohistochemical (IHC) assay, dot blot assay, or slot blot assay.
  • the expression level of GITR and/or GITRL is determined using an IHC assay.
  • the assay uses an antibody (e.g., an anti-GITR antibody or an anti-GITRL antibody).
  • the antibody is detectably labeled.
  • the label is selected from the group consisting of an immunofluorescent label, a chemiluminescent label, a phosphorescent label, an enzyme label, a radiolabel, an avidin/biotin label, colloidal gold particles, colored particles, and magnetic particles.
  • the expression level of GITR or GITRL is determined using an immunohistochemistry (IHC) assay. In some embodiments, the expression level of GITR and/or GITRL is determined using an assay which comprises an H-score evaluation. In some embodiments, the expression level of GITR is determined using an antibody that specifically binds GITR. In some embodiments, the expression level of GITRL is determined using an antibody that specifically binds GITRL. In some embodiments, GITR is detected on tumor cells. In some embodiments, GITR is detected at the plasma membrane of tumor cells. In some embodiments, GITR is detected on tumor- infiltrating immune cells and/or in the tumor microenvironment. In some embodiments, GITR is detected on T-cells. In some embodiments, GITR is detected on regulatory T-cells (Tregs). In some embodiments, GITRL is detected on tumor-infiltrating immune cells and/or in the tumor
  • GITRL is detected on antigen-presenting cells. In some embodiments, GITRL is detected on macrophages and/or dendritic cells.
  • the determining of the level of GITR and/or GITRL expression is done prior to treatment with a GITR-binding agent.
  • a GITR-binding agent is administered to the subject.
  • cells in a tumor sample or cancer sample e.g., tumor cells and/or tumor-infiltrating immune cells
  • a GITR- binding agent is administered to the subject.
  • a method comprises (i) obtaining a tumor sample from the subject; (ii) measuring the expression level of GITR and/or GITRL in the sample; and (iii) administering an effective amount of a GITR-binding agent to the subject if the tumor or cancer has an elevated or high expression level of GITR and/or GITRL.
  • a method comprises (i) obtaining a tumor sample from the subject; (ii) measuring the expression level of GITR and/or GITRL in the sample; and (iii) administering an effective amount of a GITR-binding agent to the subject if the sample has an elevated or high expression level of GITR and/or GITRL.
  • the sample is taken from a subject having a tumor or cancer.
  • the sample is a fresh tumor/cancer sample.
  • the sample is a frozen tumor/cancer sample.
  • the sample is a formalin-fixed paraffin-embedded sample.
  • the sample is a blood sample.
  • the sample is a plasma sample.
  • the sample is processed to a cell lysate.
  • the sample is processed to DNA or RNA.
  • the sample is a biopsy sample.
  • the sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, and any combination thereof.
  • the sample is a formalin-fixed paraffin embedded (FFPE) sample.
  • FFPE formalin-fixed paraffin embedded
  • the sample is archival, fresh, or frozen tissue.
  • the present invention provides compositions comprising a GITR-binding agent for use in the methods described herein.
  • the present invention also provides pharmaceutical compositions comprising a GITR-binding agent described herein and a pharmaceutically acceptable vehicle.
  • the pharmaceutical compositions find use in immunotherapy.
  • the pharmaceutical compositions find use in immuno-oncology.
  • the compositions find use in inhibiting tumor growth.
  • the pharmaceutical compositions find use in inhibiting tumor growth in a subject (e.g., a human patient).
  • the compositions find use in treating cancer.
  • the pharmaceutical compositions find use in treating cancer in a subject (e.g., a human patient).
  • Formulations are prepared for storage and use by combining a purified agent of the present invention with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient).
  • a pharmaceutically acceptable vehicle e.g., a carrier or excipient.
  • pharmaceutically acceptable carriers, excipients, and/or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.
  • the agents described herein are formulated in a buffer comprising of 20mM histidine, 40mM NaCl, 5% sucrose, and 0.01% polysorbate 20. In some embodiments, the agents described herein are formulated in a buffer comprising of 20mM histidine, 40mM NaCl, 5% sucrose, and 0.01% polysorbate 20 at pH 5.5. In some embodiments, the agents described herein are formulated in a buffer comprising of 20mM histidine, 40mM NaCl, 5% sucrose, and 0.01% polysorbate 20 at pH 6.0.
  • the agents described herein are formulated in a buffer comprising of 20mM histidine, 40mM NaCl, 5% sucrose, and 0.01% polysorbate 20 at pH 6.5. In some embodiments, the agents described herein are formulated in a buffer comprising of 20mM histidine, lOOmM NaCl, 150mM sucrose, and 0.01% polysorbate 20 at pH 6.0. In some
  • the agents described herein are formulated in a buffer comprising of lOmM potassium phosphate and 0.04% polysorbate 20 at pH 7.5.
  • compositions or pharmaceutical compositions comprising a GITR-binding agent described herein and further comprising about 20mM histidine, about 40mM NaCl, about 5% sucrose, and about 0.01% polysorbate 20.
  • pH of the composition is about pH 5.5, about pH 6.0, or about pH 6.5.
  • a GITR-binding agent described herein is lyophilized and/or stored in a lyophilized form.
  • a formulation comprising a GITR-binding agent described herein is lyophilized.
  • Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, hist
  • compositions of the present invention can be administered in any number of ways for either local or systemic treatment.
  • Administration can be topical by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, and intranasal; oral; or parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular).
  • parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular).
  • the therapeutic formulation can be in unit dosage form.
  • Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories.
  • solid compositions such as tablets the principal active ingredient is mixed with a pharmaceutical carrier.
  • Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and diluents (e.g., water). These can be used to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof.
  • the solid preformulation composition is then subdivided into unit dosage forms of a type described above.
  • the tablets, pills, etc. of the formulation or composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner composition covered by an outer component.
  • the two components can be separated by an enteric layer that serves to resist disintegration and permits the inner component to pass intact through the stomach or to be delayed in release.
  • enteric layers or coatings such materials include a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
  • microcapsules can also be entrapped in microcapsules.
  • microcapsules are prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions as described in Remington: The Science and Practice of Pharmacy, 22 nd Edition, 2012, Pharmaceutical Press, London.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • macroemulsions as described in Remington: The Science and Practice of Pharmacy, 22 nd Edition, 2012, Pharmaceutical Press, London.
  • compositions include a GITR-binding agent of the present invention complexed with liposomes.
  • Methods to produce liposomes are known to those of skill in the art.
  • some liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized
  • sustained-release preparations comprising the GITR-binding agents described herein can be produced. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing an agent, where the matrices are in the form of shaped articles (e.g., films or microcapsules).
  • sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
  • polyesters such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)
  • polylactides copolymers of L-glutamic acid and 7 ethyl-L-glutamate
  • non-degradable ethylene-vinyl acetate non-degradable ethylene-vinyl a
  • the method or treatment further comprises administering at least one additional immunotherapeutic agent.
  • the additional immunotherapeutic agent includes, but is not limited to, a colony stimulating factor (e.g., granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF)), an interleukin (e.g., IL-1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), a checkpoint inhibitor, an antibody that blocks immunosuppressive functions (e.g., an anti-CTLA-4 antibody, anti- CD28 antibody, anti-CD3 antibody), a toll-like receptor (e.g., TLR4, TLR7, TLR9), or a member of the B7 family (e.g., CD80, CD86).
  • a colony stimulating factor e.g., granulocyte-macrophage colony stimulating factor (GM
  • An additional immunotherapeutic agent can be administered prior to, concurrently with, and/or subsequently to, administration of the agent.
  • Pharmaceutical compositions comprising a GITR-binding agent and the immunotherapeutic agent(s) are also provided.
  • the immunotherapeutic agent comprises 1, 2, 3, or more immunotherapeutic agents.
  • the method or treatment further comprises administering at least one additional therapeutic agent.
  • An additional therapeutic agent can be administered prior to, concurrently with, and/or subsequently to, administration of the agent.
  • Pharmaceutical compositions comprising an agent and the additional therapeutic agent(s) are also provided.
  • the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.
  • Combination therapy with two or more therapeutic agents often uses agents that work by different mechanisms of action, although this is not required. Combination therapy using agents with different mechanisms of action may result in additive or synergetic effects. Combination therapy may allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent(s). Combination therapy may decrease the likelihood that resistant cancer cells will develop.
  • combination therapy comprises a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) the tumor/cancer cells.
  • the combination of a GITR-binding agent described herein and at least one additional therapeutic agent results in additive or synergistic results.
  • the combination therapy results in an increase in the therapeutic index of the agent.
  • the combination therapy results in an increase in the therapeutic index of the additional therapeutic agent(s).
  • the combination therapy results in a decrease in the toxicity and/or side effects of the agent.
  • the combination therapy results in a decrease in the toxicity and/or side effects of the additional therapeutic agent(s).
  • Useful classes of therapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, anti-folates, anti-metabolites, chemotherapy sensitizers, duocarmycins, etoposides, fhiorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like.
  • the second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, or an an anthracyclines
  • Therapeutic agents that may be administered in combination with the agents described herein include chemotherapeutic agents.
  • the method or treatment involves the administration of a GITR-binding agent of the present invention in combination with a
  • chemotherapeutic agent or in combination with a cocktail of chemotherapeutic agents. Treatment with an agent can occur prior to, concurrently with, or subsequent to administration of
  • chemotherapies Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.
  • Preparation and dosing schedules for such chemotherapeutic agents can be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in The Chemotherapy Source Book, 4 th Edition, 2008, M. C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, PA.
  • Chemotherapeutic agents useful in the present invention include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil
  • paclitaxel TAXOL
  • docetaxel TXOTERE
  • chlorambucil gemcitabine
  • 6- thioguanine mercaptopurine
  • platinum analogs such as cisplatin and carboplatin
  • vinblastine vinblastine
  • navelbine novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT 11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine (XELODA); and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • DMFO difluoromethylornithine
  • XELODA capecitabine
  • Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the additional therapeutic agent is cisplatin.
