WO2024077099A1 - Perk inhibitor hc-5404 in combination with anti-pd-1 antibody and/or an antiangiogenetic agent for use in the treatment of cancer - Google Patents

Perk inhibitor hc-5404 in combination with anti-pd-1 antibody and/or an antiangiogenetic agent for use in the treatment of cancer Download PDF

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Publication number
WO2024077099A1
WO2024077099A1 PCT/US2023/076006 US2023076006W WO2024077099A1 WO 2024077099 A1 WO2024077099 A1 WO 2024077099A1 US 2023076006 W US2023076006 W US 2023076006W WO 2024077099 A1 WO2024077099 A1 WO 2024077099A1
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effective amount
therapeutically effective
cancer
pharmaceutically acceptable
compound
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PCT/US2023/076006
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French (fr)
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Nandita BOSE
Jeremy DREES
David Surguladze
Michael Stokes
Mark J. Mulvihill
Veronica CALVO VIDAL
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Hibercell, Inc.
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Publication of WO2024077099A1 publication Critical patent/WO2024077099A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens

Definitions

  • Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020 (World Health Organization). Targeted therapy and immunotherapy have expanded the horizons for treatment of cancers by improving prognosis drastically.
  • tumor recurrence, drug resistance, and drug intolerance continue to be major challenges in the management of cancer (Wang et al., “Drug resistance and combating drug resistance in cancer,” Cancer Drug Resistance, 2019, 2(2): 141-160; Chakraborty et al., “The difficulties in cancer treatment” Ecancermedicalscience, 2012, 6:edl6). Cancer shows extensive proliferation of tumor cells which requires well-functioning protein machinery (Waczak et al, 2019).
  • the endoplasmic reticulum (ER) plays a crucial role in regulating protein folding, synthesis, and maturation required for tumor cells, maintaining tumor homeostasis (Yoo et al, 2017).
  • the accumulation of unfolded proteins in the ER lumen activates three main pathways, 1) PERK, 2) inositol -requiring enzyme-1 alpha (IREla), and 3) activating transcription factor 6 (ATF6) — together known as the unfolded protein response (UPR) — which allows cells to correct and survive this stress (Walter and Ron, 2011; Urra et al, 2016).
  • UPR activation is a mechanism that allows tumor cells to adapt to demands on the ER and oxidative conditions imposed by conditions inherent to their biology such as an enhanced translational load caused by oncogenes and loss of tumor suppressor genes, and by environmental conditions common in tumor/metastasis microenvironments such as hypoxia among other signals (Blais et al, 2004; Chevet et al, 2015; Tameire et al, 2015; Hart et al, 2012; Martin-Perez et al, 2014; Rajasekhar and Holland, 2004;
  • PERK As a member of the UPR pathways, PERK is involved in pro-tumorigenesis processes, and this role makes it an attractive target for anticancer therapy. More specifically, PERK phosphorylates the eukaryotic translation initiation factor 2 alpha (eIF2a), nuclear factor (erythroid- derived 2)-like-2 (Nrf2), and forkhead box protein 01 (FOXO) proteins. Phosphorylation of eIF2a inhibits general protein synthesis and lowers the protein load, while inducing the targeted translation of uORF containing mRNAs such as ATF4.
  • eIF2a eukaryotic translation initiation factor 2 alpha
  • Nrf2 nuclear factor- derived 2
  • FOXO forkhead box protein 01
  • ATF4 then triggers a gene expression program consisting of antioxidant, chaperones, and autophagy genes among others aimed at restoring protein homeostasis and inducing cell survival in the context of ER stress.
  • Cancer cells are, by definition, characterized by cellular activities and microenvironmental factors that contribute to the activation of the UPR (including oncogenic protein synthesis, hypoxic environments, dissemination and lodging into a different microenvironment, etc.) and, in addition, the action of many therapeutic agents further contribute to the activation of the UPR in the cells that are not eliminated by the therapy.
  • the UPR pathways, including PERK, have not been a commonly investigated pathway / target for cancer therapy.
  • kits for activating the immune system of a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD- 1 antibody.
  • kits for activating the immune system of a subject having cancer comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD- 1 antibody.
  • the method inhibits the activity of myeloid-derived suppressor cells (MDSCs). In certain embodiments, the method increases the infiltration of T cells and NK cells into a tumor.
  • MDSCs myeloid-derived suppressor cells
  • the tumor is selected from the group consisting of bladder cancer, squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer.
  • the method increases the frequency of dendritic cells in a draining lymph node.
  • kits for treating a cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD- 1 antibody.
  • the cancer is a solid tumor.
  • the cancer is selected from the group consisting of bladder cancer, squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer.
  • the cancer is a liquid tumor.
  • the cancer is selected from the group consisting of Classical Hodgkin’s lymphoma, primary thymic mediastinal lymphoma, multiple myeloma, and B cell malignancies (e.g., non -Hodgkin lymphomas or chronic lymphocytic leukemia).
  • the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, and cemiplimab. In certain embodiments, the anti-PD-1 antibody is pembrolizumab. In certain embodiments, the anti-PD-1 antibody is nivolumab.
  • the therapeutically effective amount of the anti-PD-1 antibody is administered once daily. In certain embodiments, the therapeutically effective amount of the anti- PD-1 antibody is administered twice daily.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered simultaneously. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered sequentially.
  • the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered in an amount of about 22 mg to about 451 mg twice daily. In certain embodiments, the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount of about 177 mg to about 900 mg twice daily.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered in the form of a capsule.
  • the method increases the tumor cell expression of type 1 interferon receptor (INF ARI) in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
  • the method increases the peripheral blood monocyte surface expression of INF ARI in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
  • the method increases tumor cell expression of calreticulin in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
  • the method increases polymorphonuclear myeloid-derived suppressor cell (MDSC) and/or tumor associated macrophage (TAM) expression of INF ARI in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
  • MDSC polymorphonuclear myeloid-derived suppressor cell
  • TAM tumor associated macrophage
  • the method increases TAM expression of PD-L1 in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody. In certain embodiments, the method increases the infiltration of CD8 T cells and/or NK cells in a tumor in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
  • the method increases expression of CD69 on T cells in a tumor draining lymph node in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
  • the method reduces suppressive activity of MDSCs in the subject.
  • the method increases dendritic cell frequency in a tumor draining lymph node in the subject.
  • the method increases Ki67, Granzyme B (GzmB), and/or memory phenotypic markers on CD4 T-cells in the subject.
  • the compound of formula (I) is administered as a pharmaceutically acceptable salt.
  • the pharmaceutically acceptable salt is a hemifumarate salt.
  • provided herein are methods of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I): or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent.
  • methods of treating a cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I): or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent, wherein the subject has been treated with an anti-cancer therapy.
  • the subject is resistant or has acquired resistance to the anti-cancer therapy.
  • the anti-cancer therapy is selected from the group consisting of: administration of an immunotherapeutic agent, a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, an anti angiogenic agent, or a combination thereof; radiation therapy; surgery; and combinations thereof.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti angiogenic agent are administered simultaneously.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti angiogenic agent are administered sequentially.
  • the therapeutically effective amount of the anti angiogenic agent is administered to the subject daily.
  • the therapeutically effective amount of the anti angiogenic agent is administered to the subject once daily.
  • the therapeutically effective amount of the anti angiogenic agent is administered to the subject twice daily.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti angiogenic agent reduces p-PERK levels in the subject relative to administering the therapeutically effective amount of the anti angiogenic agent alone.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti angiogenic agent increases ATF4 levels in the subject relative to administering the therapeutically effective amount of the anti angiogenic agent alone.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti angiogenic agent modulates the activation of unfolded protein response (UPR) relative to administering the therapeutically effective amount of the anti angiogenic agent alone.
  • URR unfolded protein response
  • kits for treating a cancer in a subject receiving a therapeutically effective amount of an anti angiogenic agent therapy comprising administering to the subject a therapeutically effective amount of a compound of formula (I): or a pharmaceutically acceptable salt thereof.
  • the subject is resistant or has acquired resistance to the anti angiogenic agent therapy.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof reduces p-PERK levels in the subject relative to administering the therapeutically effective amount of the anti angiogenic agent alone.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof increases ATF4 levels in the subject relative to administering the therapeutically effective amount of the anti angiogenic agent alone.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof modulates the activation of unfolded protein response (UPR) relative to administering the therapeutically effective amount of the anti angiogenic agent alone.
  • URR unfolded protein response
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof decreases tumor vasculature in the subject relative to administering the therapeutically effective amount of the anti angiogenic agent alone.
  • the anti angiogenic agent is a vascular endothelial growth factor receptor (VEGFR) modulator.
  • VEGFR vascular endothelial growth factor receptor
  • the anti angiogenic agent is a vascular endothelial growth factor (VEGF) targeting antibody.
  • VEGF vascular endothelial growth factor
  • the anti angiogenic agent is a tyrosine kinase inhibitor (TKI).
  • TKI tyrosine kinase inhibitor
  • the anti angiogenic agent is a VEGF receptor tyrosine kinase inhibitor (VEGFR- TKI).
  • the anti angiogenic agent is selected from the group consisting of: apatinib, axitinib, brivanib, cabozantinib, canertinib, cediranib, dasatinib, erlotinib, gefitinib, leflunomide, lenvatinib, motesanib, nilotinib, nintedanib, pazopanib, regorafenib, semaxinib, sorafenib, sunitinib, tivozanib, vandetanib, vatalanib, XL0192, bevacizumab, ramucirumab, and pharmaceutically acceptable salts or biosimilars thereof.
  • the anti angiogenic agent is selected from the group consisting of: bevacizumab, ramucirumab, and biosimilars thereof.
  • the anti angiogenic agent is selected from the group consisting of: apatinib, axitinib, brivanib, cabozantinib, canertinib, cediranib, dasatinib, erlotinib, gefitinib, leflunomide, lenvatinib, motesanib, nilotinib, nintedanib, pazopanib, regorafenib, semaxinib, sorafenib, sunitinib, tivozanib, vandetanib, vatalanib, XL0192, and pharmaceutically acceptable salts thereof.
  • the cancer is a metastatic cancer or a locally advanced cancer.
  • the cancer is a solid tumor.
  • the cancer is selected from the group consisting of: a bladder cancer, a breast cancer, a carcinoma, a cervical cancer, a colorectal cancer, a gastric cancer, a hepatocellular cancer, a kidney cancer, a lung cancer, a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer, and a thyroid cancer.
  • the cancer is selected from the group consisting of: a breast cancer, a gastric cancer, a kidney cancer, and a lung cancer.
  • the breast cancer is triple negative breast cancer or metastatic breast cancer;
  • the carcinoma is carcinoma of unknown primary (CUP), endometrial carcinoma, head and neck squamous cell carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma;
  • the gastric cancer is adenocarcinoma or gastrointestinal stromal tumor;
  • the kidney cancer is a renal cell carcinoma (RCC); or
  • the lung cancer is small cell lung cancer (SCLC) or nonsmall cell lung cancer.
  • the breast cancer is triple negative breast cancer or metastatic breast cancer.
  • the carcinoma is carcinoma of unknown primary (CUP), endometrial carcinoma, head and neck squamous cell carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma.
  • CUP carcinoma of unknown primary
  • endometrial carcinoma head and neck squamous cell carcinoma
  • Merkel cell carcinoma Merkel cell carcinoma
  • squamous cell carcinoma or urothelial carcinoma.
  • the gastric cancer is adenocarcinoma or gastrointestinal stromal tumor.
  • the kidney cancer is a renal cell carcinoma (RCC).
  • RRC renal cell carcinoma
  • the lung cancer is small cell lung cancer (SCLC) or non-small cell lung cancer.
  • the renal cell carcinoma is clear cell renal cell carcinoma (ccRCC).
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, daily.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, once daily.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, twice daily.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, daily.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, once daily.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, twice daily.
  • the subject is in a fasting state.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered with food.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered about 30 minutes to about 1 hour after food.
  • the therapeutically effective amount of the compound of formula (I) is administered as a pharmaceutically acceptable salt.
  • the pharmaceutically acceptable salt is a hemifumarate salt.
  • the method further comprises administering to the subject a therapeutically effective amount of an immunotherapy.
  • kits for treating a cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I): or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent, and a therapeutically effective amount of an immunotherapy.
  • kits for treating a cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of formula (I): or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent, and a therapeutically effective amount of an immunotherapy, wherein the subject has been treated with an anti -cancer therapy.
  • the subject is resistant or has acquired resistance to the anti-cancer therapy.
  • the anti-cancer therapy is selected from the group consisting of: administration of an immunotherapeutic agent, a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, an anti angiogenic agent, or a combination thereof; radiation therapy; surgery; and combinations thereof.
  • kits for treating a cancer in a subject receiving a therapeutically effective amount of an anti angiogenic agent therapy comprising administering to the subject a therapeutically effective amount of a compound of formula (I): or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy.
  • the immunotherapy is an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody.
  • the immunotherapy is a therapeutically effective amount of an anti- PD-1 antibody.
  • the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, and cemiplimab.
  • the anti-PD-1 antibody is pembrolizumab.
  • the anti-PD-1 antibody is nivolumab.
  • the therapeutically effective amount of the immunotherapy is administered daily.
  • the therapeutically effective amount of the immunotherapy is administered once daily.
  • the therapeutically effective amount of the immunotherapy is administered twice daily.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the immunotherapy are administered simultaneously.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of an immunotherapy are administered sequentially.
  • the therapeutically effective amount of the anti angiogenic agent and the therapeutically effective amount of an immunotherapy are administered simultaneously.
  • the therapeutically effective amount of the anti angiogenic agent and the therapeutically effective amount of an immunotherapy are administered sequentially.
  • FIG. 1A is a graph showing the effects on tumor volume of single agent 10 mg/kg HC- 5404 twice daily or 30 mg/kg HC-5404 twice daily compared to vehicle in a renal cell carcinoma model.
  • FIG. IB is a graph showing the effects on tumor volume of single agent 30 mg/kg HC- 5404 twice daily compared to vehicle in a gastric cancer model.
  • FIG. 2A is a scheme showing a study design for the MB49 studies described in Example 2.
  • FIG. 2B is a graph showing the tumor volume post-MB49 cell inoculation in MB49 tumor-bearing mice administered with vehicle orally twice daily, 3 mg/kg HC-5404-FU orally twice daily, 10 mg/kg HC-5404-FU orally twice daily, 30 mg/kg HC-5404-FU orally twice daily, 5 mg/kg anti-PD-1 antibody intraperitoneally biweekly, 3 mg/kg HC-5404-FU orally twice daily + 5 mg/kg anti-PD-1 antibody intraperitoneally biweekly, 10 mg/kg HC-5404-FU orally twice daily + 5 mg/kg anti-PD-1 antibody intraperitoneally biweekly, or 30 mg/kg HC-5404-FU orally twice daily + 5 mg/kg anti-PD-1 antibody intraperitoneally biweekly.
  • FIG. 2C is a graph showing the tumor volume post-MB49 cell inoculation in MB49 tumor-bearing mice administered with vehicle, 30 mg/kg HC-5404-FU orally twice daily, 5 mg/kg anti-PD-1 antibody intraperitoneally biweekly, or 30 mg/kg HC-5404-FU orally twice daily + 5 mg/kg anti-PD-1 antibody intraperitoneally biweekly.