  • the additional therapeutic agent is carboplatin.
  • the chemotherapeutic agent is a topoisomerase inhibitor.
  • Topoisomerase inhibitors are chemotherapy agents that interfere with the action of a topoisomerase enzyme (e.g., topoisomerase I or II).
  • Topoisomerase inhibitors include, but are not limited to, doxorubicin HC1, daunorubicin citrate, mitoxantrone HC1, actinomycin D, etoposide, topotecan HC1, teniposide (VM-26), and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.
  • the additional therapeutic agent is irinotecan.
  • the chemotherapeutic agent is an anti-metabolite.
  • An anti-metabolite is a chemical with a structure that is similar to a metabolite required for normal biochemical reactions, yet different enough to interfere with one or more normal functions of cells, such as cell division.
  • Anti-metabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine,
  • the additional therapeutic agent is gemcitabine.
  • the chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind tubulin.
  • the agent is a taxane.
  • the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel.
  • the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (nab-paclitaxel;
  • the antimitotic agent comprises a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or pharmaceutically acceptable salts, acids, or derivatives thereof.
  • the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of a mitotic kinase such as Aurora A or Plkl.
  • the additional therapeutic agent is paclitaxel. In certain embodiments, the additional therapeutic agent is nab-paclitaxel.
  • an additional therapeutic agent comprises an agent such as a small molecule.
  • treatment can involve the combined administration of a GITR-binding agent of the present invention with a small molecule that acts as an inhibitor against tumor-associated antigens including, but not limited to, EGFR, HER2 (ErbB2), and/or VEGF.
  • a GITR-binding agent of the present invention is administered in combination with a protein kinase inhibitor selected from the group consisting of: gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatanib, vandetanib (ZACTIMA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B).
  • IRESSA gefitinib
  • TARCEVA sunitinib
  • ZACTIMA ZACTIMA
  • AEE788, CI-1033 cediranib
  • sorafenib NEXAVAR
  • GW786034B pazopanib
  • the additional therapeutic agent is a small molecule that inhibits a cancer stem cell pathway.
  • the additional therapeutic agent is an inhibitor of the Notch pathway.
  • the additional therapeutic agent is an inhibitor of the Wnt pathway.
  • the additional therapeutic agent is an inhibitor of the BMP pathway.
  • the additional therapeutic agent is an inhibitor of the Hippo pathway.
  • the additional therapeutic agent is an inhibitor of the mTOR/AKR pathway.
  • the additional therapeutic agent is an inhibitor of the RSPO/LGR pathway.
  • an additional therapeutic agent comprises a biological molecule, such as an antibody.
  • treatment can involve the combined administration of a GITR-binding agent of the present invention with antibodies against tumor-associated antigens including, but not limited to, antibodies that bind EGFR, HER2/ErbB2, and/or VEGF.
  • the additional therapeutic agent is an antibody specific for a cancer stem cell marker.
  • the additional therapeutic agent is an antibody that binds a component of the Notch pathway.
  • the additional therapeutic agent is an antibody that binds a component of the Wnt pathway.
  • the additional therapeutic agent is an antibody that inhibits a cancer stem cell pathway.
  • the additional therapeutic agent is an inhibitor of the Notch pathway.
  • the additional therapeutic agent is an inhibitor of the Wnt pathway.
  • the additional therapeutic agent is an inhibitor of the BMP pathway.
  • the additional therapeutic agent is an antibody that inhibits ⁇ -catenin signaling.
  • the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor antibody).
  • the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX).
  • the additional therapeutic agent is an antibody that modulates the immune response.
  • the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTLA-4 antibody, or an anti-TIGIT antibody.
  • treatment with a GITR-binding agent described herein can include combination treatment with other biologic molecules, such as one or more cytokines (e.g., lymphokines, interleukins, tumor necrosis factors, and/or growth factors) or can be accompanied by surgical removal of tumors, removal of cancer cells, or any other therapy deemed necessary by a treating physician.
  • the additional therapeutic agent is an immunotherapeutic agent.
  • the GITR-binding agent can be combined with a growth factor selected from the group consisting of: adrenomedullin (AM), angiopoietin (Ang), BMPs, BDNF, EGF, erythropoietin (EPO), FGF, GDNF, G-CSF, GM-CSF, GDF9, HGF, HDGF, IGF, migration-stimulating factor, myostatin (GDF-8), NGF, neurotrophins, PDGF, thrombopoietin, TGF-a, TGF- ⁇ , TNF-a, VEGF, PIGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18.
  • a growth factor selected from the group consisting of: adrenomedullin (AM), angiopoietin (Ang), BMPs, BDNF, EGF, erythrop
  • the additional therapeutic agent is an immunotherapeutic agent.
  • the immunotherapeutic agent is selected from the group consisting of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3 (IL- 3), interleukin 12 (IL-12), interleukin 1 (IL-1), interleukin 2 (IL-2), B7-1 (CD80), B7-2 (CD86), 4- 1BB ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-Ll antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • IL- 3 interleukin 12
  • IL-1 interleukin 1
  • IL-2 inter
  • an immunotherapeutic agent is selected from the group consisting of: a modulator of PD-1 activity, a modulator of PD-L1 activity, a modulator of PD-L2 activity, a modulator of CTLA-4 activity, a modulator of CD28 activity, a modulator of CD80 activity, a modulator of CD86 activity, a modulator of 4-1BB activity, an modulator of OX40 activity, a modulator of KIR activity, a modulator of Tim-3 activity, a modulator of LAG3 activity, a modulator of CD27 activity, a modulator of CD40 activity, a modulator of GITR activity, a modulator of TIGIT activity, a modulator of CD20 activity, a modulator of CD96 activity, a modulator of IDOl activity, a cytokine, a chemokine, an interferon, an interleukin, a lymphokine, a member of the tumor necrosis factor (
  • an immunotherapeutic agent is selected from the group consisting of: a PD-1 antagonist, a PD-L1 antagonist, a PD-L2 antagonist, a CTLA-4 antagonist, a CD80 antagonist, a CD86 antagonist, a KIR antagonist, a Tim-3 antagonist, a LAG3 antagonist, a TIGIT antagonist, a CD20 antagonist, a CD96 antagonist, and/or an IDOl antagonist.
  • the PD-1 antagonist is an antibody that specifically binds PD-1.
  • the antibody that binds PD-1 is pembrolizumab (KEYTRUDA, MK-3475), pidilizumab (CT-011), nivolumab (OPDIVO, BMS-936558, MDX-1106), MEDI0680 (AMP-514), REGN2810, BGB-A317, PDR-001, or STI-A1110.
  • the antibody that binds PD-1 is described in PCT Publication WO 2014/179664, for example, an antibody identified as APE2058, APE1922, APE1923, APE1924, APE 1950, or APE1963, or an antibody containing the CDR regions of any of these antibodies.
  • the PD-1 antagonist is a fusion protein that includes PD-L2, for example, AMP-224.
  • the PD-1 antagonist is a peptide inhibitor, for example, AU P-12.
  • the PD-L1 antagonist is an antibody that specifically binds PD-L1.
  • the antibody that binds PD-L1 is atezolizumab (RG7446, MPDL3280A),
  • MEDI4736 BMS-936559 (MDX-1105), avelumab (MSB0010718C), KD033, the antibody portion of KD033, or STI-A1014.
  • the antibody that binds PD-L1 is described in PCT Publication WO 2014/055897, for example, Ab-14, Ab-16, Ab-30, Ab-31, Ab-42, Ab-50, Ab-52, or Ab-55, or an antibody that contains the CDR regions of any of these antibodies.
  • the CTLA-4 antagonist is an antibody that specifically binds CTLA-4.
  • the antibody that binds CTLA-4 is ipilimumab (YERVOY) or tremelimumab (CP-675,206).
  • the CTLA-4 antagonist a CTLA-4 fusion protein, for example, KAHR-102.
  • the LAG3 antagonist is an antibody that specifically binds LAG3.
  • the antibody that binds LAG3 is ⁇ 701, IMP731, BMS-986016, LAG525, and GSK2831781.
  • the LAG3 antagonist includes a soluble LAG3 receptor, for example, IMP321.
  • the KIR antagonist is an antibody that specifically binds KIR.
  • the antibody that binds KIR is lirilumab.
  • an immunotherapeutic agent is selected from the group consisting of: a CD28 agonist, a 4-lBB agonist, an OX40 agonist, a CD27 agonist, a CD80 agonist, a CD86 agonist, a CD40 agonist, and a GITR agonist.
  • the OX40 agonist includes OX40 ligand, or an OX40-binding portion thereof.
  • the OX40 agonist may be MEDI6383.
  • the OX40 agonist is an antibody that specifically binds OX40.
  • the antibody that binds OX40 is MEDI6469, MEDI0562, or MOXR0916 (RG7888).
  • the OX40 agonist is a vector (e.g., an expression vector or virus, such as an adenovirus) capable of expressing OX40 ligand.
  • the OX40-expressing vector is Delta -24 -RGDOX or DNX2401.
  • the 4-lBB (CD137) agonist is a binding molecule, such as an anticalin.
  • the anticalin is PRS-343.
  • the 4-lBB agonist is an antibody that specifically binds 4-lBB.
  • antibody that binds 4-lBB is PF- 2566 (PF-05082566) or urelumab (BMS-663513).
  • the CD27 agonist is an antibody that specifically binds CD27.
  • the antibody that binds CD27 is varlilumab (CDX-1127).
  • the GITR agonist comprises a GITR ligand or a GITR-binding portion thereof.
  • the GITR agonist is an antibody that specifically binds GITR.
  • the antibody that binds GITR is TRX518, MK-4166, or INBRX-110.