  • FIG. 2D is a graph showing the tumor volume of individual subjects and number of responders post-cell inoculation in the vehicle treatment group described in FIG. 2C.
  • FIG. 2E is a graph showing the tumor volume of individual subjects and number of responders post-cell inoculation in the 30 mg/kg HC-5404-FU treatment group described in FIG.
  • FIG. 2F is a graph showing the tumor volume of individual subjects and number of responders post-cell inoculation in the anti-PD-1 antibody treatment group described in FIG. 2C.
  • FIG. 2G is a graph showing the tumor volume of individual subjects and number of responders post-cell inoculation in the 30 mg/kg HC-5404-FU + anti-PD-1 antibody treatment group described in FIG. 2C.
  • FIG. 3A is a graph showing the IFNAR1 expression on Ml macrophages from the treatment groups described in FIG. 2B 14 days post-inoculation (7 days post treatment), as determined by flow cytometry.
  • FIG. 3B is a graph showing the IFNAR1 expression on M2 macrophages from the treatment groups described in FIG. 2B 14 days post-inoculation (7 days post treatment), as determined by flow cytometry.
  • FIG. 3C is a graph showing the IFNAR1 expression on polymorphonuclear MDSCs from the treatment groups described in FIG. 2B 14 days post-inoculation (7 days post treatment), as determined by flow cytometry.
  • FIG. 3D is a graph showing the IFNAR1 expression on tumor cells from the treatment groups described in FIG. 2B 14 days post-inoculation (7 days post treatment), as determined by flow cytometry.
  • FIG. 3E is a graph showing the PD-L1 expression on macrophages from the treatment groups described in FIG. 2B 14 days post-inoculation (7 days post treatment), as determined by flow cytometry.
  • FIG. 4A is a diagram showing proteins involved in immunogenic cell death in cancer therapy.
  • FIG. 4B is a graph showing the surface calreticulin expression on tumor cells from the treatment groups described in FIG. 2B 14 days post-inoculation (7 days post treatment), as determined by flow cytometry.
  • FIG. 4C is a comparison of images of 786-0 cells from a xenograft model treated with HC-5404 or vehicle showing HMGB1 in the cytoplasm detected by immunohistochemistry.
  • FIG. 4D is a graph showing HMGB1 cytoplasmic expression induced by 30 mg/kg HC- 5404 treatment compared to vehicle in 786-0 xenograft model on day 15 post treatment.
  • FIG. 5A is a graph showing the frequency of tumor-infiltrated CD8 T cells in the treatment groups described in FIG. 2B on day 7 post treatment, as determined by flow cytometry.
  • FIG. 5B is a graph showing the frequency of tumor-infiltrated CD8 NK cells in the treatment groups described in FIG. 2B on day 7 post treatment, as determined by flow cytometry.
  • FIG. 5C is a graph showing the frequency of CD69 expression on CD4 T cells from lymph nodes of the treatment groups described in FIG. 2B on day 7 post treatment, as determined by flow cytometry.
  • FIG. 5D is a graph showing the frequency of CD69 expression on CD8 T cells from lymph nodes of the treatment groups described in FIG. 2B on day 7 post treatment, as determined by flow cytometry.
  • FIG. 6A is a graph showing the Ki67 expression on CD4 T-cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
  • FIG. 6B is a graph showing the EOMES expression on CD4 T-cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
  • FIG. 6C is a graph showing the Granzyme B expression on CD4 T-cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
  • FIG. 6D is a graph showing the effector memory populations of CD4 T-cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
  • FIG. 6E is a graph showing the TCF1 expression on CD4 T-cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
  • FIG. 7A is a graph showing the total dendritic cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
  • FIG. 7B is a graph showing the XCR1+ cDCl cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
  • FIG. 7C is a graph showing the cDC2 cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
  • FIG. 8 is a comparison of graphs showing the expression of IFNAR1 on monocytes from whole blood of the vehicle, 10 mg/kg HC-5404, and 30 mg/kg HC-5404 treatment groups described in FIG. 2B on days 7, 11, and 14 post treatment, as determined by flow cytometry.
  • FIG. 9 is a heat map showing the expression of myeloid (Ml- and M2-related), lymphoid (T-cell related), and interferon stimulated genes (ISG) in the vehicle, 10 mg/kg HC-5404, and 30 mg/kg HC-5404 treatment groups described in FIG. 2B on day 7 post treatment.
  • Ml- and M2-related myeloid
  • T-cell related lymphoid
  • ISG interferon stimulated genes
  • FIG. 10A shows the results of a T cell suppression assay performed using human CD3 T cells co-cultured with human MDSCs and treated with vehicle, 500 nM HC-5404, or 750 nM HC- 5404 (1 : 1 MDSC:T cell ratio).
  • FIG. 10B is a scheme showing the preparation of differentiated human MDSCs from cord blood.
  • FIG. 10C is a histogram showing the results of the T cell suppression assay described in
  • FIG. 11A shows the results of a T cell suppression assay performed using mouse CD8 T cells co-cultured with MDSCs and treated with RPMI medium, LSLG, or LSLG + 500 nM HC- 5404 (8: 1 MDSGT cell ratio).
  • FIG. 11B is a scheme showing the preparation of mouse MDSC from bone marrow of non-tumor bearing mouse.
  • FIG. 11C is a set of histograms showing the results of the suppression assay described in FIG. 11A and a similar assay performed for mouse CD4 T cells.
  • FIG. 12 is a graph showing the FRET -based biochemical assays used to evaluate HC-5404 inhibition of GCN2, an ISR kinase, across concentration series ranging from 0.2 nM to 10 pM.
  • FIG. 13 is a graph showing the FRET -based biochemical assays used to evaluate HC-5404 inhibition of PERK, an ISR kinase, across concentration series ranging from 0.2 nM to 10 pM.
  • FIG. 14 is a graph showing the FRET -based biochemical assays used to evaluate HC-5404 inhibition of HRI, an ISR kinase, across concentration series ranging from 0.2 nM to 10 pM.
  • FIG. 15 is a graph showing the FRET -based biochemical assays used to evaluate HC-5404 inhibition of GCN2, an ISR kinase, across concentration series ranging from 0.2 nM to 10 pM.
  • FIG. 16 is a table demonstrating that HC-5404 is selective for PERK relative to four closely -related ISR kinases.
  • FIG. 17 is a Biochemical TreeSpot kinome panel assay demonstrating the selectivity of HC-5404 (100 nM, 1000 nM, 10,000 nM) against over 400 kinases. PERK is not included in the panel, and no interactions were observed when HC-5404 assayed at 100 nM.
  • FIG. 18 shows free drug levels of HC-5404 in plasma following single oral administration. Plasma sampled across a 24 h period and quantified by LC-MS/MS.
  • FIG. 19 shows PK/PD relationship of plasma exposure following oral administration of HC-5404 at 30 mg/kg and pPERK levels in mouse pancreas.
  • FIG. 20 shows PK/PD relationship of plasma exposure following oral administration of HC-5404 at 100 mg/kg and pPERK levels in mouse pancreas.
  • FIG. 21 shows PD effect of HC-5404 on pPERK in 786-0 tumors sampled 1, 4, 8, 12 h post last dose following 15 days of BID dosing.
  • FIG. 22 is a graph showing the tumor volumes for 786-0 xenografts treated with HC-5404 at multiple dose levels and treatment regimens for 28 days. Values represent mean tumor volume ⁇ SEM.
  • FIG. 23 is a graph showing the tumor volumes for mice harboring subcutaneous CAPAN- 2 pancreatic tumor xenografts treated with HC-5404 at 30 mg/kg and 100 mg/kg PO, BID for 48 days. Values represent mean tumor volume ⁇ SEM.
  • FIG. 24 Mouse pancreas sections following three weeks of treatment with HC-5404 at either 30 mg/kg, PO, BID or 100 mg/kg PO, BID. The pancreas on the right was given a two-week washout period following treatment with 100 mg/kg PO BID, to demonstrate reversibility of the effect.
  • FIG. 25 is a graph demonstrating the pPERK/PERK protein ratio from 786-0 xenografts treated with sunitinib (40 mg/kg; PO; QD) for one or two weeks.
  • FIG. 26 A, D, G 786-0 tumor xenografts treated with cabozantinib (15, 30, 45 mg/kg; PO; QD), lenvatinib (5 and 10 mg/kg; PO; QD), or axitinib (15 and 30 mg/kg; PO; BID) for 21 days.
  • B, E, H pPERK relative to total PERK were evaluated from tumor samples taken at the end of study, following 21 days of treatment. ANOVA statistical analysis compared differences between protein abundance. Bars highlight significant differences between groups, including p- values.
  • C, F, I Mouse body weights measured twice weekly across the course of treatment.
  • FIG. 27 shows a SimpleWestem protein analysis of pPERK and PERK in 786-0 xenografts treated with VEGFR-TKIs in panel B, sampled after seven days of treatment.
  • FIG. 28 is a graph demonstrating the tumor volume of 786-0 tumor xenografts treated with HC-5404 (30 mg/kg; BID), sunitinib (40 mg/kg; QD), lenvatinib (10 mg/kg; BID), axitinib (30 mg/kg; BID), or cabozantinib (30 mg/kg; QD) and combinations as indicated for 28 days.
  • FIG. 29 demonstrates that HC-5404 induces accumulation of ASNS, CBS, and CTH.
  • FIG. 30 shows pPERK and total PERK abundance evaluated by SimpleWestem in RCC tumor xenografts treated for seven days with HC-5404 (30 mg/kg; PO; BID), sunitinib (20 or 40 mg/kg as indicated; PO; QD), or a combination thereof.
  • FIG. 31 shows A498 RCC tumor xenografts treated with HC-5404 (30 mg/kg; PO; BID) and sunitinib (20 mg/kg; PO; QD) for 30 days.
  • FIG. 32 shows Caki-1 RCC tumor xenografts treated with HC-5404 (30 mg/kg; PO; BID) and sunitinib (20 mg/kg; PO; QD) for 30 days.
  • FIG. 33 is a growth curve of a representative RCC PDX model across 28 days of treatment with either axitinib (30 mg/kg; PO; BID), HC-5404 (30 mg/kg; PO; BID), or a combination thereof.
  • FIG. 34 is a growth curve of a representative RCC PDX model across 28 days of treatment with either axitinib (30 mg/kg; PO; BID), HC-5404 (30 mg/kg; PO; BID), or a combination thereof.
  • FIG. 36 shows the sensitivity across three RCC xenograft models of HC-5404 administered at 10 and 30 mg/kg, PO, BID for 28 days.
  • 786-0 and A498 are VHL mutant models; Caki-1 VHL wild type.
  • FIG. 37 shows RCC tumor models treated with HC-5404 (30 mg/kg; PO; BID) and DC- 101 (15 mg/kg; IP; BIW) for 28 days. Shown here are mean tumor volumes ⁇ SEM. B, D, F) Ratio of pPERK to total PERK across four treatment groups in three RCC models, evaluated by SimpleWestern protein analysis system. Note: low signal to noise ratio in Caki-1 model.
  • FIG. 39 shows quantification of IHC staining of xenograft sections. Graphs indicate proportion of cells stained positive for Meca32+, positive for CD31 in absence of SMA (%CD31+SMA-), or cells that stain positive for both CD31 and SMA (%CD31+SMA+).
  • One-way ANOVA statistical analysis evaluated differences between treatment groups; significant differences between treatment vs vehicle indicated by bars and p-values.
  • FIG. 40 shows 786-0 tumor growth across the study period. Xenografts that progressed in presence of axitinib (30 mg/kg; PO; BID) for 14 days were rerandomized and transferred to indicated treatment groups for an additional 28 days.
  • axitinib (30 mg/kg; PO; BID)
  • FIG. 41 shows IHC images of xenograft sections stained with antibodies specific for CD31 and SMA.
  • FIG. 42 shows quantification of proportion of cells that stained positive for SMA, CD31, or Meca32.
  • One-way ANOVA was used to evaluate the statistical significance between groups. P- value indicated for significant differences between treatments and vehicle.
  • Quantification of % positive cells stained for CD31 a marker of the vascular endothelium.
  • FIG. 43 shows quantification of IHC staining of tumor sections using antibodies specific for the pericyte markers NG2 and MCAM.
  • One-way ANOVA was used to evaluate the statistical significance between groups. P-value indicated for significant differences between treatment groups versus either baseline or vehicle.
  • FIG. 44 shows images and quantification of IHC staining of tumor sections using antibodies specific for the pericyte markers NG2 and MCAM.
  • One-way ANOVA was used to evaluate the statistical significance between groups. P-value indicated for significant differences between treatment groups versus either baseline or vehicle.
  • FIG. 45 shows a tumor growth curve and pPERK/PERK levels relative to total PERK in 786-0 tumor xenografts treated with cabozantinib (15, 30, 45 mg/kg; PO; QD) for 21 days.
  • ANOVA statistical analysis compared differences between protein abundance. Bars highlight significant differences between groups, including p-values.
  • FIG. 46 shows a tumor growth curve and pPERK/PERK levels relative to total PERK in 786-0 tumor xenografts treated with lenvatinib (5 and 10 mg/kg; PO; QD) for 21 days.
  • ANOVA statistical analysis compared differences between protein abundance. Bars highlight significant differences between groups, including p-values.
  • FIG. 47 shows a tumor growth curve and pPERK/PERK levels relative to total PERK in 786-0 tumor xenografts treated with axitinib (15 and 30 mg/kg; PO; BID) for 21 days.
  • ANOVA statistical analysis compared differences between protein abundance. Bars highlight significant differences between groups, including p-values.
  • FIG. 48 shows a tumor growth curve for 786-0 tumor xenografts treated with once daily oral administration of lenvatinib (10 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof.
  • FIG. 49 shows a tumor growth curve for 786-0 tumor xenografts treated with twice daily oral administration of axitinib (30 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof.
  • FIG. 50 shows a tumor growth curve for A498 tumor xenografts treated with once daily oral administration of sunitinib (20 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof.
  • FIG. 51 shows a tumor growth curve for A498 tumor xenografts treated with twice weekly intraperitoneal injection of DC-101 (15 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof.
  • FIG. 52 shows a tumor growth curve for 786-0 tumor xenografts treated with twice daily oral administration of HC-5404 at 3, 10, or 30 mg/kg as a single agent or in combination with once daily oral dosing of cabozantinib at 30 mg/kg.
  • HC-5404 at 10 or 30 mg/kg in combination with cabozantinib resulted in tumor regression -66%.
  • FIG. 53 shows a body mass curve for 786-0 tumor xenografts treated with twice daily oral administration of HC-5404 at 3, 10, or 30 mg/kg as a single agent or in combination with once daily oral dosing of cabozantinib at 30 mg/kg.
  • FIG. 54 shows images of IHC staining of CD31+SMA- cells treated with cabozantinib in combination with HC-5404.
  • FIG. 55 shows quantification of IHC staining of CD31+SMA- cells treated with cabozantinib in combination with HC-5404.
  • FIG. 56 shows images of IHC staining of pericyte cells treated with cabozantinib in combination with HC-5404.
  • FIG. 57 shows quantification of IHC staining of pericyte cells treated with cabozantinib in combination with HC-5404. Quantification of IHC staining of 786-0 RCC tumor xenografts treated with HC-5404 at 3, 10, or 30 mg/kg (PO; BID) as a single agent or in combination with cabozantinib (30 mg/kg; PO; QD).
  • FIG. 58 shows a tumor growth curve for RENCA (VHL w t) RCC tumor xenografts treated with HC-5404, axitinib, anti-PD-1, or a combination thereof.