  • immunotherapeutic agents include, but are not limited to, cytokines such as chemokines, interferons, interleukins, lymphokines, and members of the tumor necrosis factor (TNF) family.
  • immunotherapeutic agents include immunostimulatory oligonucleotides, such as CpG dinucleotides.
  • an immunotherapeutic agent includes, but is not limited to, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-CD28 antibodies, anti-CD80 antibodies, anti-CD86 antibodies, anti-4-lBB antibodies, anti-OX40 antibodies, anti-KIR antibod [0153] ies, anti-Tim-3 antibodies, anti-LAG3 antibodies, anti-CD27 antibodies, anti-CD40 antibodies, anti-GITR antibodies, anti-TIGIT antibodies, anti-CD20 antibodies, anti-CD96 antibodies, or anti-IDOl antibodies.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein in combination with a checkpoint inhibitor.
  • the checkpoint inhibitor is an anti-PD-1 antibody.
  • the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is melanoma.
  • the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is lung cancer.
  • the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is bladder cancer.
  • the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is a hematologic cancer.
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding agent described herein in combination with a checkpoint inhibitor.
  • the checkpoint inhibitor is an anti-PD-Ll antibody.
  • the checkpoint inhibitor is an anti-PD-Ll antibody and the cancer is melanoma.
  • the checkpoint inhibitor is an anti-PD-Ll antibody and the cancer is lung cancer.
  • the checkpoint inhibitor is an anti-PD-Ll antibody and the cancer is bladder cancer.
  • the checkpoint inhibitor is an anti-PD-Ll antibody and the cancer is breast cancer.
  • the checkpoint inhibitor is an anti-PD-Ll antibody and the cancer is a hematologic cancer.
  • the treatment involves the administration of a GITR-binding agent of the present invention in combination with radiation therapy.
  • Treatment with an agent can occur prior to, concurrently with, or subsequent to
  • Dosing schedules for such radiation therapy can be determined by the skilled medical practitioner.
  • Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.
  • a GITR-binding agent described herein and at least one additional therapeutic agent may be administered in any order or concurrently.
  • the GITR-binding agent will be administered to patients that have previously undergone treatment with a second therapeutic agent.
  • the GITR- binding agent and a second therapeutic agent will be administered substantially simultaneously or concurrently.
  • a subject may be given an agent while undergoing a course of treatment with a second therapeutic agent (e.g., chemotherapy).
  • a GITR-binding agent will be administered within 1 year of the treatment with a second therapeutic agent.
  • a GITR-binding agent will be administered within 10, 8, 6, 4, or 2 months of any treatment with a second therapeutic agent. In certain other embodiments, a GITR-binding agent will be administered within 4, 3, 2, or 1 weeks of any treatment with a second therapeutic agent. In some embodiments, a GITR-binding agent will be administered within 5, 4, 3, 2, or 1 days of any treatment with a second therapeutic agent. It will further be appreciated that the two (or more) agents or treatments may be administered to the subject within a matter of hours or minutes (i.e., substantially simultaneously).
  • a GITR-binding agent of the present invention depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the agent is administered for therapeutic or preventative purposes, previous therapy, the patient's clinical history, and so on, all at the discretion of the treating physician.
  • the GITR-binding agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved (e.g., reduction in tumor size).
  • dosage is from O.O ⁇ g to lOOmg/kg of body weight, from O.
  • the dosage of the agent is from about 0. lmg to about 20mg/kg of body weight. In some embodiments, the dosage of the agent is about 0.00 lmg to lOmg/kg of body weight.
  • the dosage of the agent is about 0.001, 0.0025, 0.005, 0.010, 0.015, 0.020, or 0.025mg/kg of body weight. In some embodiments, the dosage of the agent is about 0.030, 0.040, 0.050, 0.10, 0.15, 0.20, or 0.25mg/kg of body weight. In some embodiments, the dosage of the agent is about 0.30, 0.40, 0.45, 0.50, 0.60, 0.75, or 0.9mg/kg of body weight. In some embodiments, the dosage of the agent is about 0.005, 0.015, 0.05, 0.15, 0.45, 0.9, 1.8, or 3.6mg/kg of body weight. In some embodiments, the dosage of the agent is about 0.
  • the dosage of the agent is about 0.25mg/kg of body weight. In some embodiments, the dosage of the agent is about 0.5mg/kg of body weight. In some embodiments, the dosage of the agent is about lmg/kg of body weight. In some embodiments, the dosage of the agent is about 1.5mg/kg of body weight. In some embodiments, the dosage of the agent is about 2mg/kg of body weight. In some embodiments, the dosage of the agent is about 2.5mg/kg of body weight. In some
  • the dosage of the agent is about 5mg/kg of body weight. In some embodiments, the dosage of the agent is about 7.5mg/kg of body weight. In some embodiments, the dosage of the agent is about lOmg/kg of body weight. In some embodiments, the dosage of the agent is about 12.5mg/kg of body weight. In some embodiments, the dosage of the agent is about 15mg/kg of body weight. In certain embodiments, the dosage can be given once or more daily, weekly, monthly, or yearly. In certain embodiments, the agent is given once every week, once every two weeks, once every three weeks, or once every four weeks. In certain embodiments, the agent is given once a week. In certain embodiments, the agent is given once every two weeks. In certain embodiments, the agent is given once every three weeks. In certain embodiments, the agent is given once every four weeks. In certain embodiments, the agent is given once a month.
  • an agent may be administered at an initial higher "loading" dose, followed by one or more lower doses.
  • the frequency of administration may also change.
  • a dosing regimen may comprise administering an initial dose, followed by additional doses (or "maintenance" doses) once a week, once every two weeks, once every three weeks, or once every month.
  • a dosing regimen may comprise administering an initial loading dose, followed by a weekly maintenance dose of, for example, one-half of the initial dose.
  • a dosing regimen may comprise administering an initial loading dose, followed by maintenance doses of, for example one-half of the initial dose every other week.
  • a dosing regimen may comprise administering three initial doses for 3 weeks, followed by maintenance doses of, for example, the same amount every other week.
  • any therapeutic agent may lead to side effects and/or toxicities.
  • the side effects and/or toxicities are so severe as to preclude administration of the particular agent at a therapeutically effective dose.
  • drug therapy must be discontinued, and other agents may be tried.
  • many agents in the same therapeutic class often display similar side effects and/or toxicities, meaning that the patient either has to stop therapy, or if possible, suffer from the unpleasant side effects associated with the therapeutic agent.
  • the dosing schedule may be limited to a specific number of administrations or "cycles".
  • the agent is administered for 3, 4, 5, 6, 7, 8, or more cycles.
  • the agent is administered only once or only twice.
  • the agent is administered every 2 weeks for 6 cycles, the agent is administered every 3 weeks for 6 cycles, the agent is administered every 2 weeks for 4 cycles, the agent is administered every 3 weeks for 4 cycles, etc.
  • Dosing schedules can be decided upon and subsequently modified by those skilled in the art.
  • the agent is administered only once or only twice.
  • the present invention provides methods of administering to a subject the GITR-binding agents described herein comprising using an intermittent dosing strategy for administering one or more agents, which may reduce side effects and/or toxicities associated with administration of an agent, chemotherapeutic agent, etc.
  • a method for treating cancer in a human subject comprises administering to the subject a therapeutically effective dose of an agent in combination with a therapeutically effective dose of a chemotherapeutic agent, wherein one or both of the agents are administered according to an intermittent dosing strategy.
  • the intermittent dosing strategy comprises administering an initial dose of an agent to the subject, and administering subsequent doses of the agent about once every 2 weeks.
  • the intermittent dosing strategy comprises administering an initial dose of an agent to the subject, and administering subsequent doses of the agent about once every 3 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of an agent to the subject, and administering subsequent doses of the agent about once every 4 weeks. In some embodiments, the agent is administered using an intermittent dosing strategy and the chemotherapeutic agent is administered weekly. III. GITR-binding agents
  • the present invention provides methods comprising agents that bind glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR). These agents may be referred to herein as "GITR-binding agents".
  • GITR-binding agents may be referred to herein as "GITR-binding agents”.
  • the GITR-binding agent is a GITR agonist.
  • the GITR-binding agent induces, activates, enhances, increases, and/or prolongs GITR signaling.
  • the agent is a polypeptide.
  • the agent is an antibody.
  • the agent is a soluble protein.
  • the agent is a fusion polypeptide.
  • the agent is a soluble ligand or soluble "co-receptor".
  • the GITR-binding agent comprises a fragment of human GITRL.
  • a fragment of the extracellular domain of human GITRL can demonstrate altered biological activity (e.g., increased protein half -life) compared to a soluble agent comprising the entire extracellular domain.
  • a GITR-binding agent is an agonist antibody that specifically binds GITR.
  • Non-limiting examples of antibodies that bind GITR have been described in, for example, International Application Publication Nos. WO2006/105021,
  • Antibodies that bind GITR include, but are not limited to, INCAGN01876, TRX518, GWN323, AMG 228, BMS-986156, MK-4166, and MK1248.
  • a GITR-binding agent comprises a fusion protein comprising at least one copy of a soluble GITRL polypeptide.
  • a GITR-binding agent comprises a fusion protein comprising at least one copy of the extracellular domain of GITRL or a GITR-binding fragment thereof.
  • the extracellular domain of GITRL lacks a stalk region.
  • the extracellular domain of GITRL comprises a stalk region.
  • the fusion protein comprises one, two, or three copies of the extracellular domain of GITRL.
  • the fusion protein comprises a single chain polypeptide comprising a trimer of the extracellular domain of GITRL. In some embodiments, the fusion protein comprises a single chain polypeptide comprising (a) a trimer of the extracellular domain of GITRL and (b) a human Fc region. In some embodiments, the fusion protein comprises peptides linkers. In some embodiments, the fusion protein does not comprise any peptide linkers.
  • a GlTR-binding agent comprises a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof capable of binding human GITR. In some embodiments, a GlTR-binding agent comprises a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof capable of binding human GITR, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises the stalk region of GITRL.
  • a GlTR-binding agent comprises at least one copy of the extracellular domain of GITRL or a GlTR-binding fragment thereof.
  • the "extracellular domain" of GITRL is approximately amino acids 71-199 of SEQ ID NO:l.
  • the N-terminus and/or C-terminus of the extracellular domain described herein may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.
  • the extracellular domain of GITRL generally comprises the "stalk region” and the "TNF family domain".
  • the copy of the extracellular domain of GITRL in the agents described herein comprises the "stalk region" of GITRL.
  • the "stalk region” of GITRL is approximately amino acids 71-77 of SEQ ID NO: 1.
  • the stalk region comprises approximately amino acids LQLETAK (SEQ ID NO:4).
  • the "TNF homology domain” or “TNF family domain” of GITRL is approximately amino acids 89-192 of SEQ ID NO:l .
  • a GlTR-binding agent comprises a first, second, and third copy of the extracellular domain of GITRL or a GlTR-binding fragment thereof.
  • a GlTR-binding agent comprises at least one copy of SEQ ID NO:3. In some embodiments, a GlTR-binding agent comprises at least two copies of SEQ ID NO:3. In some embodiments, a GlTR-binding agent comprises three copies of SEQ ID NO:3. In certain embodiments, a GlTR-binding agent comprises at least a first, second, and third copy of the extracellular domain of GITRL or a GlTR-binding fragment thereof as a single chain polypeptide. In certain embodiments, a GlTR-binding agent comprises SEQ ID NO: 6. In certain embodiments, a GlTR-binding agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO: 6.
  • a GlTR-binding agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO: 6. In certain embodiments, a GlTR-binding agent comprises a polypeptide having at least 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6. In some embodiments, a GlTR-binding agent comprises a polypeptide consisting essentially of SEQ ID NO:6. In some embodiments, a GlTR-binding agent comprises a polypeptide consisting of SEQ ID NO: 6. In certain embodiments, a GITR-binding agent comprises at least a first, second, and third copy of a fragment of the extracellular domain of GITRL.
  • the copies of the extracellular domain of GITRL consist of the same amino acid sequence. In some embodiments, the copies of the extracellular domain of GITRL are not identical. In some embodiments, the copies of the extracellular domain of GITRL comprise substitutions, deletions, and/or additions to the amino acid sequence of human GITRL as compared to the wild-type sequence.
  • a GITR-binding agent comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof. In some embodiments, a GITR-binding agent comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof, wherein at least one of the extracellular domains comprises the stalk region of GITRL.
  • a GITR-binding agent comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof, wherein at least two of the extracellular domains comprise the stalk region of GITRL. In some embodiments, a GITR-binding agent comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof, wherein each extracellular domain comprises the stalk region of GITRL.
  • a GITR- binding agent comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL, wherein the polypeptide does not comprise any peptide linkers (i.e., exogenous peptide linkers).
  • a GITR-binding agent comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL, wherein the polypeptide does not comprise an exogenous peptide linker between any of the copies of the extracellular domain or a fragment thereof.
  • a GITR- binding agent comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL, wherein each extracellular domain comprises the stalk region and the polypeptide does not comprise any peptide linkers (i.e., exogenous peptide linkers).
  • a GITR-binding agent comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a GITR-binding fragment thereof, wherein the second and third copies of the extracellular domain comprise a stalk.
  • the extracellular domain of GITRL comprises amino acids 71-199 of SEQ ID NO: l.
  • the extracellular domain of GITRL comprises SEQ ID NO:3.
  • the single chain fusion polypeptide comprises SEQ ID NO:6.
  • a GITR-binding agent comprises a variant of the extracellular domain GITRL amino acid sequence or a fragment thereof that comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) conservative substitutions and is capable of binding GITR.
  • the agent is a polypeptide.
  • the polypeptide is a fusion protein.
  • the fusion protein comprises at least one copy of the extracellular domain of human GITRL or a fragment thereof, and further comprises a non-GITRL polypeptide.
  • the fusion protein may include an extracellular domain or fragment thereof linked to a heterologous functional and structural polypeptide including, but not limited to, a human Fc region, one or more protein tags (e.g., myc, FLAG, GST), other endogenous proteins or protein fragments, or any other useful protein sequence including any peptide sequence between the extracellular domain and the non-GITRL polypeptide.
  • the non- GITRL polypeptide comprises a human Fc region.
  • the Fc region can be obtained from any of the classes of immunoglobulin, IgG, IgA, IgM, IgD and IgE.
  • the Fc region is a human IgGl Fc region.
  • the Fc region is a human IgG2 Fc region.
  • the Fc region is a wild-type Fc region.
  • the Fc region is a natural variant of a wild-type Fc region.
  • the Fc region is a mutated Fc region.
  • the Fc region is truncated at the N-terminal end by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, (e.g., in the hinge domain). In some embodiments, the Fc region is truncated at the C- terminal end (e.g., lysine is absent). In some embodiments, an amino acid in the hinge domain is changed to hinder undesirable disulfide bond formation. In some embodiments, a cysteine is replaced with a different amino acid to hinder undesirable disulfide bond formation. In some embodiments, a cysteine is replaced with a serine to hinder undesirable disulfide bond formation. In some embodiments, the Fc region comprises SEQ ID NO:9.
  • the GITR-binding agent comprises a single chain GITRL trimer-Fc protein. In some embodiments, the GITR-binding agent is a single chain GITRL trimer-Fc protein. In some embodiments, the GITR-binding agent is a single chain GITRL trimer-IgGl Fc protein. In some embodiments, the GITR-binding agent comprises SEQ ID NO:7 or SEQ ID NO:8. In some embodiments, the GITR-binding agent comprises SEQ ID NO:7. In some embodiments, the GITR- binding agent comprises SEQ ID NO: 8. In some embodiments, the GITR-binding agent consists essentially of SEQ ID NO: 7 or SEQ ID NO: 8.
  • the GITR-binding agent consists of SEQ ID NO:8.
  • the GITR-binding agent comprises a polypeptide encoded by the "hGITRL-hlgGl" plasmid deposited with ATCC and assigned designation number PTA-122112.
  • the GITR-binding agent is OMP-336B11.
  • the GITR-binding agent is a single chain GITRL trimer-IgG2 Fc protein.
  • the non-GITRL polypeptide comprises SEQ ID NO:9. In certain embodiments, the non-GITRL polypeptide consists essentially of SEQ ID NO:9. In certain embodiments, the non-GITRL polypeptide consists of SEQ ID NO:9. [0175] In certain embodiments, the non-GITRL polypeptide comprises an immunoglobulin heavy chain. In certain embodiments, the immunoglobulin heavy chain is associated with an
  • the non-GITRL polypeptide comprises an antibody. In certain embodiments, the non-GITRL polypeptide comprises a single chain antibody or Fab.
  • a fusion protein comprises a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof and a non-GITRL polypeptide, wherein the C-terminal end of the non-GITRL polypeptide is linked to the extracellular domain(s) of GITRL.
  • a fusion protein comprises a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof and a non-GITRL polypeptide, wherein the N- terminal end of the non-GITRL polypeptide is linked to the extracellular domain(s) of GITRL.
  • the first copy of the extracellular domain of GITRL is linked to the C-terminal end of the non-GITRL polypeptide.
  • the third copy of the extracellular domain of GITRL is linked to the N-terminal end of the non-GITRL polypeptide.
  • the extracellular domain(s) of GITRL is linked to the C-terminal end of a Fc region.
  • the extracellular domain(s) of GITRL is linked to the N-terminal end of a Fc region.
  • the extracellular domain(s) of GITRL is directly linked to the Fc region (i.e. without an intervening peptide linker).
  • the extracellular domain(s) of GITRL is linked to the Fc region via a peptide linker.
  • linker refers to a linker inserted between a first polypeptide (e.g., a extracellular domain of GITRL or a fragment thereof) and a second polypeptide (e.g., a Fc region).
  • the linker is a peptide linker.
  • Linkers should not adversely affect the expression, secretion, or bioactivity of the fusion protein. Linkers should not be antigenic and should not elicit an immune response. Suitable linkers are known to those of skill in the art and often include mixtures of glycine and serine residues and often include amino acids that are sterically unhindered.
  • linkers can range in length, for example from 1-50 amino acids in length, 1-22 amino acids in length, 1-10 amino acids in length, 1-5 amino acids in length, or 1-3 amino acids in length.
  • the linker may comprise a cleavage site.
  • the linker may comprise an enzyme cleavage site, so that the second polypeptide may be separated from the first polypeptide.
  • a linker is an intervening peptide sequence that does not include amino acid residues from either the C-terminus of the first polypeptide (e.g., an extracellular domain of GITRL) or the N- terminus of the second polypeptide (e.g., the Fc region).
  • a GITR-binding agent described herein specifically binds GITR and acts as a GITR agonist. In some embodiments, a GITR-binding agent described herein specifically binds GITR and activates GITR signaling. In some embodiments, a GITR-binding agent described herein specifically binds GITR and induces, activates, promotes, increases, enhances, or prolongs GITR activity.
  • a GITR-binding agent described herein specifically binds GITR and modulates an immune response. In some embodiments, a GITR-binding agent described herein specifically binds GITR and induces, augments, increases, and/or prolongs an immune response.
  • a GITR-binding agent described herein specifically binds GITR with a dissociation constant (K D ) of about ⁇ or less, about lOOnM or less, about 40nM or less, about 20nM or less, about ⁇ or less, about InM or less, or about O. lnM or less.
  • K D dissociation constant
  • a GITR-binding agent binds GITR with a K D of about InM or less.
  • a GITR- binding agent binds GITR with a K D of about 0. InM or less.