  • FIG. 59 shows a body mass curve for RENCA (VHL w t) RCC tumor xenografts treated with HC-5404, axitinib, anti-PD-1, or a combination thereof.
  • FIG. 60 shows graphs demonstrating the effect of HC-5404 in combination with axitinib on RENCA RCC model.
  • FIG. 61 shows graphs demonstrating the effect of HC-5404, axitinib, or HC-5404 in combination with axitinib on RENCA RCC model.
  • FIG. 62 shows graphs demonstrating the effect of HC-5404, axitinib, or HC-5404 in combination with axitinib on CD4 T-Cells in tumor-draining lymph nodes.
  • Top left CD4 T-cells that expressed the activation marker CD69 were quantified as a % positive of total CD4 T-cells.
  • Top right Effector memory CD4 T-cells (CD4+, CD44+, CD62L-) in TDLN were quantified as a percentage of total CD4-cells.
  • Middle right; bottom left; bottom right Multiple exhaustion markers were measured as % positive of total CD4 T-cells.
  • Graphs represent individual animals +/- SD for each treatment group. Significance indicators are based on ordinary one-way ANOVA and Tukey’s multiple comparisons test.
  • FIG. 63 demonstrates the effect of HC-5404, axitinib, and HC-5404 in combination with axitinib on myeloid cells in tumor microenvironment in RENCA RCC tumors.
  • FIG. 64 demonstrates the effect of HC-5404, axitinib, and HC-5404 in combination with axitinib on myeloid cells in tumor microenvironment in RENCA RCC tumors. Fluorescence microscopy for the following markers on macrophages are shown: M2 marker CD206 (top left), and the Ml markers CD86 (top right) and PD-L1 (bottom). Values indicate the percentage of macrophages that express each marker. Dots represent individual animals, and error bars indicate the SD of each group. Significance indicators are based on ordinary one-way ANOVA and Tukey’s multiple comparisons test.
  • FIG. 65 demonstrates the effect of HC-5404, axitinib, aPD-1, or a combination of HC- 5404 with axitinib and aPD-1 on macrophages in RENCA RCC model.
  • Macrophages with an Ml- like phenotype (MHCII+, CD206-) (left) or an M2 -like phenotype (MHCII-, CD206+) (right) were quantified as a percentage of total F480+ macrophages.
  • Individual animals +/- SEM are displayed. Significance is based on a One-way ANOVA with Dunnett’s multiple comparisons.
  • FIG. 66 demonstrates the effect of HC-5404 in combination with axitinib and aPD-1 on Renca RCC model.
  • FIG. 67 demonstrates the effect of HC-5404, axitinib, aPD-1, and HC-5404 in combination with axitinib and aPD-1 on T-cell infiltration.
  • FIG. 68 demonstrates the effect of HC-5404 in combination with aPD-1 on myeloid cells in tumor microenvironment and T-cell activation. Tumor volume was measured over time and is displayed for each group (mean +/- SEM).
  • FIG. 69 demonstrates the effect of HC-5404 in combination with aPD-1 on myeloid cells in tumor microenvironment and T-cell activation in syngeneic MB49 model.
  • Top Ml Macs, M2 Macs, PMN-MDSCs
  • IFNAR1 type 1 interferon receptor
  • MFI geometric mean fluorescence intensity
  • TDLN tumor-draining lymph nodes
  • FIG. 70 shows the efficacy of HC-5404 on RT4 bladder cells.
  • FIG. 71 shows the efficacy of HC-5404 on HCT116 CRC cells.
  • FIG. 72 shows the efficacy of HC-5404 on A549 NSCLC cells.
  • FIG. 73 shows the efficacy of HC-5404 on A375 melanoma cells.
  • FIG. 74 shows a tumor growth curve for MFE280 MSS model of endometrial cancer treated with twice daily oral administration of HC-5404 (30 mg/kg), once daily oral administration of lenvatinib (10 mg/kg), or a combination thereof.
  • FIG. 75 shows a tumor growth curve for GAO 114 PDX model of gastric cancer treated with HC-5404 and DC-101.
  • FIG. 76 shows a tumor growth curve for TM01125 PDX model of pancreatic cancer treated with HC-5404 and sunitinib.
  • FIG. 77 shows a tumor growth curve for MFE280 model of human endometrial cancer model treated with twice daily oral administration of HC-5404, once daily oral administration of lenvatinib (10 mg/kg), or a combination thereof.
  • FIG. 78 shows a body mass curve for MFE280 model of human endometrial cancer model treated with twice daily oral administration of HC-5404, once daily oral administration of lenvatinib (10 mg/kg), or a combination thereof.
  • FIG. 79 demonstrates the effect of HC-5404 in combination with lenvatinib on MFE280 human endometrial cancer model.
  • FIG. 80 demonstrates the effect of HC-5404 in combination with envatinib on MFE280 human endometrial cancer model.
  • FIG. 81 demonstrates the effect of HC-5404 in combination with DC-101 in gastric PDX model.
  • ASNS asparagine synthetase
  • PSGDH phosphoglycerate dehydrogenase
  • PSAT1 phosphoserine aminotransferase 1
  • PSPH phosphoserine phosphatase
  • SHMT1 serine hy droxymethyltransf erase 1
  • HC-5404 (30 mg/kg; PO; BID), DC101 (20 mg/kg; IP; BIW), or a combination thereof for seven days and sampled 1 h after final morning dose.
  • These proteins can serve a biomarkers of HC-5404 activity in vivo.
  • FIG. 82 demonstrates the effect of HC-5404 in combination with DC-101 in gastric PDX model. Quantification of SimpleWestem signal represented in panel FIG. 81.
  • FIG. 83 is a tumor growth curve for CAPAN2 PDAC model treated with HC-5404 in combination with gemcitabine.
  • FIG. 84 is a tumor growth curve for MB49 bladder cancer model treated with HC-5404 at 3, 10, 30 mg/kg PO BID in combination with anti-PDl (IP; BIW) antibody for 14 days.
  • FIG. 85 is a tumor growth curve for NCI-H660 NEPC model treated with HC-5404 in combination with docetaxel.
  • FIG. 86 shows plasma analysis from mice bearing 786-0 tumors, treated with HC-5404 at 3, 10, or 30 mg/kg (PO; BID) as a single agent or in combination with cabozantinib (30 mg/kg; PO; QD).
  • Vascular endothelial growth factor-A (VEGF-A) was decreased in plasma by HC-5404 as a single agent in a dose-dependent fashion.
  • FIG. 87 shows plasma analysis from mice bearing 786-0 tumors, treated with HC-5404 at 3, 10, or 30 mg/kg (PO; BID) as a single agent or in combination with cabozantinib (30 mg/kg; PO; QD).
  • Hepatocyte growth factor (HGF) was decreased by the combination of HC-5404 and cabozantinib in mouse plasma.
  • FIG. 88 shows plasma analysis from mice bearing 786-0 tumors, treated with HC-5404 at 3, 10, or 30 mg/kg (PO; BID) as a single agent or in combination with cabozantinib (30 mg/kg; PO; QD).
  • Interleukin-8 (IL-8) was decreased by the combination of HC-5404 and cabozantinib in mouse plasma.
  • FIG. 89 shows plasma analysis from mice bearing 786-0 tumors, treated with HC-5404 at 3, 10, or 30 mg/kg (PO; BID) as a single agent or in combination with cabozantinib (30 mg/kg; PO; QD).
  • Lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) was decreased by HC-5404 in a dose-dependent manner as either monotherapy or in combination with cabozantinib.
  • FIG. 90 shows plasma analysis from mice bearing 786-0 tumors, treated with HC-5404 at 3, 10, or 30 mg/kg (PO; BID) as a single agent or in combination with cabozantinib (30 mg/kg; PO; QD). Syndecan was decreased by HC-5404 as monotherapy or in combination with cabozantinib.
  • FIG. 91 is a tumor growth curve for mice harboring 786-0 xenografts that were treated for 14 days with axitinib (30 mg/kg; PO; BID), following which the mice with tumors that progressed (approximately doubled in size) were rerandomized into four groups. After enabling 786-0 xenografts to progress on axitinib for two weeks, HC-5404 was added to the regimen. Data presented are mean ⁇ SEM. [00207] FIG.
  • 92 shows quantification of IHC images from FFPE tumor sections sampled 7 days after treating mice harboring 786-0 xenografts for 14 days with axitinib (30 mg/kg; PO; BID), randomizing the mice with tumors that progressed (approximately doubled in size), and then treating with HC-5404.
  • SMA smooth muscle actin
  • Top right Proportion of cells expressing Meca32 was decreased in the combination group relative to vehicle. Meca32 is a marker of vascular endothelial cells.
  • FIG. 93 shows quantification of IHC images from FFPE tumor sections sampled 7 days after treating mice harboring 786-0 xenografts for 14 days with axitinib (30 mg/kg; PO; BID), randomizing the mice with tumors that progressed (approximately doubled in size), and then treating with HC-5404. Tumors randomized into the combination group have decreased proportion of cells expressing the pericyte markers NG2 and MCAM.
  • FIG. 94 is a tumor growth curve for 786-0 RCC tumor models treated with HC-5404 (30 mg/kg; PO; BID) and DC-101 (15 mg/kg; IP; BIW) for 28 days. Mean tumor volumes ⁇ SEM.
  • FIG. 95 is a tumor growth curve for A498 RCC tumor models treated with HC-5404 (30 mg/kg; PO; BID) and DC-101 (15 mg/kg; IP; BIW) for 28 days. Mean tumor volumes ⁇ SEM.
  • FIG. 96 is a tumor growth curve for RENCA RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID), or a combination thereof.
  • FIG. 97 is a tumor growth curve for RXF-393 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
  • FIG. 98 is a tumor growth curve for RXF-2282 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
  • FIG. 99 is a tumor growth curve for RXF-2178 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
  • FIG. 100 is a tumor growth curve for RXF-2502 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
  • FIG. 101 is a tumor growth curve for SMTCA75 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
  • FIG. 102 is a tumor growth curve for RXF-616 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
  • FIG. 103 is a tumor growth curve for RXF-2304 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
  • FIG. 104 is a tumor growth curve for RXF-488 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
  • FIG. 105 is a tumor growth curve for RXF-2667 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
  • FIG. 106 is a tumor growth curve for RXF-1220 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
  • FIG. 107 is a tumor growth curve for RXF-2783 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
  • FIG. 108 is a tumor growth curve for RXF-631 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
  • the present disclosure provides methods of treating a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
  • the present disclosure also provides methods of activating the immune system of a subject in need thereof (e.g., a subject having cancer).
  • the methods described herein generally comprise administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy (e.g., an anti-PD-1 antibody).
  • an immunotherapy e.g., an anti-PD-1 antibody
  • the present disclosure also provides methods of treating a cancer (e.g., a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer), and a thyroid cancer) in a subject in need thereof.
  • a cancer e.g., a bladder cancer,
  • the methods described herein generally comprise administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent. Also disclosed herein are methods comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent, and a therapeutically effective amount of an immunotherapy.
  • compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
  • variables or parameters are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual subcombination of the members of such groups and ranges.
  • an integer in the range of 0 to 40 is specifically intended to individually disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40
  • an integer in the range of 1 to 20 is specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
  • compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.
  • composition or “pharmaceutical formulation” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.
  • “Pharmaceutically acceptable” means approved or approvable by a regulatory agency of the federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly, in humans.
  • “pharmaceutically acceptable salt” refers to any salt of an acidic or a basic group that may be present in a compound of the present invention (e.g., a compound of formula (I)), which salt is compatible with pharmaceutical administration.
  • salts of compounds may be derived from inorganic or organic acids and bases.
  • acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene- -sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acid.
  • Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid addition salts.
  • bases include, but are not limited to, alkali metal (e.g., sodium and potassium) hydroxides, alkaline earth metal (e.g., magnesium and calcium) hydroxides, ammonia, and compounds of formula NW 4+ , wherein W is Ci-4 alkyl, and the like.
  • alkali metal e.g., sodium and potassium
  • alkaline earth metal e.g., magnesium and calcium
  • W is Ci-4 alkyl
  • salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemifumarate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, prop
  • salts include anions of the compounds of the present invention compounded with a suitable cation such as Na + , K + , Ca 2+ , NH 4+ , and NW 4+ (where W can be a Ci-4 alkyl group), and the like.
  • salts of the compounds of the present invention are contemplated as being pharmaceutically acceptable.
  • salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.
  • pharmaceutically acceptable excipient refers to a substance that aids the administration of an active agent to and/or absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient.
  • Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, such as a phosphate buffered saline solution, emulsions (e.g., such as an oil/water or water/oil emulsions), lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer’s solution), alcohols, oils, gelatins, carbohydrates, fatty acid esters, and colors, and the like.
  • emulsions e.g., such as an oil/water or water/oil emulsions
  • lactated Ringer lactated Ringer’s
  • sucrose normal glucose
  • binders such as a phosphate buffered saline solution
  • fillers such as an oil/water or water/oil emulsions
  • disintegrants e.g., such as an oil/water or water/
  • Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention.
  • a “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and/or non-human animals, e.g., mammals such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and/or dogs.
  • the subject is a human.
  • the subject is an adult human.
  • the subject is a non-human animal.
  • solid dosage form means a pharmaceutical dose(s) in solid form, e.g., tablets, capsules, granules, powders, sachets, reconstitutable powders, dry powder inhalers and chewables.
  • administering means oral administration, administration as a suppository, topical contact, intravenous administration, parenteral administration, intraperitoneal administration, intramuscular administration, intralesional administration, intrathecal administration, intracranial administration, intranasal administration or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject.
  • Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).
  • Parenteral administration includes, e.g., intravenous, intramuscular, intra-arterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.
  • co-administer it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies (e.g., anti-cancer agent, chemotherapeutic, or immunotherapy).
  • additional therapies e.g., anti-cancer agent, chemotherapeutic, or immunotherapy.
  • the compound of formula (I), or a pharmaceutically acceptable salt thereof can be administered alone or can be co-administered to the patient.
  • Coadministration is meant to include simultaneous or sequential administration of the compound individually or in combination (more than one compound or agent).
  • the preparations can also be combined, when desired, with other active substances (e.g., to
  • fasting state means at least 1 hour before food or at least 2 hours after food is consumed by a subject.
  • the terms “treat,” “treating,” and “treatment” contemplate an action that occurs while a subject is suffering from the specified disease, disorder or condition, which reduces the severity of the disease, disorder or condition, or retards or slows the progression of the disease, disorder or condition (e.g., “therapeutic treatment”).
  • an “effective amount” or a “therapeutically effective amount” of a compound refers to an amount sufficient to elicit the desired biological response, e.g., to treat a cancer or activate the immune system in a subject in need thereof.
  • the therapeutically effective amount of a compound of the disclosure may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, weight, health, and condition of the subject.
  • activating refers to stimulation of an immune cell (e.g., a T cell, NK cell, B cell, dendritic cell, or macrophage) that results in cellular proliferation, maturation, cytokine production and/or induction of regulatory or effector functions.
  • an immune cell e.g., a T cell, NK cell, B cell, dendritic cell, or macrophage
  • Methods of evaluating immune cell activation or function include, but are not limited to, proliferation assays such as BRDU and thymidine incorporation, cytotoxicity assays such as chromium release, cytokine secretion assays such as intracellular cytokine staining ELISPOT and ELISA, and expression assays of activation markers such as CD25 and CD69 using flow cytometry and multimer (e.g., tetramer) assays.