  • a GITR-binding agent binds human GITR and/or mouse GITR with a K D of about ⁇ or less. In some embodiments, a GITR-binding agent binds human GITR with a K D of about ⁇ or less.
  • a GITR-binding agent binds human GITR and/or mouse GITR with a K D of about ⁇ or less. In some embodiments, GITR-binding agent binds human GITR and/or mouse GITR with a K D of about InM or less. In some embodiments, a GITR-binding agent binds human GITR and/or mouse GITR with a K D of about 0. InM or less. In some embodiments, a GITR- binding agent binds human GITR and does not bind mouse GITR. In some embodiments, a GITR- binding agent binds human GITR with a K D of about ⁇ or less.
  • a GITR- binding agent binds human GITR with a K D of about InM or less. In some embodiments, a GITR- binding agent binds human GITR with a K D of about O.lnM or less.
  • the dissociation constant of the GITR-binding agent to GITR is the dissociation constant determined using a GITR fusion protein comprising at least a portion of a GITR extracellular domain immobilized on a Biacore chip.
  • a GITR-binding agent binds human GITR with a half maximal effective concentration (EC 50 ) of about ⁇ or less, about lOOnM or less, about 40nM or less, about 20nM or less, about ⁇ or less, about InM or less, or about O. lnM or less.
  • EC 50 half maximal effective concentration
  • fusion polypeptides are made using recombinant DNA techniques as known to one skilled in the art.
  • polynucleotides encoding a specific protein or a fragment thereof are isolated from mammalian cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the gene encoding the protein, and the nucleotide sequence is determined using conventional techniques.
  • the isolated polynucleotides encoding the protein may be cloned into suitable expression vectors which produce the polypeptide when transfected into host cells such as E. coli, simian COS cells, or Chinese hamster ovary (CHO) cells.
  • recombinant proteins, or fragments thereof can be isolated from phage display libraries or using other cell surface display techniques.
  • the polynucleotide(s) encoding a protein can be modified in a number of different manners using recombinant DNA technology to generate alternative or variant proteins. Site-directed or high- density mutagenesis of a protein can be used to optimize specificity, affinity, stability, etc. of a recombinant protein.
  • Proteins generally contain a signal sequence that directs the transport of the proteins.
  • Signal sequences also referred to as signal peptides or leader sequences
  • Signal sequences are located at the N-terminus of nascent polypeptides. They target the polypeptide to the endoplasmic reticulum and the proteins are sorted to their destinations, for example, to the inner space of an organelle, to an interior membrane, to the cell outer membrane, or to the cell exterior via secretion. Most signal sequences are cleaved from the protein by a signal peptidase after the proteins are transported to the endoplasmic reticulum.
  • the cleavage of the signal sequence from the polypeptide usually occurs at a specific site in the amino acid sequence and is dependent upon amino acid residues within the signal sequence. Although there is usually one specific cleavage site, more than one cleavage site may be recognized and/or used by a signal peptidase resulting in a non-homogenous N-terminus of the polypeptide. For example, the use of different cleavage sites within a signal sequence can result in a polypeptide expressed with different N-terminal amino acids. Accordingly, in some embodiments, the polypeptides as described herein may comprise a mixture of polypeptides with different N-termini.
  • the N-termini differ in length by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In some embodiments, the N-termini differ in length by 1, 2, 3, 4, or 5 amino acids.
  • the polypeptide is substantially homogeneous, i.e., the polypeptides have the same N-terminus.
  • the signal sequence of the polypeptide comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) amino acid substitutions and/or deletions as compared to the native sequence of the protein.
  • the signal sequence of the polypeptide comprises amino acid substitutions and/or deletions that allow one cleavage site to be dominant, thereby resulting in a substantially homogeneous polypeptide with one N-terminus.
  • the signal sequence of a fusion polypeptide is not the native signal sequence of the protein(s) contained within the fusion polypeptide.
  • a polypeptide, agent, or fusion polypeptide described herein comprises the Fc region of an immunoglobulin.
  • agents of this invention will comprise fusion proteins in which at least a portion of the Fc region has been deleted or otherwise altered so as to provide desired biochemical characteristics, such as increased cancer cell localization, increased tumor penetration, reduced serum half-life, or increased serum half -life, when compared with a fusion protein of approximately the same immunogenicity comprising a native or unaltered Fc region.
  • Modifications to the Fc region may include additions, deletions, or substitutions of one or more amino acids in one or more domains.
  • the modified fusion proteins disclosed herein may comprise alterations or modifications to one or more of the two heavy chain constant domains (CH2 or CH3) or to the hinge region.
  • CH2 or CH3 the entire CH2 domain may be removed (ACH2 constructs).
  • the omitted constant region domain is replaced by a short amino acid spacer (e.g., 10 aa residues) that provides some of the molecular flexibility typically imparted by the absent constant region domain.
  • the modified fusion proteins are engineered to link the CH3 domain directly to the hinge region or to the first polypeptide.
  • a peptide spacer or linker is inserted between the hinge region or the first polypeptide and the modified CH2 and/or CH3 domains.
  • constructs may be expressed wherein the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region or first polypeptide with a 5-20 amino acid spacer.
  • Such a spacer may be added to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible.
  • amino acid spacers may, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct. Accordingly, in certain embodiments, any spacer added to the construct will be relatively non-immunogenic so as to maintain the desired biological qualities of the fusion protein.
  • the modified fusion proteins may have only a partial deletion of a constant domain or substitution of a few or even a single amino acid.
  • the mutation of a single amino acid in selected areas of the CH2 domain may be enough to substantially reduce Fc binding and thereby increase cancer cell localization and/or tumor penetration.
  • Such partial deletions of the constant regions may improve selected characteristics of the agent (e.g., serum half-life) while leaving other desirable functions associated with the subject constant region domain intact.
  • the constant regions of the disclosed fusion proteins may be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct.
  • the modified fusion proteins comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as decreasing or increasing effector function, or provide for more cytotoxin or carbohydrate attachment sites.
  • the constant region mediates several effector functions. For example, binding of the CI component of complement to the Fc region of IgG or IgM antibodies (bound to antigen) activates the complement system. Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can also be involved in autoimmune hypersensitivity.
  • the Fc region can bind to a cell expressing a Fc receptor (FcR).
  • Fc receptors There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors).
  • the modified fusion proteins provide for altered effector functions that, in turn, affect the biological profile of the agent.
  • the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified agent, thereby increasing cancer cell localization and/or tumor penetration.
  • the constant region modifications increase or reduce the serum half -life of the agent.
  • the constant region is modified to eliminate disulfide linkages or oligosaccharide moiety attachment sites.
  • a modified fusion protein does not have one or more effector functions normally associated with an Fc region.
  • the agent has no antibody- dependent cell-mediated cytotoxicity (ADCC) activity, and/or no complement-dependent cytotoxicity (CDC) activity.
  • ADCC antibody- dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the agent does not bind to the Fc receptor and/or complement factors.
  • the agent has no effector function normally associated with an Fc region.
  • polypeptides and agents of the present invention can be assayed for specific binding to a target by any method known in the art.
  • the immunoassays which can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Biacore analyses, FACS analyses, immunofluorescence, immunocytochemistry, Western blot analyses,
  • radioimmunoassays ELISAs, "sandwich” immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays.
  • assays are routine and well-known in the art.
  • test agent e.g., a polypeptide
  • An ELISA assay comprises preparing GITR protein, coating wells of a 96- well microtiter plate with the GITR, adding the test agent conjugated to a detectable compound such as an enzymatic substrate (e.g. horseradish peroxidase or alkaline phosphatase) to the well, incubating for a period of time and detecting the presence of the agent bound to GITR.
  • a detectable compound such as an enzymatic substrate (e.g. horseradish peroxidase or alkaline phosphatase)
  • the test agent is not conjugated to a detectable compound, but instead a labeled secondary antibody that recognizes the agent is added to the well.
  • the test agent can be coated to the well, GITR is added, and a second antibody conjugated to a detectable compound that recognizes GITR can be used to detect binding.
  • a detectable compound that recognizes GITR can be used to detect binding.
  • the specific binding of a test agent e.g., a polypeptide) to human GITR may be determined using FACS.
  • a FACS screening assay may comprise generating a cDNA construct that expresses GITR, transfecting the construct into cells, expressing GITR on the surface of the cells, mixing the test agent with the transfected cells, and incubating for a period of time.
  • the cells bound by the test agent may be identified by using a secondary antibody conjugated to a detectable compound (e.g., PE-conjugated anti-Fc antibody) and a flow cytometer.
  • a detectable compound e.g., PE-conjugated anti-Fc antibody
  • the binding affinity of a test agent to a target (e.g., human GITR) and the off-rate of an agent- target interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled target (e.g., 3 H or 125 I- labeled GITR), or fragment or variant thereof, with the agent of interest in the presence of increasing amounts of unlabeled target followed by the detection of the agent bound to the labeled target.
  • labeled target e.g., 3 H or 125 I- labeled GITR
  • the affinity of the agent for a target (e.g., human GITR) and the binding off-rates can be determined from the data by Scatchard plot analysis.
  • Biacore kinetic analysis is used to determine the binding on and off rates of agents that bind a target (e.g., human GITR).
  • Biacore kinetic analysis comprises analyzing the binding and dissociation of agents from chips with immobilized target (e.g., human GITR) on the chip surface.
  • this invention also encompasses homodimeric agents and heterodimeric agents/molecules.
  • the homodimeric agents are polypeptides.
  • the heterodimeric molecules are polypeptides. Generally the homodimeric molecule comprises two identical polypeptides. Generally the heterodimeric molecule comprises two non-identical polypeptides.
  • a heterodimeric molecule is capable of binding at least two targets, e.g., a bispecific agent.
  • the targets may be, for example, two different proteins on a single cell or two different proteins on two separate cells.