  • proliferation assays such as BRDU and thymidine incorporation
  • cytotoxicity assays such as chromium release
  • cytokine secretion assays such as intracellular cytokine staining ELISPOT and ELISA
  • expression assays of activation markers such as CD25 and CD69 using flow cytometry and multimer (e.g., tetramer) assays.
  • myeloid derived suppressor cell refers to a collection of immature myeloid cells that modulate the activity of a variety of effector cells and antigen-presenting cells, such as T cells, NK cells, dendritic cells, and macrophages, among others.
  • Myeloid derived suppressor cells are distinguished by their gene expression profile, and express all or a subset of proteins and small molecules selected from the group consisting of B7-1 (CD80), B7- H1 (PD-L1 ), CCR2, CDld, CDldl , CD2, CD31 (PECAM-1), CD43, CD44, complement component C5aRl , F4/80 (EMR1), Fey RIII (CD16), Fey RII (CD32), Fey RIIA (CD32a), Fey RUB (CD32b), Fey RIIB/C (CD32b/c), Fey RIIC (CD32c), Fey RIIIA (CD16A), Fey RIIIB (CD16b), galectin-3, GP130, Gr-1 (Ly-6G), ICAM-1 (CD54), IL-1RI, IL-4Ra, IL- 6Ra, integrin aL (CD49d), integrin aL (CD1 la), integrin aM (
  • MDSCs do not express proteins selected from the group consisting of B7-2 (CD86), B7-H4, CDl lc, CD14, CD21 , CD23 (FCERII), CD34, CD35, CD40 (TNFRSF5), CD117 (c-kit), HLA-DR, and Sca-1 (Ly6).
  • tumor-infiltrating lymphocytes refers to white blood cells (e.g., T cells and/or NK cells) of a subject afflicted with a cancer (e.g., a cancer described herein), that are resident in a tumor or otherwise have left the circulation (blood or lymphatic fluid) and have migrated into a tumor.
  • a cancer e.g., a cancer described herein
  • immunotherapy refers to a treatment of disease by inducing, enhancing, or suppressing an immune response. Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress an immune response are classified as suppression immunotherapies.
  • the phrase “immune checkpoint inhibitor” refers to an agent that blocks certain proteins made by some types of immune system cells, such as T cells, and some cancer cells. These proteins help keep immune responses in check and can prevent T cells from killing cancer cells. When these proteins are blocked, immune system function is restored and the immune system is released enabling T cells to kill cancer cells.
  • the term “anti angiogenic agent” refers an agent that modulates (e.g., inhibits) angiogenesis, vasculogenesis, or vascular permeability. Anti angiogenic agents include agents that block the angiogenic activity of the angiogenic factor or its receptor.
  • the term “antibody” refers to a functional component of serum and is often referred to either as a collection of molecules (antibodies or immunoglobulins) or as one molecule (the antibody molecule or immunoglobulin molecule).
  • An antibody is capable of binding to or reacting with a specific antigenic determinant (the antigen or the antigenic epitope), which in turn may lead to induction of immunological effector mechanisms.
  • An individual antibody is usually regarded as monospecific, and a composition of antibodies may be monoclonal (i.e., consisting of identical antibody molecules) or polyclonal (i.e., consisting of two or more different antibodies reacting with the same or different epitopes on the same antigen or on distinct, different antigens).
  • Each antibody has a unique structure that enables it to bind specifically to its corresponding antigen, and all natural antibodies have the same overall basic structure of two identical light chains and two identical heavy chains.
  • Antibodies are also known collectively as immunoglobulins.
  • An antibody may be of human or non-human (for example, rodent such as murine, dog, camel, etc.) origin (e.g., may have a sequence originally developed in a human or non-human cell or organism), or may be or comprise a chimeric, humanized, reshaped, or reformatted antibody based, e.g., on a such a human or non-human antibody (or, in some embodiments, on an antigen-binding portion thereof).
  • biosimilar is used to describe a biological agent that is highly similar to a reference biological agent.
  • the term is generally used to describe a subsequent version of a biological agent (e.g., from a different source) and is unlikely to be exactly the same as the reference biological agent.
  • an antagonist refers to an agent that (i) decreases or suppresses one or more effects of another agent; and/or (ii) decreases or suppresses one or more biological events.
  • an antagonist may reduce the level and/or activity of one or more agents that it targets.
  • an antagonist may be a receptor antagonist, e.g., a receptor ligand or drug that blocks or dampens a biological response by binding to and blocking a receptor rather than activating it like an agonist.
  • agonist refers to an agent that (i) increases or induces one or more effects of another agent; and/or (ii) increases or induces one or more biological events.
  • an agonist may increase the level and/or activity of one or more agents that it targets.
  • the compound of formula (I), as depicted below, is a selective PERK inhibitor, and is also known as (A)-2-amino-5-(4-(2-(3,5-difluorophenyl)-2-hydroxyacetamido)-2-methylphenyl)-N- i sopropy Ini cotinami de :
  • the compound of formula (I) may also be referred to as HC-5404 throughout the present disclosure.
  • the hemifumarate salt of the compound of formula (I) may be referred to as HC-5404- FU throughout the present disclosure.
  • a method of chemically synthesizing the compound of formula (I) and its hemifumarate salt is described in Example 1.
  • a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for the treatment of a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endo
  • a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for the treatment of a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine
  • a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD-1 antibody for the treatment of a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial
  • a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD-1 antibody for the treatment of a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer
  • a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
  • an immunotherapy e.g., an immune checkpoint inhibitor
  • a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD-1 antibody for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
  • a method of administering a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for the treatment of a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial
  • a method of administering a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for the treatment of a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer
  • a method of administering a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and a therapeutically effective amount of an anti-PD-1 antibody for the treatment of a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma,
  • a method of administering a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and a therapeutically effective amount of an anti-PD-1 antibody for the treatment of a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer
  • a method of administering a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
  • an immunotherapy e.g., an immune checkpoint inhibitor
  • a method of administering a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and a therapeutically effective amount of an anti-PD-1 antibody for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
  • the immunotherapy is a PD-1 or PD-L1 inhibitor. In certain embodiment, the immunotherapy is an anti-PD-1 antibody.
  • a cancer e.g., a solid tumor, which, in some embodiment
  • a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (VEGFR-TKI))
  • a cancer
  • a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma
  • a cancer e.g., a solid tumor
  • a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma
  • a cancer e.g., a solid tumor
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroen
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g.,
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroen
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g.,
  • the method further comprises administering to the subject a therapeutically effective amount of an immunotherapy (e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody).
  • an immunotherapy e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody.
  • a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)), and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti- CTLA-4 antibody) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal
  • an anti angiogenic agent e.
  • a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)), and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti- CTLA-4 antibody) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal
  • an anti angiogenic agent e.
  • a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)), and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti- CTLA-4 antibody) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal
  • an anti angiogenic agent e.
  • a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)), and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti- CTLA-4 antibody) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal
  • an anti angiogenic agent e.
  • a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy e.g., an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody
  • an immunotherapy e.g., an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gas
  • a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy e.g., an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody
  • an immunotherapy e.g., an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gas
  • the pharmaceutically acceptable salt of the compound of formula (I) is a fumarate salt. In certain embodiments, the pharmaceutically acceptable salt of the compound of formula (I) is a hemifumarate salt.
  • Antiangiogenic agents are limited by primary and secondary resistance mechanisms. As part of their mechanism, VEGFR-TKIs induce hypoxia and nutrient-deprivation that drives ER stress. Tumors evade deleterious ER stress by activating the PERK branch of the integrated stress response, which arrests global translation and restores protein homeostasis.
  • an antiangiogenic agent is an antibody or a biosimilar thereof. In some embodiments, an antiangiogenic agent is a small molecule or a pharmaceutically acceptable salt thereof. In some embodiments, an antiangiogenic agent is a vascular endothelial growth factor receptor (VEGFR) modulator. In some embodiments, an antiangiogenic agent is a vascular endothelial growth factor (VEGF) targeting antibody. In some embodiments, an antiangiogenic agent is a tyrosine kinase inhibitor (TKI). In some embodiments, an antiangiogenic agent is a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI).
  • antiangiogenic agents include, but are not limited to, apatinib, axitinib, brivanib, cabozantinib, canertinib, cediranib, dasatinib, erlotinib, gefitinib, leflunomide, 42envatinib, motesanib, nilotinib, nintedanib, pazopanib, regorafenib, semaxinib, sorafenib, sunitinib, tivozanib, vandetanib, vatalanib, XL0192, bevacizumab, ramucirumab, and pharmaceutically acceptable salts or biosimilars thereof.
  • compositions generally comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.
  • a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
  • an immunotherapy e.g., an immune checkpoint inhibitor
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanom
  • a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
  • an immunotherapy e.g., an immune checkpoint inhibitor
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma
  • a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an anti-PD-1 antibody for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer,
  • a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an anti-PD-1 antibody for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma,
  • a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
  • an immunotherapy e.g., an immune checkpoint inhibitor
  • a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an anti-PD-1 antibody for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
  • a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor), for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, and urothelial carcinoma) in a subject in need thereof.
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer,
  • a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor), for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, and urothelial carcinoma) in a subject in need thereof.
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma,
  • a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients and a therapeutically effective amount of an anti-PD-1 antibody, for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-
  • a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients and a therapeutically effective amount of an anti-PD-1 antibody, for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
  • a cancer e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endo
  • a method of administering a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
  • an immunotherapy e.g., an immune checkpoint inhibitor
  • a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an anti-PD-1 antibody for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
  • a pharmaceutical composition for treating a cancer e.g., a cancer described herein
  • the pharmaceutical composition comprises a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and wherein the pharmaceutical composition is to be administered with a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor).
  • an immunotherapy e.g., an immune checkpoint inhibitor
  • a pharmaceutical composition for treating a cancer e.g., a cancer described herein
  • the pharmaceutical composition comprises a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and wherein the pharmaceutical composition is to be administered with a therapeutically effective amount of an anti-PD-1 antibody.
  • the immunotherapy is an inhibitor of PD-1 or PD-L1.
  • the immunotherapy is an anti-PD-1 antibody.
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.
  • a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients.
  • the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 22 mg to about 226 mg, about 35 mg to about 226 mg, about 45 mg to about 226 mg, about 55 mg to about 226 mg, about 65 mg to about 226 mg, about 75 mg to about 226 mg, about 85 mg to about 226 mg, about 95 mg to about 226 mg, about 105 mg to about 226 mg, about 115 mg to about 226 mg, about 125 mg to 226 200 mg, about 135 mg to about 226 mg, about 145 mg to about 226 mg, about 155 mg to about 226 mg, about 165 mg to about 226 mg, about 175 mg to about 226 mg, about 185 mg to about 226 mg, about 195 mg to about 226 mg, about 22 mg to about 190 mg, about 22 mg to about 180 mg, about 22 mg to about 170 mg, about 22 mg to about 160 mg, about 22 mg to about 150 mg, about 22 mg
  • the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 177 mg to about 900 mg, about 250 mg to about 900 mg, about 300 mg to about 900 mg, about 350 mg to about 900 mg, about 400 mg to about 900 mg, about 450 mg to about 900 mg, about 500 mg to about 900 mg, about 550 mg to about 900 mg, about 600 mg to about 900 mg, about 650 mg to about 900 mg, about 700 mg to about 900 mg, about 750 mg to about 900 mg, about 800 mg to about 900 mg, about 850 mg to about 900 mg, about 177 mg to about 850 mg, about 177 mg to about 800 mg, about 177 mg to about 750 mg, about 177 mg to about 700 mg, about 177 mg to about 650 mg, about 177 mg to about 600 mg, about 177 mg to about 550 mg, about 177 mg to about 500 mg, about 177 mg to about 450 mg,
  • the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 22 mg to about 451 mg, about 50 mg to about 451 mg, about 75 mg to about 451 mg, about 100 mg to about 451 mg, about 125 mg to about 451 mg, about 150 mg to about 451 mg, about 175 mg to about 451 mg, about 200 mg to about 451 mg, about 225 mg to about 451 mg, about 250 mg to about 451 mg, about 275 mg to about 451 mg, about 300 mg to about 451 mg, about 325 mg to about 451 mg, about 350 mg to about 451 mg, about 375 mg to about 451 mg, about 22 mg to about 375 mg, about 22 mg to about 350 mg, about 22 mg to about 325 mg, about 22 mg to about 300 mg, about 22 mg to about 275 mg, about 22 mg to about 250 mg, about 22 mg to about 225 mg, about 22 mg to about 200 mg, about 22 mg to about 175 mg,
  • the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 22 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330
  • the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 22 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 50 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 75 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 100 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 125 mg.
  • the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 150 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 175 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 200 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 250 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 300 mg.
  • the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 350 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 400 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 450 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 500 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 550 mg.
  • the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 600 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 650 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 700 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 750 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 800 mg.
  • the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 850 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 900 mg.
  • the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 22 mg to about 226 mg, about 35 mg to about 226 mg, about 45 mg to about 226 mg, about 55 mg to about 226 mg, about 65 mg to about 226 mg, about 75 mg to about 226 mg, about 85 mg to about 226 mg, about 95 mg to about 226 mg, about 105 mg to about 226 mg, about 115 mg to about 226 mg, about 125 mg to about 226 mg, about 135 mg to about 226 mg, about 145 mg to about 226 mg, about 155 mg to about 226 mg, about 165 mg to about 226 mg, about 175 mg to about 226 mg, about 185 mg to about 226 mg, about 195 mg to about 226 mg, about 22 mg to about 190 mg, about 22 mg to about 180 mg, about 22 mg to about 170 mg, about 22 mg to about 160 mg, about 22 mg to about 150 mg, about 22 mg to about 140
  • the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 177 mg to about 900 mg, about 250 mg to about 900 mg, about 300 mg to about 900 mg, about 350 mg to about 900 mg, about 400 mg to about 900 mg, about 450 mg to about 900 mg, about 500 mg to about 900 mg, about 550 mg to about 900 mg, about 600 mg to about 900 mg, about 650 mg to about 900 mg, about 700 mg to about 900 mg, about 750 mg to about 900 mg, about 800 mg to about 900 mg, about 850 mg to about 900 mg, about 177 mg to about 850 mg, about 177 mg to about 800 mg, about 177 mg to about 750 mg, about 177 mg to about 700 mg, about 177 mg to about 650 mg, about 177 mg to about 600 mg, about 177 mg to about 550 mg, about 177 mg to about 500 mg, about 177 mg to about 450 mg, about 177
  • the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 22 mg to about 451 mg, about 50 mg to about 451 mg, about 75 mg to about 451 mg, about 100 mg to about 451 mg, about 125 mg to about 451 mg, about 150 mg to about 451 mg, about 175 mg to about 451 mg, about 200 mg to about 451 mg, about 225 mg to about 451 mg, about 250 mg to about 451 mg, about 275 mg to about 451 mg, about 300 mg to about 451 mg, about 325 mg to about 451 mg, about 350 mg to about 451 mg, about 375 mg to about 451 mg, about 22 mg to about 375 mg, about 22 mg to about 350 mg, about 22 mg to about 325 mg, about 22 mg to about 300 mg, about 22 mg to about 275 mg, about 22 mg to about 250 mg, about 22 mg to about 225 mg, about 22 mg to about 200 mg, about 22 mg to about 175 mg, about 22 mg to about 451 mg, about 300
  • the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 22 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about
  • the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 22 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 50 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 75 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 100 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 125 mg.