  • the bispecific agents are polypeptides.
  • one polypeptide of the heterodimeric molecule comprises a polypeptide described herein (e.g., a single chain trimer-Fc protein that binds GITR) and one polypeptide of the heterodimeric molecule is an antibody.
  • the term “arm” may be used herein to describe the structure of a homodimeric agent, a heterodimeric agent, and/or a bispecific agent. As used herein, each "arm" is directed against a target. In some embodiments, one "arm” may comprise an antigen-binding site from an antibody. In some embodiments, one "arm” may comprise a binding portion of a receptor.
  • a homodimeric agent comprises two identical arms. In some embodiments, a heterodimeric agent comprises two different arms. In some embodiments, a bispecific agent comprises two different arms.
  • a bispecific agent comprises the agents described herein.
  • the bispecific agent is a homodimeric protein.
  • the homodimer bispecific agent comprises a polypeptide comprising a heavy chain immunoglobulin and a GITRL trimer.
  • the heavy chain immunoglobulin is associated with a light chain to form an antigen-binding site.
  • the homodimeric bispecific agent comprises a polypeptide comprising an antibody and a single chain GITRL trimer.
  • the homodimeric bispecific agent comprises a polypeptide comprising a single-chain antibody and a single chain GITRL trimer.
  • the homodimeric bispecific agent comprises an antibody that specifically binds a tumor antigen. In some embodiments, the homodimeric bispecific agent comprises an antibody that specifically binds an antigen on an immune cell. In some embodiments, the homodimeric bispecific agent comprises an antibody that specifically binds PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, or TIM3. In some embodiments, the homodimeric bispecific agent binds GITR and PD-1. In some embodiments, the homodimeric bispecific agent binds GITR and PD- Ll.
  • the bispecific agent is a heterodimeric protein.
  • the heterodimeric bispecific agent comprises an antigen-binding site from an antibody (e.g., an antigen-binding site formed by an immunoglobulin heavy chain and an immunoglobulin light chain) and a GITRL trimer.
  • a bispecific agent comprises an immunotherapeutic agent or functional fragment thereof and a GITRL trimer.
  • a heterodimeric bispecific agent is capable of binding one target and also comprises a "non-binding" function.
  • one polypeptide of the heterodimeric bispecific agent comprises a polypeptide described herein (e.g., binds GITR) and one polypeptide of the heterodimeric agent is an additional immunotherapeutic.
  • immunotherapeutic is used in the broadest sense and refers to a substance that directly or indirectly stimulates the immune system by inducing activation or increasing activity of any of the immune system's components.
  • immunotherapeutic s may include cytokines, as well as various antigens including tumor antigens, and antigens derived from pathogens.
  • the immunotherapeutic includes, but is not limited to, a colony stimulating factor (e.g., granulocyte- macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF)), an interleukin (e.g., IL-1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), an antibody that blocks immunosuppressive functions (e.g., an anti- CTLA-4 antibody, anti-CD28 antibody, anti-PD-1 antibody, anti-PD-Ll antibody), a toll-like receptor (e.g., TLR4, TLR7, TLR9), or a member of the B7 family (e.g., CD80, CD86).
  • GM-CSF granulocyte- macro
  • a heterodimeric bispecific agent comprises a first polypeptide comprising a GITRL trimer and a second polypeptide comprising an antibody that specifically binds a tumor antigen.
  • a bispecific agent with a binding specificity for a tumor antigen can be used to direct the GITRL trimer polypeptide to a tumor.
  • the bispecific agent may be used to direct the GITRL trimer polypeptide to a tumor that expresses the tumor antigen or overexpresses the tumor antigen. This may be useful to induce and/or enhance an immune response near or within the tumor microenvironment.
  • a bispecific agent may be used to induce or enhance the activity of tumor infiltrating immune cells.
  • a heterodimeric bispecific agent comprises a first polypeptide comprising a GITRL trimer and a second polypeptide comprising an antibody that specifically binds an immune response molecule.
  • a heterodimeric bispecific agent comprises a first polypeptide comprising a GITRL trimer and a second polypeptide comprising an antibody that specifically binds an immune checkpoint protein.
  • a heterodimeric bispecific agent comprises a first polypeptide comprising a GITRL trimer and a second polypeptide comprising an antibody that specifically binds PD-1.
  • a heterodimeric bispecific agent comprises a first polypeptide comprising a GITRL trimer and a second polypeptide comprising an antibody that specifically binds PD-L1.
  • the heterodimeric molecule (e.g., a bispecific agent) can bind a first target, (e.g., GITR) as well as a second target, such as an effector molecule on a leukocyte (e.g., CD2, CD3, CD28, or CD80) or a Fc receptor (e.g., CD64, CD32, or CD 16) so as to elicit a stronger cellular immune response.
  • a first target e.g., GITR
  • a second target such as an effector molecule on a leukocyte (e.g., CD2, CD3, CD28, or CD80) or a Fc receptor (e.g., CD64, CD32, or CD 16)
  • a bispecific agent either heterodimeric or homodimeric, has enhanced potency as compared to an individual agent. It is known to those of skill in the art that any agent (e.g., a soluble protein or a cytokine) may have unique pharmacokinetics (PK) (e.g., circulating half -life). In some embodiments, a bispecific agent has the ability to synchronize the PK of two active agents and/or polypeptides wherein the two individual agents and/or polypeptides have different PK profiles.
  • PK pharmacokinetics
  • a bispecific molecule has the ability to concentrate the actions of two agents and/or polypeptides in a common area (e.g., a tumor and/or tumor microenvironment). In some embodiments, a bispecific molecule has the ability to concentrate the actions of two agents and/or polypeptides to a common target (e.g., a tumor or a tumor cell). In some embodiments, a bispecific agent has the ability to target the actions of two agents and/or polypeptides to more than one biological pathway or more than one aspect of the immune response. In some embodiments, the bispecific agent has decreased toxicity and/or side effects than either of the polypeptides and/or agents alone.
  • the bispecific agent has decreased toxicity and/or side effects as compared to a mixture of the two individual polypeptides and/or agents. In some embodiments, the bispecific agent has an increased therapeutic index. In some embodiments, the bispecific agent has an increased therapeutic index as compared to a mixture of the two individual agents or the agents used as single entities.
  • a heterodimeric bispecific molecule comprises a first polypeptide comprising a single chain GITRL trimer and a second polypeptide comprising an antibody. In some embodiments, a heterodimeric bispecific molecule comprises a first polypeptide comprising a single chain GITRL trimer and a second polypeptide comprising an antagonist antibody. [0206] In some embodiments, a heterodimeric bispecific agent comprises: (a) a first arm comprising a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of GITRL or a GITR-binding fragment thereof, and (b) a second arm comprising an antigen- binding site from an antibody.
  • a heterodimeric bispecific agent comprises: (a) a first arm comprising a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of GITRL or a GITR-binding fragment thereof, and (b) a second arm comprising an immunotherapeutic agent.
  • at least one copy of the extracellular domain of GITRL of the first arm comprises SEQ ID NO:3.
  • the heterodimeric bispecific agent comprises a first arm comprising SEQ ID NO:6.
  • the heterodimeric bispecific agent comprises a first arm which further comprises a non-GITRL polypeptide.
  • the heterodimeric bispecific agent comprises a single chain fusion GITRL polypeptide described herein which is directly linked to a non-GITRL polypeptide.
  • the single chain fusion polypeptide is connected to the non-GITRL polypeptide by a linker.
  • the non-GITRL polypeptide comprises a human Fc region. .
  • a heterodimeric bispecific molecule comprises a first polypeptide comprising a single chain GITRL trimer and a second polypeptide comprising an immunotherapeutic agent.
  • the multimeric molecule (e.g., a bispecific agent) comprises a first CH3 domain and a second CH3 domain, each of which is modified to promote formation of heteromultimers or heterodimers.
  • the first and second CH3 domains are modified using a knobs-into-holes technique.
  • the first and second CH3 domains comprise changes in amino acids that result in altered electrostatic interactions.
  • the first and second CH3 domains comprise changes in amino acids that result in altered hydrophobic/hydrophilic interactions (see, for example, U.S. Patent App. Publication No. 2011/0123532).
  • the agents are monovalent. In some embodiments, the agent is a soluble protein that is monovalent. In some embodiments, the agents described herein are bivalent. In some embodiments, the agents described herein are trivalent. In some embodiments, the agents described herein are monospecific. In some embodiments, the agents described herein are bispecific. In some embodiments, the agents described herein are multispecific. In some embodiments, the agent is a heterodimeric protein that comprises two arms wherein at least one arm is monovalent. In some embodiments, the agent is a heterodimeric protein that comprises two arms wherein at least one arm is bivalent. In some embodiments, the agent is a heterodimeric protein that comprises two arms wherein at least one arm is trivalent (i.e., binds three target molecules).
  • the agents comprise polypeptides that are substantially homologous to the fusion proteins and/or polypeptides described herein.
  • These agents can contain, for example, conservative substitution mutations, i.e. the substitution of one or more amino acids by similar amino acids.
  • conservative substitution refers to the substitution of an amino acid with another within the same general class such as, for example, one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid by another neutral amino acid. What is intended by a conservative amino acid substitution is well known in the art and described herein.
  • a GITR-binding agent described herein binds GITR and modulates an immune response. In some embodiments, a GITR-binding agent described herein activates and/or increases an immune response. In some embodiments, a GITR-binding agent described herein increases, promotes, or enhances cell-mediated immunity. In some embodiments, a GITR-binding agent described herein increases, promotes, or enhances innate cell-mediated immunity. In some embodiments, a GITR-binding agent described herein increases, promotes, or enhances adaptive cell- mediated immunity. In some embodiments, a GITR-binding agent described herein increases, promotes, or enhances T-cell activity.