  • the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 150 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 175 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 200 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 250 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 300 mg.
  • the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 350 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 400 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 450 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 500 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 550 mg.
  • the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 600 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 650 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 700 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 750 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 800 mg.
  • the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 850 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 900 mg.
  • compositions comprising:
  • compositions comprising:
  • compositions comprising about 22 mg to about 177 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
  • a cancer described herein e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric
  • compositions comprising about 22 mg to about 177 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
  • a cancer described herein e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and
  • compositions comprising about 177 mg to about 900 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
  • a cancer described herein e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gas
  • compositions comprising about 177 mg to about 900 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
  • a cancer described herein e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma,
  • compositions comprising about 22 mg to about 226 mg of a pharmaceutically acceptable salt of a compound of formula (I), and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, M cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
  • a cancer described herein e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, M cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma,
  • compositions comprising about 22 mg to about 226 mg of a pharmaceutically acceptable salt of a compound of formula (I), and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, M cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
  • a cancer described herein e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, M cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroen
  • compositions comprising about 177 mg to about 900 mg of a pharmaceutically acceptable salt of a compound of formula (I), and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, M cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
  • a cancer described herein e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, M cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma
  • compositions comprising about 177 mg to about 900 mg of a pharmaceutically acceptable salt of a compound of formula (I), and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, M cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
  • a cancer described herein e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, M cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuro
  • compositions comprising about 22 mg to about 226 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
  • compositions comprising about 177 mg to about 900 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
  • compositions comprising about 22 mg to about 226 mg of a pharmaceutically acceptable salt of a compound of formula (I), and one or more pharmaceutically acceptable excipients, for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
  • compositions comprising about 177 mg to about 900 mg of a pharmaceutically acceptable salt of a compound of formula (I), and one or more pharmaceutically acceptable excipients, for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
  • a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I), a therapeutically effective amount of an anti angiogenic agent, and one or more pharmaceutically acceptable excipients.
  • the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 22 mg to about 798 mg, about 35 mg to about 798 mg, about 44 mg to about 798 mg, about 66 mg to about 798 mg, about 88 mg to about 798 mg, about 110 mg to about 798 mg, about 113 mg to about 798 mg, about 155 mg to about 798 mg, about 177 mg to about 798 mg, about 199 mg to about 798 mg, about 221 mg to 798 mg, about 244 mg to about 798 mg, about 266 mg to about 798 mg, about 310 mg to about 798 mg, about 355 mg to about 798 mg, about 399 mg to about 798 mg, about 443 mg to about 798 mg, about 488 mg to about 798 mg, about 532 mg to about 798 mg, about 576 mg to about 798 mg, about 621 mg to about 798 mg, about 665 mg to about 798 mg, about 7
  • compositions comprising:
  • compositions comprising:
  • compositions comprising:
  • compositions comprising about 22 mg to about 798 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer,
  • a cancer described herein e.g.
  • compositions comprising about 22 mg to about 798 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer,
  • a cancer described herein e.g.
  • compositions comprising about 177 mg to about 798 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer
  • a cancer described herein e.g.
  • compositions comprising about 177 mg to about 798 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer
  • a cancer described herein e.g.
  • compositions comprising about 44 mg to about 177 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer,
  • a cancer described herein e.g.
  • compositions comprising about 44 mg to about 177 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer,
  • a cancer described herein e.g.
  • the pharmaceutically acceptable salt of the compound of formula (I) is a fumarate salt. In certain embodiments, the pharmaceutically acceptable salt of the compound of formula (I) is a hemifumarate salt.
  • compositions described herein can be administered by a variety of routes including, but not limited to, oral (enteral) administration, parenteral (by injection) administration, rectal administration, transdermal administration, intradermal administration, intrathecal administration, subcutaneous (SC) administration, intravenous (IV) administration, intramuscular (IM) administration, and intranasal administration.
  • oral (enteral) administration parenteral (by injection) administration
  • rectal administration transdermal administration
  • intradermal administration intrathecal administration
  • SC subcutaneous
  • IV intravenous
  • IM intramuscular
  • intranasal administration intranasal administration.
  • the pharmaceutical compositions described herein are administered orally.
  • the pharmaceutical compositions described herein may also be administered chronically (“chronic administration”).
  • Chronic administration refers to administration of the compound or pharmaceutical composition thereof over an extended period of time, e.g., for example, over 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or may be continued indefinitely, for example, for the rest of the subject’s life.
  • the chronic administration is intended to provide a constant level of the compound in the blood, e.g., within the therapeutic window over the extended period of time.
  • unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.
  • the pharmaceutical compositions provided herein are administered to the patient as a solid dosage form.
  • the solid dosage form is a capsule.
  • the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation. General considerations in the formulation and/or manufacture of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005.
  • the methods generally comprise administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor).
  • an immunotherapy e.g., an immune checkpoint inhibitor
  • kits for activating the immune system of a subject in need thereof generally comprise administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, and a therapeutically effective amount of an anti-PD- 1 antibody.
  • Also provided herein are methods of activating the immune system of a subject having a cancer comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor).
  • a cancer e.g., a cancer described herein
  • an immunotherapy e.g., an immune checkpoint inhibitor
  • Also provided herein are methods of activating the immune system of a subject having a cancer comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, and a therapeutically effective amount of an anti-PD- 1 antibody.
  • the methods of activating the immune system described herein inhibit the activity of myeloid derived suppressor cells. In some embodiments, the methods of activating the immune system described herein increase the infiltration of T cells and NK cells in a tumor. In some embodiments, the methods of activating the immune system described herein increase the frequency of dendritic cells in a draining lymph node.
  • kits for treating a cancer in a subject in need thereof generally comprise administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor).
  • an immunotherapy e.g., an immune checkpoint inhibitor
  • the cancer is a solid tumor.
  • kits for treating a cancer in a subject in need thereof generally comprise administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein, and a therapeutically effective amount of an anti-PD-1 antibody.
  • the cancer is a solid tumor.
  • the compound of formula (I), or a pharmaceutically acceptable salt thereof, in combination with an immunotherapy is contemplated to be useful in treating cancers including, but not limited to, pancreatic cancer; bladder cancer; colorectal cancer; breast cancer, including metastatic breast cancer; prostate cancer, including androgen-dependent and androgen-independent prostate cancer; kidney or renal cancer, including, e.g., metastatic renal cell carcinoma; hepatocellular cancer; lung cancer, including, e.g., NSCLC, bronchioloalveolar carcinoma (BAC), and adenocarcinoma of the lung; ovarian cancer, including, e.g., progressive epithelial or primary peritoneal cancer; cervical cancer; gastric cancer; esophageal cancer; head and neck cancer, including, e.g., squamous cell carcinoma of the head and neck; melanoma; neuroendocrine cancer, including metastatic neuroendocrine tumors; brain tumor
  • an immunotherapy e.g., an anti-PD-1 antibody
  • the cancer is selected from the group consisting of bladder cancer, squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer.
  • the cancer is selected from the group consisting of bladder cancer, squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer.
  • the cancer is a liquid tumor.
  • the cancer is selected from the group consisting of multiple myeloma, leukemia, and lymphoma.
  • the leukemia is selected from the group consisting of chronic lymphocytic leukemia, chronic myelocytic leukemia, acute lymphoblastic leukemia, and acute myeloid leukemia (AML).
  • the lymphoma is selected from the group consisting of classical Hodgkin’s lymphoma, primary thymic mediastinal lymphoma, and non-Hodgkin’s lymphoma.
  • the lymphoma is selected from classical Hodgkin’s lymphoma and primary thymic mediastinal lymphoma.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 22 mg to about 226 mg, about 35 mg to about 226 mg, about 45 mg to about 226 mg, about 55 mg to about 226 mg, about 65 mg to about 226 mg, about 75 mg to about 226 mg, about 85 mg to about 226 mg, about 95 mg to about 226 mg, about 105 mg to about 226 mg, about 115 mg to about 226 mg, about 125 mg to about 226 mg, about 135 mg to about 226 mg, about 145 mg to about 226 mg, about 155 mg to about 226 mg, about 165 mg to about 226 mg, about 175 mg to about 226 mg, about 185 mg to about 226 mg, about 195 mg to about 226 mg, about 22 mg to about 190 mg, about 22 mg to about 180 mg, about 22 mg to about 170 mg, about 22 mg to about
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 177 mg to about 900 mg, about 250 mg to about 900 mg, about 300 mg to about 900 mg, about 350 mg to about 900 mg, about 400 mg to about 900 mg, about 450 mg to about 900 mg, about 500 mg to about 900 mg, about 550 mg to about 900 mg, about 600 mg to about 900 mg, about 650 mg to about 900 mg, about 700 mg to about 900 mg, about 750 mg to about 900 mg, about 800 mg to about 900 mg, about 850 mg to about 900 mg, about 177 mg to about 850 mg, about 177 mg to about 800 mg, about 177 mg to about 750 mg, about 177 mg to about 700 mg, about 177 mg to about 650 mg, about 177 mg to about 600 mg, about 177 mg to about 550 mg, about 177 mg to about 500 mg, about 177 mg to about 177 mg to about 750 mg, about
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 22 mg to about 451 mg, about 50 mg to about 451 mg, about 75 mg to about 451 mg, about 100 mg to about 451 mg, about 125 mg to about 451 mg, about 150 mg to about 451 mg, about 175 mg to about 451 mg, about 200 mg to about 451 mg, about 225 mg to about 451 mg, about 250 mg to about 451 mg, about 275 mg to about 451 mg, about 300 mg to about 451 mg, about 325 mg to about 451 mg, about 350 mg to about 451 mg, about 375 mg to about 451 mg, about 22 mg to about 375 mg, about 22 mg to about 350 mg, about 22 mg to about 325 mg, about 22 mg to about 300 mg, about 22 mg to about 275 mg, about 22 mg to about 250 mg, about 22 mg to about 225 mg, about 22 mg to about 200 mg, about 22 mg to about
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 22 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg,
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 22 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 50 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 75 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 100 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 125 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 150 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 175 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 200 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 250 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 300 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 350 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 400 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 450 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 500 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 550 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 600 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 650 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 700 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 750 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 800 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 850 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 900 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 22 mg to about 226 mg, about 35 mg to about 226 mg, about 45 mg to about 226 mg, about 55 mg to about 226 mg, about 65 mg to about 226 mg, about 75 mg to about 226 mg, about 85 mg to about 226 mg, about 95 mg to about 226 mg, about 105 mg to about 226 mg, about 115 mg to about 226 mg, about 125 mg to about 226 mg, about 135 mg to about 226 mg, about 145 mg to about 226 mg, about 155 mg to about 226 mg, about 165 mg to about 226 mg, about 175 mg to about 226 mg, about 185 mg to about 226 mg, about 195 mg to about 226 mg, about 22 mg to about 190 mg, about 22 mg to about 180 mg, about 22 mg to about 170 mg, about 22 mg to about 160 mg, about 22 mg to about 150 mg, about 22 mg to about 190 mg, about 22
  • administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 177 mg to about 900 mg, about 250 mg to about 900 mg, about 300 mg to about 900 mg, about 350 mg to about 900 mg, about 400 mg to about 900 mg, about 450 mg to about 900 mg, about 500 mg to about 900 mg, about 550 mg to about 900 mg, about 600 mg to about 900 mg, about 650 mg to about 900 mg, about 700 mg to about 900 mg, about 750 mg to about 900 mg, about 800 mg to about 900 mg, about 850 mg to about 900 mg, about 177 mg to about 850 mg, about 177 mg to about 800 mg, about 177 mg to about 750 mg, about 177 mg to about 700 mg, about 177 mg to about 650 mg, about 177 mg to about 600 mg, about 177 mg to about 550 mg, about 177 mg to about 500 mg, about 177 mg to about 450
  • administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 22 mg to about 451 mg, about 50 mg to about 451 mg, about 75 mg to about 451 mg, about 100 mg to about 451 mg, about 125 mg to about 451 mg, about 150 mg to about 451 mg, about 175 mg to about 451 mg, about 200 mg to about 451 mg, about 225 mg to about 451 mg, about 250 mg to about 451 mg, about 275 mg to about 451 mg, about 300 mg to about 451 mg, about 325 mg to about 451 mg, about 350 mg to about 451 mg, about 375 mg to about 451 mg, about 22 mg to about 375 mg, about 22 mg to about 350 mg, about 22 mg to about 325 mg, about 22 mg to about 300 mg, about 22 mg to about 275 mg, about 22 mg to about 250 mg, about 22 mg to about 225 mg, about 22 mg to about 200 mg, about 22 mg to about 175
  • administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 22 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about
  • administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 22 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 50 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 75 mg of the pharmaceutically acceptable salt of the compound of formula (I).
  • administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 100 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 125 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 150 mg of the pharmaceutically acceptable salt of the compound of formula (I).
  • administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 175 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 200 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 250 mg of the pharmaceutically acceptable salt of the compound of formula (I).
  • administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 300 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 350 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 400 mg of the pharmaceutically acceptable salt of the compound of formula (I).
  • administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 450 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 500 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 550 mg of the pharmaceutically acceptable salt of the compound of formula (I).
  • administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 600 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 650 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 700 mg of the pharmaceutically acceptable salt of the compound of formula (I).
  • administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 750 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 800 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 850 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 900 mg of the pharmaceutically acceptable salt of the compound of formula (I).
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject daily. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive days. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for 21 consecutive days.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for at least 21 consecutive days.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject twice daily for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject twice daily for 21 consecutive days.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject twice daily for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive days. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for at least 21 consecutive days.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for 1 year.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for 2 years.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for at least 1 year.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for at least 2 years.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject twice daily for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject twice daily for 1 year.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject twice daily for 2 years.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject twice daily for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject twice daily for at least 1 year.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject twice daily for at least 2 years.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 22 mg to about 451 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 22 mg to about 451 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 22 mg to about 451 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, daily.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 22 mg to about 451 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, once daily.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 22 mg to about 451 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, twice daily.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject the effective amount within about 30 minutes of completing a meal.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 22 mg to about 451 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, within about 30 minutes of completing a meal.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 177 mg to about 900 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 177 mg to about 900 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 177 mg to about 900 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, daily.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 177 mg to about 900 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, once daily.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 177 mg to about 900 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, twice daily.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject the therapeutically effective amount within about 30 minutes of completing a meal.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 177 mg to about 900 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, within about 30 minutes of completing a meal.
  • the immunotherapy is selected from the group consisting of an immune checkpoint inhibitor, an adoptive cell therapy, and a monoclonal antibody. In some embodiments, the immunotherapy is selected from an immune checkpoint inhibitor and an adoptive cell therapy.
  • the immunotherapy is an adoptive cell therapy.
  • the adoptive cell therapy is selected from the group consisting of tumor-infiltrating lymphocyte therapy, engineered T-cell receptor therapy, CAR T-cell therapy, and natural killer cell therapy.
  • the immunotherapy is an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor is selected from the group consisting of a PD-1 (programmed cell death protein 1) antagonist, a PD-L1 (programmed cell death protein ligand 1) antagonist, a PD-L2 antagonist, a CD27 (cluster of differentiation 27) antagonist, a CD28 antagonist, a CD70 antagonist, a CD80 antagonist, a CD86 antagonist, a CD137 antagonist, a CD276 antagonist, a KIRs (killer-cell immunoglobulin-like receptors) antagonist, a LAG3 (lymphocyte-activation gene 3) antagonist, a TNFRSF4 (tumor necrosis factor receptor superfamily, member 4) antagonist, a GITR (glucocorticoid-induced TNFR-related protein) antagonist, a GITRL (GITR ligand) antagonist, a 4-1BBL (4-1BB ligand) antagonist, a CTLA-4 (cytotoxic T
  • the immune checkpoint inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, lambrolizumab, BMS-936559, atezolizumab, AMP-224, AMP224, AUNP12, BGB108, MCLA134, MEDI0680, spartalizumab, cemiplimab, SHR1210, STIA110X, STIA1110, TSR042, MPDL3280A, MEDI- 4736, MSB0010718C, ALN-PDL, BGBA317, KD033, KY1003, STIA100X, STIA1010, STIA1011, STIA1012, STIA101, BMS-663513, and PF-05082566.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the immunotherapy are administered simultaneously. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the immunotherapy are administered separately. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the immunotherapy are administered sequentially.
  • the therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) and the therapeutically effective amount of the immunotherapy are administered simultaneously. In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) and the therapeutically effective amount of the immunotherapy are administered separately. In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) and the therapeutically effective amount of the immunotherapy are administered sequentially.
  • the therapeutically effective amount of the immunotherapy is administered over a course of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months.
  • the therapeutically effective amount of the immunotherapy is administered over a course of 1 year.
  • the therapeutically effective amount of the immunotherapy is administered over a course of 2 years.
  • the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti- PD-L1 antibody, or an anti-CTLA-4 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody.
  • the anti-PD-1 antibody is a PD-1 antagonist. In some embodiments, the anti-PD-1 antibody is a PD-1 agonist. In some embodiments, the anti-PD-1 antibody is a PD-1 inhibitor.
  • the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, spartalizumab, tislelizumab, or cemiplimab. In some embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, and cemiplimab. In certain embodiments, the anti-PD-1 antibody is selected from pembrolizumab and nivolumab.
  • the anti-PD-1 antibody is pembrolizumab. In certain embodiments, the anti-PD-1 antibody is nivolumab. In certain embodiments, the anti-PD-1 antibody is cemiplimab.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered simultaneously. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered separately. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered sequentially.
  • the therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) and the therapeutically effective amount of the anti- PD-1 antibody are administered simultaneously. In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) and the therapeutically effective amount of the anti-PD-1 antibody are administered separately. In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) and the therapeutically effective amount of the anti-PD-1 antibody are administered sequentially.
  • the therapeutically effective amount of the anti-PD-1 antibody is administered over a course of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months.
  • the therapeutically effective amount of the anti-PD-1 antibody is administered over a course of 1 year. In some embodiments, the therapeutically effective amount of the anti-PD-1 antibody is administered over a course of 2 years.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered over a course of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody is administered over a course of 1 year.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody is administered over a course of 2 years.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered over a course of at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody is administered over a course of at least 1 year.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti- PD-1 antibody is administered over a course of at least 2 years.
  • administering the therapeutically effective amount of the anti-PD-1 antibody comprises administering about 200 mg to about 400 mg of the anti-PD-1 antibody to the subject. In some embodiments, administering the therapeutically effective amount of the anti-PD-1 antibody comprises administering about 200 mg to about 400 mg of the anti-PD-1 antibody to the subject every about 2 to about 4 weeks. In some embodiments, administering the therapeutically effective amount of the anti-PD-1 antibody comprises administering about 200 mg to about 400 mg of the anti-PD-1 antibody to the subject every about 2 to about 4 weeks for about 6 months.
  • administering the therapeutically effective amount of the anti-PD-1 antibody comprises administering about 200 mg to about 400 mg of the anti-PD-1 antibody to the subject every about 2 to about 4 weeks for about 1 year. In some embodiments, administering the therapeutically effective amount of the anti-PD-1 antibody comprises administering about 200 mg to about 400 mg of the anti-PD-1 antibody to the subject every about 2 to about 4 weeks for about 2 years.
  • the anti-PD-1 antibody is pembrolizumab.
  • the pembrolizumab is administered in an amount of about 200 mg every 3 weeks. In certain embodiments, the pembrolizumab is administered in an amount of about 200 mg every 3 weeks for about 6 months. In certain embodiments, the pembrolizumab is administered in an amount of about 200 mg every 3 weeks for about 1 year. In certain embodiments, the pembrolizumab is administered in an amount of about 200 mg every 3 weeks for about 2 years. In certain embodiments, the pembrolizumab is administered in an amount of about 400 mg every 6 weeks. In certain embodiments, the pembrolizumab is administered in an amount of about 400 mg every 6 weeks for about 6 months. In certain embodiments, the pembrolizumab is administered in an amount of about 400 mg every 6 weeks for about 1 year. In certain embodiments, the pembrolizumab is administered in an amount of about 400 mg every 6 weeks for about 2 years.
  • the anti-PD-1 antibody is nivolumab.
  • the nivolumab is administered in an amount of about 240 mg every 2 weeks. In certain embodiments, the nivolumab is administered in an amount of about 240 mg every 2 weeks for about 6 months. In certain embodiments, the nivolumab is administered in an amount of about 240 mg every 2 weeks for about 1 year. In certain embodiments, the nivolumab is administered in an amount of about 240 mg every 2 weeks for about 2 years. In certain embodiments, the nivolumab is administered in an amount of about 480 mg every 4 weeks.
  • the nivolumab is administered in an amount of about 480 mg every 4 weeks for about 6 months. In certain embodiments, the nivolumab is administered in an amount of about 480 mg every 4 weeks for about 1 year. In certain embodiments, the nivolumab is administered in an amount of about 480 mg every 4 weeks for about 2 years.
  • the anti-PD-1 antibody is cemiplimab.
  • the cemiplimab is administered in an amount of about 350 mg every 3 weeks. In certain embodiments, the cemiplimab is administered in an amount of about 350 mg every 3 weeks for about 6 months. In certain embodiments, the cemiplimab is administered in an amount of about 350 mg every 3 weeks for about 1 year. In certain embodiments, the cemiplimab is administered in an amount of about 350 mg every 3 weeks for about 2 years.
  • a method of treating a cancer comprising administering to the subject a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, as described herein; or a pharmaceutical composition comprising the compound of formula (I), or a pharmaceutically acceptable salt thereof, as described herein; and a therapeutically effective amount of pembrolizumab.
  • a method of treating a cancer comprising administering to the subject a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, as described herein; or a pharmaceutical composition comprising the compound of formula (I), or a pharmaceutically acceptable salt thereof, as described herein; and a therapeutically effective amount of nivolumab.
  • a method of treating a cancer comprising administering to the subject a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, as described herein; or a pharmaceutical composition comprising the compound of formula (I), or a pharmaceutically acceptable salt thereof, as described herein; and a therapeutically effective amount of cemiplimab.
  • the subject has previously been administered at least one prior line of therapy. In certain embodiments, the subject has previously been administered fewer than five prior lines of therapy. In certain embodiments, the subject has previously been administered one, two, three, or 4 prior lines of therapy. In certain embodiments, the subject has not been administered a prior line of therapy.
  • the subject has previously been administered at least one and no more than 5 prior lines of therapy.
  • Prior lines of therapy include, but are not limited to, surgery, radiation therapy (e.g., external beam radiation therapy or internal radiation therapy), chemotherapy (e.g., alkylating agents, nitrosoureas, anti-metabolites, plant alkaloids and natural products, anti-tumor antibiotics, hormonal agents, and biological response modifiers), gene therapy, DNA therapy, viral therapy (e.g., oncolytic virus therapy), RNA therapy, adjuvant therapy, and immunotherapy (e.g., immune checkpoint inhibition, adoptive cell therapies, (e.g., tumor-infiltrating lymphocyte therapy, engineered T-cell receptor therapy, CAR T-cell therapy, natural killer cell therapy), or monoclonal antibodies).
  • radiation therapy e.g., external beam radiation therapy or internal radiation therapy
  • chemotherapy e.g., alkylating agents, nitrosoureas, anti-metabolites, plant alkaloids and natural products, anti-tumor antibiotics, hormonal agents, and biological response modifiers
  • gene therapy e.g., DNA
  • the methods include administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I).
  • the pharmaceutically acceptable salt is a fumarate salt.
  • the fumarate salt is a hemifumarate salt.
  • the methods described herein further comprise administering a therapeutically effective amount of a third therapeutic agent to the subject.
  • the second therapeutic agent is selected from the group consisting of a checkpoint inhibitor, an EGFR inhibitor, an anti angiogenic agent, venetoclax, fluorouracil, and combinations thereof.
  • the third therapeutic agent is a checkpoint inhibitor. In certain embodiments, the third therapeutic agent is a PD-L1 inhibitor. In certain embodiments, the third therapeutic agent is selected from the group consisting of atezolizumab, avelumab, and durvalumab.
  • the third therapeutic agent is an EGFR inhibitor.
  • the EGFR inhibitor is selected from the group consisting of erlotinib, gefitinib, afatinib, and osimertinib.
  • the third therapeutic agent is an anti angiogenic agent.
  • the anti angiogenic agent is a VEGFR inhibitor.
  • the anti angiogenic agent is a VEGFR tyrosine kinase inhibitor.
  • the third therapeutic agent is a VEGFR inhibitor.
  • the VEGFR inhibitor is selected from the group consisting of sunitinib, axitinib, lenvatinib, tivozanib, pazopanib, cabozantinib, and ramucirumab.
  • a cancer e.g., a cancer described herein
  • methods of treating a cancer comprising administering any one of the pharmaceutical compositions described herein and a therapeutically effective amount of an anti- PD-1 antibody (e.g., an anti-PD-1 antibody described herein) to the subject.
  • an anti- PD-1 antibody e.g., an anti-PD-1 antibody described herein
  • kits for activating the immune system of a subject in need thereof comprising administering any one of the pharmaceutical compositions described herein and a therapeutically effective amount of an anti-PD-1 antibody (e.g., an anti-PD- 1 antibody described herein) to the subject.
  • an anti-PD-1 antibody e.g., an anti-PD- 1 antibody described herein
  • kits for activating the immune system of a subject having a cancer comprising administering any one of the pharmaceutical compositions described herein and a therapeutically effective amount of an anti- PD-1 antibody (e.g., an anti-PD-1 antibody described herein) to the subject.
  • a cancer e.g., a cancer described herein
  • an anti- PD-1 antibody e.g., an anti-PD-1 antibody described herein
  • the methods described herein increase the tumor cell expression of type 1 interferon receptor (INF ARI) in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
  • INF ARI type 1 interferon receptor
  • the methods described herein increase the peripheral blood monocyte surface expression of INF ARI in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
  • the methods described herein increase tumor cell expression of calreticulin in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
  • the methods described herein increase polymorphonuclear myeloid-derived suppressor cell (MDSC) and/or tumor associated macrophage (TAM) expression of INF ARI in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
  • MDSC polymorphonuclear myeloid-derived suppressor cell
  • TAM tumor associated macrophage
  • the methods described herein increase TAM expression of PD-L1 in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
  • the methods described herein increase the infiltration of CD8 T cells and/or NK cells in a tumor in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
  • the methods described herein increase expression of CD69 on T cells in a tumor draining lymph node in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
  • the methods described herein reduce suppressive activity of MDSCs in the subject.
  • the methods described herein increase dendritic cell frequency in a tumor draining lymph node in the subject.
  • the methods described herein increase Ki67, GzmB, and memory phenotypic markers on CD4 T-cells in the subject.
  • the methods described herein increase the immuno-stimulatory phenotype of macrophages in the tumor microenvironment.
  • the methods described herein decrease the immuno-inhibitory phenotype of macrophages in the tumor microenvironment.
  • the methods described herein increase an Ml phenotype and/or function of macrophages, or decrease an M2 phenotype or function of macrophages.
  • the methods described herein increase surface expression of activation markers (e.g., MHCII, HLA-DR, and/or CD86) on macrophages or other myeloid cells.
  • activation markers e.g., MHCII, HLA-DR, and/or CD86
  • the methods described herein increase the immuno-stimulatory phenotype of dendritic cells in the draining lymph node or tumor microenvironment.
  • a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy for use in treating a cancer e.g., a cancer described herein.
  • a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD-1 antibody for use in treating a cancer e.g., a cancer described herein.
  • a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy for use in activating the immune system of a subject in need thereof e.g., a subject having a cancer described herein.
  • a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD-1 antibody for use in activating the immune system of a subject in need thereof e.g., a subject having a cancer described herein.
  • kits for treating a cancer in a subject in need thereof generally comprise administering to the subject a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, and a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)).
  • an anti angiogenic agent e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI
  • the cancer is a solid tumor.
  • kits for treating a cancer in a subject in need thereof generally comprise administering to the subject a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein, a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)), and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor).
  • an anti angiogenic agent e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)
  • an immunotherapy e.g., an immune checkpoint inhibitor
  • the cancer is a solid tumor.
  • the compound of formula (I), or a pharmaceutically acceptable salt thereof, in combination with an anti angiogenic agent is contemplated to be useful in treating cancers including, but not limited to, a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or
  • an anti angiogenic agent e.g., e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR
  • the compound of formula (I), or a pharmaceutically acceptable salt thereof, in combination with an anti angiogenic agent is contemplated to be useful in treating cancers including, but not limited to, a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or
  • an anti angiogenic agent e.g., e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR
  • the cancer is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer (e.g., malignant melanoma)), and a thyroid cancer.
  • a bladder cancer e.g., triple negative breast cancer or meta
  • the cancer is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer), and a thyroid cancer.
  • a bladder cancer e.g., triple negative breast cancer or metastatic breast cancer
  • a carcinoma e.g.,
  • the cancer is malignant melanoma.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 22 mg to about 798 mg, about 35 mg to about 798 mg, about 44 mg to about 798 mg, about 66 mg to about 798 mg, about 88 mg to about 798 mg, about 110 mg to about 798 mg, about 113 mg to about 798 mg, about 155 mg to about 798 mg, about 177 mg to about 798 mg, about 199 mg to about 798 mg, about 221 mg to 798 mg, about 244 mg to about 798 mg, about 266 mg to about 798 mg, about 310 mg to about 798 mg, about 355 mg to about 798 mg, about 399 mg to about 798 mg, about 443 mg to about 798 mg, about 488 mg to about 798 mg, about 532 mg to about 798 mg, about 576 mg to about 798 mg, about 621 mg to about 798 mg, about 665 mg to about 798 mg,
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject daily. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive days. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for 21 consecutive days.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for at least 21 consecutive days.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject twice daily for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject twice daily for 21 consecutive days.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject twice daily for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive days.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject twice daily for at least 21 consecutive days.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for 1 year.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for 2 years.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for at least 1 year.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for at least 2 years.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject twice daily for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for 1 year.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for 2 years.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject twice daily for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for at least 1 year.