  • a GITR-binding agent described herein increases, promotes, or enhances CD4+ T-cell activity. In some embodiments, a GITR-binding agent described herein increases, promotes, or enhances CD8+ T-cell activity. In some embodiments, a GITR-binding agent described herein increases, promotes, or enhances CTL activity. In some embodiments, a GITR-binding agent described herein increases, promotes, or enhances NK cell activity. In some embodiments, a GITR-binding agent described herein increases, promotes, or enhances lymphokine-activated killer cell (LAK) activity.
  • LAK lymphokine-activated killer cell
  • a GITR-binding agent described herein increases, promotes, or enhances tumor-infiltrating lymphocyte (TIL) activity. In some embodiments, a GITR-binding agent described herein inhibits or decreases Treg cell activity. In some embodiments, a GITR-binding agent described herein inhibits or decreases MDSC cell activity. In some embodiments, a GITR-binding agent described herein increases, promotes, or enhances tumor cell killing. In some embodiments, a GITR-binding agent described herein increases, promotes, or enhances the inhibition of tumor growth.
  • TIL tumor-infiltrating lymphocyte
  • a GITR-binding agent described herein increases or enhances an effective immune response without causing substantial side effects and/or immune-based toxicities. In some embodiments, a GITR-binding agent described herein increases or enhances an effective immune response without causing cytokine release syndrome (CRS) or a cytokine storm. In certain embodiments, a GITR-binding agent described herein promotes the formation of a tertiary lymphoid structure in a tumor or tumor microenvironment in a subject.
  • CRS cytokine release syndrome
  • a GITR-binding agent described herein promotes the formation of a tertiary lymphoid structure in a tumor or tumor microenvironment in a subject.
  • a GITR-binding agent described herein binds GITR and induces, enhances, increases, and/or prolongs GITR signaling.
  • a GITR-binding agent described herein is an agonist (either directly or indirectly) of human GITR.
  • a GITR-binding agent is an agonist of GITR and activates and/or increases an immune response.
  • a GITR-binding agent is an agonist of GITR and activates and/or increases activity of NK cells and/or T-cells (e.g., cytolytic activity or cytokine production).
  • a GITR-binding agent increases the activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100%.
  • a GITR-binding agent described herein increases activation of a NK cell. In certain embodiments, a GITR-binding agent increases activation of a T-cell. In certain embodiments, the activation of a NK cell and/or a T-cell by a GITR-binding agent results in an increase in the level of activation of a NK cell and/or a T-cell of at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%.
  • a GITR-binding agent described herein inhibits or decreases the suppressive activity of a Treg cell. In certain embodiments, a GITR-binding agent inhibits activity of a Treg cell. In certain embodiments, the inhibition of suppressive activity of a Treg cell by a GITR- binding agent results in an inhibition of suppressive activity of a Treg cell of at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%.
  • a GITR-binding agent described herein inhibits or decreases the suppressive activity of a MDSC. In certain embodiments, a GITR-binding agent inhibits activity of a MDSC. In certain embodiments, the inhibition of suppressive activity of a MDSC by a GITR-binding agent results in an inhibition of suppressive activity of a MDSC of at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%.
  • a GITR-binding agent described herein promotes the formation of a tertiary lymphoid structure in a tumor or tumor microenvironment in a subject.
  • a GITR-binding agent promotes the density of tertiary lymphoid structures in a tumor or tumor microenvironment in a subject by at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about
  • a GITR-binding agent promotes the formation of a tertiary lymphoid structure in a tumor or tumor microenvironment of a subject treated with the GITR-binding agent, whereas no tertiary lymphoid structures are detectable in the tumor or tumor microenvironment of a control subject not treated with the GITR-binding agent.
  • an immunohistochemistry assay is used to detect tertiary lymphoid structures and/or to analyze the density of a tertiary lymphoid structure in a tumor sample (e.g., a tumor biopsy) obtained from a subject.
  • a tumor sample e.g., a tumor biopsy
  • a functional assay that detects T-cell activation can be used.
  • a functional assay that detects Treg activity can be used.
  • a functional assay that detects MDSC activity can be used.
  • a functional assay that detects NK cell activity can be used.
  • a functional assay that detects cytolytic T-cell activity can be used.
  • an assay that detects cytokine production can be used.
  • an assay that detects cytokine-producing cells can be used.
  • a GITR-binding agent described herein is capable of inhibiting tumor growth.
  • the GITR-binding agent is capable of inhibiting tumor growth in vivo (e.g., in a mouse model and/or in a human having cancer).
  • a GITR-binding agent described herein is capable of reducing the tumorigenicity of a tumor.
  • the GITR-binding agent is capable of reducing the tumorigenicity of a tumor in an animal model, such as a mouse model.
  • the GITR-binding agent is capable of reducing the tumorigenicity of a tumor comprising cancer stem cells in an animal model, such as a mouse model.
  • the number or frequency of cancer stem cells in a tumor is reduced by at least about two-fold, about three-fold, about five-fold, about ten-fold, about 50-fold, about 100-fold, or about 1000-fold.
  • the reduction in the number or frequency of cancer stem cells is determined by limiting dilution assay using an animal model. Additional examples and guidance regarding the use of limiting dilution assays to determine a reduction in the number or frequency of cancer stem cells in a tumor can be found, e.g., in International Publication Number WO 2008/042236; U.S. Patent Publication No. 2008/0064049; and U.S. Patent Publication No. 2008/0178305.
  • a GITR-binding agent described herein has one or more of the following effects: inhibits proliferation of tumor cells, inhibits tumor growth, reduces the tumorigenicity of a tumor, reduces the tumorigenicity of a tumor by reducing the frequency of cancer stem cells in the tumor, triggers cell death of tumor cells, increases cell contact-dependent growth inhibition, increases tumor cell apoptosis, reduces epithelial mesenchymal transition (EMT), or decreases survival of tumor cells.
  • EMT epithelial mesenchymal transition
  • a GITR-binding agent described herein has a circulating half -life in mice, rats, cynomolgus monkeys, or humans of at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, at least about 2 weeks, or at least 3 weeks.
  • the GITR-binding agent is an IgG (e.g., IgGl or IgG2) fusion protein that has a circulating half -life in mice, rats, cynomolgus monkeys, or humans of at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 3 days, at least about 1 week, at least about 2 weeks, or at least 3 weeks.
  • IgG e.g., IgGl or IgG2
  • known methods of increasing the circulating half-life of IgG fusion proteins include the introduction of mutations in the Fc region which increase the pH-dependent binding of the antibody to the neonatal Fc receptor (FcRn) at pH 6.0.
  • Known methods of increasing the circulating half-life of soluble receptors lacking a Fc region include such techniques as PEGylation.
  • the agent is a polypeptide.
  • the polypeptide can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide that binds GITR. It will be recognized in the art that some amino acid sequences of the invention can be varied without significant effect of the structure or function of the protein. Thus, the invention further includes variations of the polypeptides which show substantial binding activity to GITR. In some embodiments, amino acid sequence variations of the polypeptides include deletions, insertions, inversions, repeats, and/or other types of substitutions.
  • polypeptides, analogs and variants thereof can be further modified to contain additional chemical moieties not normally part of the polypeptide.
  • the derivatized moieties can improve the solubility, the biological half-life, and/or absorption of the polypeptide.
  • the moieties can also reduce or eliminate undesirable side effects of the polypeptides and variants.
  • An overview for chemical moieties can be found in Remington: The Science and Practice of Pharmacy, 22 nd Edition, 2012, Pharmaceutical Press, London.
  • polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide sequences and expressing those sequences in a suitable host.
  • a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest.
  • the sequence can be mutagenized by site-specific mutagenesis to provide functional analogs thereof.
  • a DNA sequence encoding a polypeptide of interest may be constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced.
  • Standard methods can be applied to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
  • the polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and/or expression of a biologically active polypeptide in a suitable host. As is well-known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.
  • a recombinant expression vector is used to amplify and express DNA encoding a GITR-binding agent described herein.
  • a recombinant expression vector can be a replicable DNA construct which has synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of an agent operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes.
  • a transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences. Regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are "operatively linked" when they are functionally related to each other.
  • DNA for a signal peptide is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation.
  • structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • recombinant protein is expressed without a leader or transport sequence, it can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus.
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCRl, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single-stranded DNA phages.
  • Suitable host cells for expression of a polypeptide include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters.
  • Prokaryotes include gram-negative or gram-positive organisms, for example E. coli or Bacillus.
  • Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems may also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are well known by those skilled in the art.
  • Suitable mammalian host cell lines include COS-7 (monkey kidney -derived), L-929 (murine fibroblast- derived), C127 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary -derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast- derived), and HEK-293 (human embryonic kidney -derived) cell lines and variants thereof.
  • Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.
  • the present invention provides cells comprising the polypeptides and agents described herein.
  • the cells produce the polypeptides and agents described herein.
  • the cells produce a fusion protein.
  • the cells produce a soluble receptor/ligand.
  • the cells produce an antibody.
  • the cells produce a bispecific agent.
  • the cells produce a bispecific antibody.
  • the cells produce a homodimeric bispecific agent.
  • the cells produce a heterodimeric bispecific agent.
  • the proteins produced by a transformed host can be purified according to any suitable method.
  • Standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification.
  • Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column.
  • Isolated proteins can also be physically characterized using such techniques as proteolysis, mass spectrometry (MS), nuclear magnetic resonance (MR), high performance liquid chromatography (HPLC), and x-ray crystallography.
  • supernatants from expression systems which secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix.
  • a suitable purification matrix for example, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
  • the matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification.
  • a cation exchange step can be employed.
  • Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.
  • a hydroxyapatite media can be employed, including but not limited to, ceramic hydroxyapatite (CHT).
  • CHT ceramic hydroxyapatite
  • one or more reverse-phase HPLC steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups can be employed to further purify an agent. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.
  • recombinant protein produced in bacterial culture can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange, or size exclusion chromatography steps. HPLC can be employed for final purification steps.
  • Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
  • a GITR-binding agent described herein is a polypeptide that does not comprise an immunoglobulin Fc region.
  • the polypeptide comprises a protein scaffold of a type selected from the group consisting of protein A, protein G, a lipocalin, a fibronectin domain, an ankyrin consensus repeat domain, and thioredoxin.
  • phage display technology may be used to produce and/or identify a binding polypeptide.
  • mammalian cell display technology may be used to produce and/or identify a binding polypeptide.
  • a polypeptide in order to increase (or decrease) its serum half-life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the polypeptide by mutation of the appropriate region in the polypeptide or by incorporating the epitope into a peptide tag that is then fused to the polypeptide at either end or in the middle (e.g., by DNA or peptide synthesis).
  • Heteroconjugate molecules are also within the scope of the present invention.
  • Heteroconjugate molecules are composed of two covalently joined polypeptides. Such molecules have, for example, been proposed to target immune cells to unwanted cells, such as tumor cells. It is also contemplated that the heteroconjugate molecules can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4- mercaptobutyrimidate .
  • a GITR-binding agent described herein can be used in any one of a number of conjugated (i.e. an immunoconjugate or radioconjugate) or non-conjugated forms.
  • the agents can be used in a non-conjugated form to harness the subject's natural defense mechanisms including CDC and ADCC to eliminate malignant or cancer cells.
  • an agent described herein is a small molecule.
  • small molecule generally refers to a low molecular weight organic compound which is by definition not a peptide/protein.
  • a GITR-binding agent described herein is conjugated to a cytotoxic agent.
  • the cytotoxic agent is a chemotherapeutic agent including, but not limited to, methotrexate, adriamicin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents.
  • the cytotoxic agent is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • diphtheria A chain nonbinding active fragments of diphtheria toxin
  • exotoxin A chain ricin A chain
  • abrin A chain abrin A chain
  • modeccin A chain alpha-s
  • the cytotoxic agent is a radioisotope to produce a radioconjugate or a radioconjugated agent.
  • radioconjugated agents include, but not limited to, 90 Y, 125 I, m I, 123 I, m In, m In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re, 188 Re, and 212 Bi.
  • Conjugates of an agent and one or more small molecule toxins, such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, can also be used.
  • Conjugates of an agent and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N- succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)
  • SPDP N- succinimidyl-3-(2-pyridyidithiol) propionate
  • IT iminothiolane
  • imidoesters such as dimethyl adipimidate HCL
  • active esters such as disuccinimidyl suberate
  • aldehydes such as glutareldehyde
  • bis-azido compounds such as bis(p-azidobenz
  • bis-diazonium derivatives such as bis-(p-diazoniumbenzoyl)-ethylenediamine
  • diisocyanates such as toluene 2,6-diisocyanate
  • bis-active fluorine compounds such as 1,5- difluoro-2,4-dinitrobenzene
  • Kits comprising agents described herein
  • kits that comprise the agents described herein and that can be used to perform the methods described herein.
  • a kit comprises at least one purified agent in one or more containers.
  • the kits contain all of the components necessary and/or sufficient to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results.
  • the disclosed agents of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.
  • kits that comprise a GITR-binding agent as well as at least one additional therapeutic agent.
  • the second (or more) therapeutic agent is a chemotherapeutic agent.
  • the second (or more) therapeutic agent is an additional immunotherapeutic agent.
  • Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail preparation of certain antibodies of the present disclosure and methods for using antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the present disclosure. EXAMPLES
  • a soluble single chain human GITRL trimer construct was generated and linked to a human IgGl Fc region (OMP-336B11; SEQ ID NO:7 with signal sequence and SEQ ID NO:8 without signal sequence).
  • OMP-336B11 was shown to bind human GITR, activate T-cells, and have anti-tumor activity in a humanized mouse model.
  • RNA-Seq data analyses of 33 tumor types in The Cancer Genome Atlas were undertaken. These analyses showed that GITR is highly expressed in a subset of solid tumors, including head & neck cancer, lung cancer, cervical cancer, uterine cancer, breast cancer, esophageal cancer, and bladder cancer ( Figure 1). Further analysis showed that in most tumors, GITR expression correlated poorly with T-cell markers, implying that GITR may not be exclusive to immune -related cells and may be expressed in tumor cells as well. Similar findings were observed in RNA-Seq data analyses of patient-derived xenograft samples from 24 tumor types ( Figure 2).
  • GITR immunohistochemistry (IHC) assay was developed and optimized using a proprietary mouse anti-hGITR monoclonal antibody.
  • IHC GITR immunohistochemistry
  • Four-micron FFPE sections were cut and mounted onto positively -charged capillary gap slides. Tissues were dewaxed through four, 5-minute changes of xylene followed by a graded alcohol series to distilled water.
  • Steam heat-induced epitope recovery (SHIER) was performed with SHIER 2 solution (citrate- based buffer pH 6.0-6.2) for 20 minutes in the upper chamber of a Black and Decker Steamer.
  • Slides were pre-treated with proteinase K enzyme (1:40 dilution) for 10 minutes and were incubated in a blocking solution for 15 minutes to reduce non-specific background staining. Slides were incubated with the anti-hGITR antibody overnight at 1 ⁇ g/ml. To block endogenous peroxidase activity slides were incubated in hydrogen peroxide 3 times for 2.5 minutes and washed in PBS. Specific binding was detected using a Polink-2 Plus Detection Kit designed for mouse antibodies and using DAB and hematoxylin. Positive staining is indicated by the presence of a brown chromogen reaction product of the horseradish peroxidase and DAB substrate. Hematoxylin counterstain provides a blue nuclear stain to assess cell and tissue morphology.
  • the scoring data for GITR expression in tumor cells was evaluated using a standard H-score approach and a Percent Score approach. The H-score is calculated using the percentage of cells with intensity of expression on a three-point semi-quantitative scale (0, 1+, 2+, or 3+). Thus, scores range from 0 to 300.
  • H-score [(% at 1+) x 1] + [(% at 2+) x 2] + [(% at 3+) x 3].
  • Range 10-30% of cases NSCLC adenocarcinoma 20
  • Negative Bladder cancer 0 Range 0% of cases with Colon cancer 0
  • Range 44-100% of Melanoma 55 cases > 2+ TNBC 48
  • 336B3 was administered to immunocompetent mice bearing CT26.WT colon, 4T1 breast, or B16F10 melanoma tumors. These tumors are considered to have varying levels of immunogenicity - CT26.WT tumors are highly immunogenic and cytotoxic T-cell rich; 4T1 tumors are highly immunogenic and immunosuppressive; and B16F10 tumors are poorly immunogenic and immunologically silent. Mice were treated once a week in each of the models.
  • Global gene expression levels were profiled by microarray on treated and control tissues.
  • Immune cell-related gene changes with emphasis on T-cell, NK cell, and B-cell markers were examined since these cell populations express GITR. Changes of immune cell populations and cytokine secretions were monitored by flow cytometry, Luminex, and IHC.
  • Murine breast carcinoma EMT6 tumors are characterized by high infiltration of myeloid cells and low infiltration of T cells.
  • EMT6 tumors are commonly viewed as exemplifying immune "cold” or “non-(T-cell) inflamed” tumors.
  • Non-inflamed tumors are generally expected to be more resistant to immune check point inhibitors, such as antagonistic anti-PD-1 or anti-PD-Ll antibodies, than "hot" tumors with high T cell infiltration.
  • EMT6 tumor bearing mice were dosed with 336B3, antagonistic anti-PDl antibody, or control antibody, and tumor growth (Example 5) and immune infiltration into tumors (Example 6) were monitored.
  • EMT6 cells were implanted subcutaneously into Balb/c mice (1.5 x 10 5 cells per mouse) and EMT6 tumors were allowed to grow to an average size of ⁇ 90 mm 3 .
  • TILs tumor infiltrating lymphocytes
  • Germinal centers can be formed in the spleen, the lymph nodes, and other secondary lymphoid tissues, as well as in peripheral tissues. When formed in peripheral tissues, germinal centers are often referred to as "tertiary lymphoid structures.” It is noteworthy that the presence of tertiary lymphoid structures is sometimes observed within tumors and that the presence of such tertiary lymphoid structures has been strongly associated with favorable outcome.
  • the results presented in this Example indicate that a GITRL-Fc fusion protein can increase lymphocyte infiltration into an immune cold and checkpoint inhibitor resistant tumor and promote the formation of tertiary lymphoid structures.
  • the results presented in Example 5 indicate that the same GITRL-Fc fusion protein can inhibit the growth of such lymphocyte infiltrated tumor.
  • OMP-336B11 Human single chain GITRL trimer-Fc (IgGl) with signal sequence nucleotide sequence (SEQ ID NO: 15)

Abstract

Méthodes comprenant l'utilisation de nouveaux agents qui se lient spécifiquement au GITR. Ces méthodes comprennent l'utilisation d'agents se liant au GITR pour induire et/ou augmenter la réponse immunitaire, ainsi que des méthodes de traitement de maladies telles que le cancer.
PCT/US2018/019970 2017-02-28 2018-02-27 Méthodes de traitement du cancer comprenant des agents se liant au gitr WO2018160572A1 (fr)

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CN112162097A (zh) * 2020-07-24 2021-01-01 广州医科大学 Gdf1作为评估pd-1单抗治疗效果的生物标志物

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WO2016126781A1 (fr) * 2015-02-03 2016-08-11 Oncomed Pharmaceuticals, Inc. Agents de liaison à la tnfrsf et leurs utilisations

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CN108289966A (zh) * 2015-09-24 2018-07-17 北卡罗来纳-查佩尔山大学 用于减少转移的方法和组合物
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