  • the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof is administered orally to the subject once daily for at least 2 years.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent comprises administering to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, daily.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, once daily.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an antiangiogenic agent comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, twice daily.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an antiangiogenic agent comprises administering to the subject the therapeutically effective amount within about 30 minutes of completing a meal.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an antiangiogenic agent comprises administering to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, within about 30 minutes of completing a meal.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an antiangiogenic agent comprises administering to the subject about 177 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an antiangiogenic agent comprises administering orally to the subject about 177 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an antiangiogenic agent comprises administering orally to the subject about 177 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, daily.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent comprises administering orally to the subject about 177 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, once daily.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent comprises administering orally to the subject about 177 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, twice daily.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent comprises administering to the subject the therapeutically effective amount within about 30 minutes of completing a meal.
  • administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent comprises administering to the subject about 177 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, within about 30 minutes of completing a meal.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis, daily.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis, once daily.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis, twice daily.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis, daily.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis, once daily.
  • administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis, twice daily.
  • the subject is a human. In certain embodiments, the subject is an adult human.
  • IP A isopropyl alcohol
  • TGI tumor growth inhibition Example 1: Synthesis of (7?)-2-amino-5-(4-(2-(3,5-difluorophenyl)-2-hydro yacetamido)-2- methylphenyl)-N-isopropylnicotinamide (Compound of Formula (I)) and hemifumarate salt
  • Lipase PS Amano (see Mendiola, J. et al, Org. Process Res. Dev. 2012, 16, 1312-1316) was supported in diatomaceous earth prior to use by mixing 200 g of diatomaceous earth and 200 g of lipase PS Amano SD. H2O was added to cover the solid, and the mixture was stirred. H2O was removed in an oven at 4 mbar and 40 °C for 16 h. H2O was below 1% through Karl Fischer titration for water determination.
  • Step 3 Synthesis of (R)-2-amino-5-(4-(2-(3,5-dijluorophenyl)-2-hydroxyacetamido)-2- methylphenyl)-N-isopropylnicotinamide
  • Step 4 Synthesis of (R)-2-amino-5-(4-(2-(3,5-difluorophenyl)-2-hydroxyacetamido)-2- methylphenyl)-N-isopropylnicotinamide hemifumarate salt
  • Example 2 Studies of HC-5404 + anti-PD-1 antibody (RMP1-14 mouse antibody) combination therapy in murine MB49 syngeneic bladder cancer model
  • C57BL/6 mice were subcutaneously inoculated with MB49 cells, and treatment started on day 8 post cell inoculation.
  • PO, BID 3 mg/kg, 10 mg/kg, or 30 mg/kg
  • aPD-1 antibody RMP1-14
  • IP BIW
  • HC-5404 alone showed only a modest anti-tumor effect (32% tumors growth inhibition (TGI)
  • TGI tumors growth inhibition
  • the addition of HC-5404 to aPD-1 provided combination antitumor benefits (75% TGI) and significantly improved the effects of aPD-1 alone (53% TGI) (FIGS. 2B, 2C, 2D, 2E, 2F, and 2G).
  • HC-5404 + aPD-1 treatment efficacy was correlated with increased expression of type 1 interferon receptor (IFNAR1) and increased surface calreticulin on tumor cells (FIG. 3D and 4B). Additionally, IFNAR1 expression was also significantly increased on polymorphonuclear myeloid- derived suppressor cells (PMN-MDSCs) and tumor-associated macrophages (TAM) (FIGS. 3A, 3B, and 3C). TAMs also showed increased expression of PD-L1 with combination treatment (FIG. 3E). Concomitant to the activation of myeloid cells, combination treatment increased the frequency of CD8 T-cells in the tumor (FIGS.
  • IFNAR1 type 1 interferon receptor
  • PMN-MDSCs polymorphonuclear myeloid- derived suppressor cells
  • TAM tumor-associated macrophages
  • HC-5404 + aPD-1 treatment was also correlated with increased CD4 activation and memory T cell induction (demonstrated by Ki67 expression, EOMES expression, Granzyme B expression, TCF-1 expression, and/or effector memory population) (FIGS. 6A, 6B, 6C, 6D, and 6E), and expansion of DC populations in the lymph node (LN) of the treated mice (FIGS. 7A, 7B, and 7C).
  • mice Female C57BL/6 mice (7-8 weeks of age animals, 20-25 grams, from Envigo) were used.
  • MB49 (lot # 18B016) mouse urothelial carcinoma cell line was purchased from Sigma - The European Collection of Authenticated Cell Cultures (EC ACC). Cells were free of human and animal pathogens as tested by h-IMPACT-I and IMPACT-III (IDEXX Bioresearch, Columbia, MO). Additionally, the genetic profile of the cells matched with the ECACC reference standard as tested by STR based DNA fingerprinting assay. Cells were maintained in DMEM with 10% FBS, and cultured up to 4 passages prior to implantation. Cells were harvested at -80% confluence, washed twice with DMEM without FBS and suspended in DMEM without FBS at 5 x 10 6 cells/mL for implantation. Establishing MB49 Model
  • mice Female Balb/c mice (7-8 week of age) were implanted into the right flank area subcutaneously under Isoflurane anesthesia with 0.5 x 10 6 MB49 cells/mouse in a total volume of 100 mL on Day 0. Animals were randomized into treatment groups when the average tumor volume reached ⁇ 50 mm 3 on Day 5 post inoculation.
  • Tumor volume was measured twice weekly.
  • the %T/C ratio between the tumor volume in the treated group and in the control group was calculated by the formula 100 x AT/AC, if AT > 0 of the mean values.
  • AT mean tumor volume of the drug-treated group on the observation day of the study - mean tumor volume of the drug-treated group on initial day of dosing;
  • AC mean tumor volume of the control group on the observation day of the study - mean tumor volume of the control group on initial day of dosing.
  • Tumor volume data were analyzed for statistics using 2-way ANOVA followed by Dunnett multiple comparisons test (GraphPad Prism).
  • Tumor tissue samples were obtained 7 days post treatment (14 days post inoculation). Single cells were prepared by enzymatic digestion of tumor tissues and subsequently analyzed by flow cytometry on a Cytek Aurora. Expression of IFNAR1 (A) and PD-L1 (B) on the myeloid populations and tumors were shown.
  • FACS buffer PBS with 2.5% FBS and 0.1% sodium azide
  • Fc receptor blocked for 5-10 minutes and then stained with the Ab cocktail in Super Bright Complete Staining Buffer (Invitrogen cat # SB-4401) for 30 min at 4 °C and protected from light.
  • the labeled cells were then fixed with Fix/Perm buffer (Tonbo Biosciences) for 30 minutes at 4 °C, washed three times with Perm buffer (Tonbo Biosciences) before staining with intracellular staining Abs for 30 minutes at 4 °C.
  • the stained cells were then washed once with Perm buffer and twice with FACS buffer sequentially. Stained cells were analyzed by flow cytometry on the Cytek Aurora (Cytek Biosciences) platform and data were analyzed by Flow Jo (BD).
  • Effector memory (EM) CD4 cells are defined as CD62L" CD44 + CD4 + .
  • Total DC is identified as CD1 lc + MHCII + CD1 lb dimA
  • cDCl is identified as XCR1 + DC
  • cDC2 is identified as XCR1" CD24 + DC.
  • IFNAR1 expression was determined by flow cytometry and gated on CD 14+ monocytes
  • HC-5404 has demonstrated single agent and combinatorial efficacy in multiple solid tumor xenograft models.
  • 786-0 tumor cells were maintained in vitro with MEM medium supplemented with 10% heat inactivated fetal bovine serum and 0.01 mM NEAA, at 37 °C in an atmosphere of 5% CO2. Each mouse was inoculated subcutaneously at the right front region with 786-0 tumor cells (5x106) in 0.1 ml of PBS (1 : 1 matrigel) for tumor development. The randomization for efficacy study began when the mean tumor size reached approximately 200-300 mm 3 , preferably to initiate with 250 mm 3 .
  • Mouse tumor, spleen and lung tissues were snap frozen and stored in -800 °C freezer.
  • RNAlater®- ICE ThermoFisher
  • RET lysis buffer Qiagen
  • RNA_02 ThermoFisher
  • Total RNA was isolated from homogenized tissue using the RNeasy Plus Mini Kit (Qiagen). RNA concentration was determined by Epoch Microplate Spectrophotometer (Biotek). QuantiGene® Plex assay
  • a custom-made 77-plex mouse Immune QuantiGene Assay kit (ThermoFisher Scientific) using a bead-based multiplex assay were used to measure the expression of 69 immune genes, 4 stress genes plus 4 reference genes with Luminex xMAP 200 (ThermoFisher Scientific) according to the manufacturer’s instructions.
  • Penicillin-streptomycin (Gibco cat# 15140122)
  • CTV Cell Trace Violet
  • IL-2 (R&D systems cat# 202-IL)
  • IMDM complete medium 100 ng/ml human GM-CSF, 100 ng/ml human G-CSF and 50 ng/ml human SCF were added to the IMDM complete medium and cells were expanded for 13-14 days in a 37 °C, 5% CO2 humidified incubator and medium was refreshed on day 7.
  • MDSC differentiation was induced by culturing the expanded CD34+ cells in a 24 well plate at 0.1-0.15 x 10 6 cells/ well in 2 ml of IMDM complete medium supplemented with 100 ng/ml human GM-CSF, 50 ng/mL G-CSF and 30 ng/ml IL-6 for additional 6-7 days with medium changed on day 3.
  • HC-5404 treatment the indicated dose of HC-5404 was added to the cell culture on day 0 of the MDSC differentiation phase, medium was refreshed on day 3 and HC-5404 was added afresh. MDSCs were harvested on day 6 or 7 and used in the T cell suppression assay.
  • PBMCs were enriched from Trima Cone obtained from Innovative Blood Resources (Protocol ID # 120160627) by Ficoll density gradient centrifugation.
  • CD3 T cells were further enriched from PBMC using EasySep Human T cell isolation kit.
  • CD3 T cells were labeled with Cell Trace Violet (CTV) according to Vendor’s protocol.
  • CTV-labeled T cells were resuspended in RPMI medium supplemented with 10% FBS, lx penicillin-streptomycin and 2 mM L-glutamax and added at 50,000 cells per well in a 96 well round-bottom plate.
  • T-activator CD3/CD28 were washed twice with PBS and then added to T cells at 1 : 1 ratio.
  • HC-5404 treated and un-treated MDSC were harvested from the plates, washed three times with RPMI medium and then added to the wells at various ratio of MDSC: T (4: 1; 2: 1; 1 : 1).
  • IL-2 (30u/ml) was added to each well and the 96 well plate was incubated in a 37 0C, 5% CO2 humidified incubator to 3-5 days.
  • T cell proliferation was analyzed by flow cytometry. Cells were washed 2-3 times in PBS, and then stained with fluorescent- conjugated anti-CD3 Ab for 30 minutes at 4 °C and protected from light.
  • the labeled cells were analyzed on a BD Fortessa cytometer and data was analyzed by FlowJo. Statistical Analyses was performed using Prism software Prism (GraphPad Software). Data were analyzed using one-way ANOVA with multiple comparison corrected with Dunnett test. *P ⁇ .05; **P ⁇ .01; ***p ⁇ .005, ****P ⁇ .0001 (FIGS. 10A, 10B, and 10C).
  • Mouse MDSC Preparation Single cell suspensions from bone marrow were prepared from the tibia and femur bone of C57BL/6 mice. Cells were cultured in RPMI complete medium (RPMI medium supplemented with 10% FBS, lOmM HEPES, 1 mM Sodium pyruvate, 50uM 2- ME, 1 mM MEM NEAA, lx penicillin-streptomycin and 2 mM L-glutamax) at 2.5 x 10 6 cells per 10 ml in a 100 mm 2 tissue culture dish for 4 days in a 37 0 °C, 5% CO2 humidified incubator.
  • RPMI complete medium RPMI medium supplemented with 10% FBS, lOmM HEPES, 1 mM Sodium pyruvate, 50uM 2- ME, 1 mM MEM NEAA, lx penicillin-streptomycin and 2 mM L-glutamax
  • Cytokines GM-CSF (10 ng/ml) and IL-6 (10 ng/mL) were added to the cell culture on day 0.
  • LSLG low serum low glucose
  • cultured cells were first harvested from the plate and washed twice with RPMI medium to rinse off residual serum and glucose, and then cultured in 5 ml of LSLG medium (glucose-free RPMI supplemented with 1% FBS, Ig/L glucose, lx penicillin-streptomycin and 2 mM L-glutamax) in a 15 ml conical tube for 6 hours with or without HC-5404 addition. Cells were harvested and used in the T cell suppression assay.
  • Mouse T cell suppression assay Spleens were harvested from C57BL/6 mice. Single cell suspensions were prepared and CD3 T cells were isolated using EasySep Mouse T cell isolation kit. CD3 T cells were labeled with Cell Trace Violet (CTV) according to Vendor’s protocol. CTV-labeled T cells were resuspended in RPMI medium supplemented with 10% FBS, lx penicillin-streptomycin and 2 mM L-glutamax and added at 100,000 cells per well in a 96 well round-bottom plate. Dynabeads mouse T-activator CD3/CD28 were washed twice with PBS and then added to T cells at 1 : 1 ratio.
  • CTV Cell Trace Violet
  • LSLG +/- HC5404- treated and un-treated MDSC were harvested from the plates, washed three times with RPMI medium and then added to the wells at various ratio of MDSC: T (8: 1, 4: 1; 2: 1).
  • IL-2 (30 ug/ml) was added to each well and the 96 well plate was incubated in a 37 °C, 5% CO2 humidified incubator to 3-5 days.
  • T cell proliferation was analyzed by flow cytometry. Cells were washed 2-3 times in PBS, and then stained with fluorescent- conjugated anti-CD4 and anti-CD8 Ab for 30 minutes at 4 °C and protected from light. The labeled cells were analyzed on a BD Fortessa cytometer and data was analyzed by FlowJo.
  • HEK293 cells were plated at 1 x 10 6 cells per well and treated with HC- 5404 30 minutes prior to addition of tunicamycin (1 pM) to induce ER stress. Cells were harvested 4 hours following tunicamycin addition and lysed for western blotting in RIPA buffer supplemented with protease and phosphatase inhibitors. Total protein concentrations were determined by BCA and 30 mg of protein were loaded on to SDS-PAGE gels, transferred to nitrocellulose and probed with a primary antibody against phosphorylated T-982 of human PERK or total ATF4 followed by incubation with an infrared labelled secondary antibody.
  • Blots were imaged on a LiCor imager and normalized to total protein in the wells using RevertTM stain. Band intensities were quantified and normalized to DMSO treated samples. Normalized band intensities were plotted as percent inhibition against 10-point; 3-fold dilution series of HC-5404. IC50 values were calculated using 4-parameter logistical fitting in XLFit.
  • TSA-conjugated Alexa488 and Alexa647 fluorophores from Invitrogen were used at 1 :500 dilution. Slides were counterstained with DAPI and cover slipped using Mowiol antifade mounting media (Sigma #D2522).
  • mice were allowed to acclimate for 7 days upon arrival in animal facility. Mice were housed in a 12-hour light/dark cycle facility under pathogen-free conditions with standard laboratory chow and water ad libitum.
  • Xenograft RCC models were generated as follows: Female NOD.SCID mice (6-8 weeks of age) were injected with A498 RCC cells (5xl0 6 cells/mouse), female Balb/c Nude mice (6-8 weeks old) were implanted with 786-0 RCC cells (5xl0 6 cells/mouse) or Caki-1 RCC cells (6xl0 6 cells/mouse), female Athymic Nude-Foxnlnu mice (7-8 weeks of age) were implanted with MFE280 endometrial cells (10xl0 6 cells/mouse). Cells were implanted into the flank of animals.
  • the treatment groups typically included the vehicle group (10 pl/g body weight, orally (PO), twice per day (BID)), HC-5404 (3, 10 or 30 mg/kg, PO, BID), cabozantinib (30 mg/kg, PO, daily (QD)), axitinib (30 mg/kg, PO, BID), 104envatinib (10 mg/kg, PO, BID), sunitinib (20 mg/kg, PO, QD) or DC101 (15 mg/kg, 15 mg/kg or 20 mg/kg, intraperitoneally (IP), twice per week (BIW)).
  • vehicle group 10 pl/g body weight, orally (PO), twice per day (BID)
  • HC-5404 3, 10 or 30 mg/kg, PO, BID
  • cabozantinib (30 mg/kg, PO, daily (QD)
  • axitinib (30 mg/kg, PO, BID
  • 104envatinib (10 mg/kg, PO, BID
  • sunitinib
  • Animals with >50% regressions were partial responders. Animals with tumor volume of 0 (no measurable tumor) were considered as complete responders, and animals with ⁇ 50% tumor regression and 30% growth over the baseline were considered as stable disease. Animals with tumors >30% increase over the baseline were considered progressive disease.
  • the % change in body weight was calculated by the formula (body weight on observation day - body weight on initial day)/body weight on initial day x 100%.
  • mice harboring 786-0 xenografts (-200 mm 3 ) began treatment with axitinib as a single agent (30 mg/kg, PO; BID).
  • axitinib as a single agent (30 mg/kg, PO; BID).
  • the remaining animals were removed from study.
  • the four treatment groups were vehicle, HC-5404 (30 mg/kg, PO; BID), axitinib (30 mg/kg, PO; BID), or the combination thereof, which continued for 28 days.
  • Example 8 HC-5404 is a Potent and Selective PERK Inhibitor
  • PERK is one of four closely related kinases of the integrated stress response (ISR) that phosphorylate eIF2a in response to cellular stress. Both tumorigenesis and treatment with many anticancer agents have been shown to activate ISR, and hypoxic stress is associated with activation of PERK signaling as an adaptive response. To understand the specific role of PERK in RCC, the aminopyridine PERK inhibitor HC-5404 was investigated.
  • ISR integrated stress response
  • HC-5404 The selectivity of HC-5404 against the broader kinome was then evaluated using a KINOMEScan biochemical panel assay that measured binding against 468 unique kinases, including 403 wild-type human kinases and 59 mutant isoforms. Notably, the kinome panel does not include PERK (also called EIF2AK3). HC-5404 was evaluated at 100 nM, 1000 nM, and 10,000 nM, which revealed a high level of selectivity across the kinome (FIG. 17).
  • HC-5404 is a potent, selective, and cell active PERK kinase inhibitor.
  • HC-5404 was quantified from mouse plasma following a single oral administration (PO) at doses ranging from 3 to 100 mg/kg. Dose-proportional increases in exposure were observed up to 100 mg/kg, with Cmax before 1 h and average half-life of 2.22 h. Mouse plasma protein binding (PPB%) was determined to be 95.6%, which enabled us to calculate the free drug plasma exposure across time (FIG. 18). Oral dosing at 30 mg/kg resulted in free drug exposure of 355 nM at Cmax, well below the 1000 nM concentration assayed in the kinome scan that revealed minimal secondary binding.
  • PO oral administration
  • HC-5404 results in a timedependent and dose-dependent inhibition of p-PERK in pancreas tissue.
  • HC-5404 reached 3668 ng/mL (355 nM, unbound) in plasma at 1 h, as shown in table below.
  • pPERK inhibition was approximately 90% (FIG. 19).
  • HC- 5404 is cleared from plasma, pPERK gradually increased until basal levels were restored by 12 h after dosing.
  • HC-5404 The ability of HC-5404 to suppress tumor growth was evaluated in the 786-0 xenograft model of RCC.
  • the 786-0 tumor model is homozygous for a frameshift mutation following Glyl04 in von Hippel -Lindau protein (VHLmut), resulting in HIF stabilization and activation of UPR, including elevated pPERK. Similar to the pancreas, administration of 30 mg/kg HC-5404 induced -90% pPERK inhibition in 786-0 tumors at 1 h post dose, which returned to baseline by 8 h (FIG. 21).
  • VHLmut von Hippel -Lindau protein
  • mice harboring 786-0 xenografts were treated with 3, 10, or 30 mg/kg PO, BID dosing alongside 6, 20, or 60 mg/kg PO, QD dosing to evaluate the effect of the dose regimen on tumor growth.
  • BID and QD dosing were not statistically significant, the general trend indicated better antitumor effects with BID dosing.
  • 30 mg/kg BID induced the greatest effect at 48% tumor growth inhibition (FIG. 22). Based on this result, 30 mg/kg BID dosing was used for subsequent in vivo tumor studies.
  • Example 10 HC-5404 Sensitizes RCC Tumor Models to VEGFR-TKIs
  • Receptor tyrosine kinase inhibitors that target the VEGF receptor are anti angiogenic agents that disrupt tumor vascular development as a critical component of their mechanism of action, driving tumor hypoxia and nutrient deprivation. Hypoxia and glucose deprivation are known drivers of ER stress and PERK pathway activation in tumors.
  • VEGFR-TKIs Receptor tyrosine kinase inhibitors that target the VEGF receptor
  • cabozantinib 15, 30 and 60 mg/kg
  • lenvatinib 5 and 10 mg/kg
  • axitinib 15 and 30 mg/kg
  • All three VEGFR-TKIs induced dose-dependent increases in pPERK that reflected the level of tumor growth inhibition (FIG. 26).
  • the doses used were well tolerated, as no treatment-associated body weight loss was observed (FIG. 26).
  • Sunitinib was evaluated in 786-0 xenografts and also demonstrated increased pPERK. This effect increased over time (FIG. 27). Based on these experiments, dose regimens were selected for follow-up combination studies.
  • HC-5404 and the second generation VEGFR-TKIs resulted in modest effects on tumor volume.
  • VEGFR-TKIs were combined with HC-5404, tumors regressed relative to baseline (FIG. 28).
  • sunitinib was a more effective inhibitor of 786-0 tumors compared to the second generation of VEGFR-TKIs.
  • Sunitinib as a single agent was an effective inhibitor of 786-0 growth, yet combination with HC-5404 still enhanced the level of TGI, underscoring the mechanistic interplay between the VEGFR-TKIs and PERK inhibitors, and highlighting the therapeutic benefit that might be achieved by combining VEGFR-TKIs with HC- 5404.
  • HC-5404 sensitizes RCC tumor models to anti angiogenic VEGFR-TKIs and enhance their antitumor effect.
  • HC-5404 was further validated in tumors by assessing the response of markers of in vivo PERK inhibition. Previous studies have highlighted a surprising induction of downstream targets of ATF4, including asparagine synthetase (ASNS) and phosphoserine aminotransferase (PSAT1). Other canonical targets of ATF4, including CBS and CTH, were investigated. Treatment with HC-5404 induced a dose-dependent accumulation of ASNS, CBS and CTH in 786-0 tumors (FIG. 29).
  • ASNS asparagine synthetase
  • PSAT1 phosphoserine aminotransferase
  • RCC is frequently characterized by mutations in and epigenetic silencing of VHL, which results in dysregulated HIF expression that drives a proangiogenic tumor environment associated with increased VEGF expression and activated UPR.
  • VHL mutation status, pPERK expression, and sensitivity to VEGFR-TKIs may be linked.
  • Three RCC tumor models were investigated: 786-0 and A498 (Glyl44 frameshift) were selected as representative VHL mutant models, and Caki-1 model was chosen as a VHL wild-type model.
  • HC-5404 The presence of basal PERK pathway engagement may predict sensitivity to PERK inhibition, and sensitivity to HC-5404 was investigated in three RCC models. Sensitivity to HC- 5404 varied across the three xenograft models in a manner that reflected their basal levels of pPERK activity and VHL status. HC-5404 induced TGI of approximately 52% and 41% in 786-0 and A498, respectively, whereas HC-5404 had minimal effect on the growth of the Caki-1 model (FIG. 36)
  • A498 and Caki-1 models were treated with HC-5404, sunitinib, or a combination thereof for a 28- day period.
  • the level of tumor response across the three models was reflective of the genetic background: the VHL mutant A498 was sensitive to HC-5404 as a single agent, although the TGI only reached -40% (FIG. 31).
  • the A498 model was sensitive to sunitinib with robust single agent activity that resulted in 80% TGI.
  • sunitinib and HC-5404 were combined in the VHL mutant model, tumor regression of 22% was observed in A498 (FIG. 31).
  • Caki-1 was far less sensitive to either HC-5404 or sunitinib as monotherapy, and although an effect of the combination treatments was observed, the TGI only reached 47% in the combination group (FIG.
  • VHL mutation status could be used as a selection criterion that predicts sensitivity to treatment.
  • 15 diverse RCC patient-derived xenografts PDXs
  • Animals were treated to axitinib, HC-5404, or a combination thereof for 28 days, and scored for the magnitude of combination benefit observed. All five VHLwt models that were included in the study responded to the combination treatment with significant improvement over monotherapy (representative growth charts in FIG. 33 and FIG. 34).
  • the models were ranked based on change in tumor volume relative to starting baseline and scored for the number of partial responses (PRs). In this case, PR was considered as a regression in volume greater than 50% compared to starting baseline. While axitinib as a monotherapy resulted in 1/15 PRs, the combination of HC-5404 with axitinib resulted in 7/15 PRs (FIG. 35). Importantly, the VHLwt models retained responsiveness to the combination treatment, although three of the models appeared to have decreased sensitivity to axitinib. From this study, it was concluded that HC-5404 improves tumor response to VEGFR-TKI across diverse tumor contexts, and this effect is independent of VHL mutation status.
  • Example 12 HC-5404 Demonstrates Combination Benefit with Other Agents Targeting Angiogenesis without Induction of Tumor pPERK
  • VEGFR-TKIs may improve antitumor activity.
  • a mouse monoclonal antibody that selectively targets VEGFR-2 was investigated.
  • DC-101 is a surrogate mAb to human anti-VEGFR2 mAb ramucirumab, that is approved for use in multiple cancer types.
  • DC-101 was tested to determine whether treatment with HC-5404 could enhance the activity of DC-101 across three models of RCC (786-0, A498, and Caki-1). Similar to the observations of HC-5404 with multi-kinase-targeted TKIs, the combination treatment significantly improved the effects of each monotherapy in 786-0 and A498 xenografts with TGI values of 82% and 78%, respectively (FIG. 37). In contrast, cotreatment of DC-101 and HC-5404 did not exhibit significant combination benefits resulting in a TGI value of only 24% in Caki-1 following 28 days of treatment (FIG. 37). The combination treatments did not result in tumor regression in any of the models tested.
  • PERK is an adaptive stress response that links hypoxia and nutrient deprivation with proangiogenic signaling.
  • H4C staining of tumor crosssections confirmed that axitinib decreased the proportion of Meca32 (also called PLVAP) expressing vascular endothelial cells, and that this effect was enhanced by the addition of HC-5404 (FIG. 38, FIG. 39).
  • HC-5404 alone did not affect this vascular endothelium marker, highlighting a context-dependent effect of HC-5404 on tumor angiogenesis.
  • axitinib as a single agent decreased the proportion of immature CD31+SMA- vessels, which was enhanced in the combination treatment group (FIG. 39).
  • axitinib did not affect the number of mature CD31+SMA+ cells at the dose and time points analyzed, consistent with the predominant role that VEGF axis plays in the production of new vasculature through sprouting angiogenesis.
  • HC-5404 as a single agent did not affect the expression of any vascular markers, whereas the combination treatments resulted in a significant decrease in mature CD31+SMA+ blood vessel cells (FIG. 39).
  • Example 14 PERK is a Vulnerability for RCC Xenografts That Have Progressed on VEGFR-TKIs
  • the pericytes form a layer of mural cells that surround blood vessels, and are important for vascular morphogenesis and branching.
  • Two pericyte markers (NG2 and MCAM) were used to evaluate the effect of axitinib and HC-5404 on this layer.
  • the proportion of cells that stained positive for NG2+ decreased only in the combination group relative to vehicle, highlighting the impact of the combination groups on this pericyte marker.
  • MCAM+ cells were decreased by axitinib, and this effect was enhanced by the addition of HC-5404 (FIG. 43, FIG. 44).
  • HC-5404 and axitinib combination inhibits markers of tumor vasculature endothelium and pericyte cells.
  • Example 15 Adaptive Stress Drives Resistance to VEGFR Inhibitors
  • Anti angiogenic therapies enhance tumor hypoxia.
  • Tumor cells adapt to hypoxia via activation of adaptive stress pathways, notably PERK, which leads to cell survival.
  • Example 16 HC-5404 Potentiates Antiangiogenic Agents in 786-0 RCC Xenograft Models
  • This example investigated the inhibition of PERK-mediated stress adaptation using HC- 5404.
  • HC-5404 combines with VEGRF-TKIs to inhibit tumor growth in 786-0 xenograft models of RCC.
  • 786-0 tumor xenografts were treated with once daily oral administration of lenvatinib (10 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof and twice daily oral administration of axitinib (30 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof following analogous procedures as described in the examples above.
  • Tumor growth curves are shown in FIG. 48 and FIG. 49.
  • A498 tumor xenografts were treated with once daily oral administration of sunitinib (20 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof and twice weekly intraperitoneal injection of DC-101 (15 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof following analogous procedures as described in the examples above. Tumor growth curves are shown in FIG. 50 and FIG. 51.
  • HC-5404 at 3, 10 and 30 mg/kg, PO, BID showed a trend for dose-dependent antitumor efficacy with the maximum efficacy at 30 mg/kg with a TGI% value of -61% while cabozantinib monotherapy resulted in tumor stasis with 2/8 PRs in this model.
  • HC-5404 significantly improved the effects of cabozantinib alone and exhibited similar efficacy resulting in average tumor regression of -66% with 8/8 PRs in each group. No treatment-related body weight loss was observed. Treatment with HC-5404 or cabozantinib, either as monotherapy or in combination, did not affect mouse body weight, suggesting the treatments are well tolerated.
  • Example 17 Cabozantinib Inhibits Formation of New Tumor Vasculature and Disrupts Pericytes
  • HC-5404 decreased mature CD31+SMA+ vascular cells as a trend only in presence of cabozantinib. New vascular formation is highly dependent on VEGF signaling. Images of IHC staining are shown in FIG. 54, and FIG. 55 shows quantification of the IHC staining.
  • Pericyte is a stabilizing cell layer only associated with mature blood vessels.
  • HC-5404 and cabozantinib combination decreased pericyte markers NG2 and MCAM.
  • Statistical comparisons only shown for significant differences between vehicle and treatment groups. MCAM trend was observed for Cabo+HC-5404 groups. Images of IHC staining are shown in FIG. 56, and FIG. 57 shows quantification of the IHC staining.
  • HC-5404 showed improved efficacy in combination with axitinib and anti-PD-1 antibody in RENCA tumor-bearing BALB/c mice.
  • mice were inoculated subcutaneously with syngeneic RENCA RCC tumor cells. The indicated treatments were initiated on Day 6 post cell inoculation following randomization for equivalent starting tumor size. Tumor volume (FIG. 58) and body weight (FIG. 59) were monitored over time and are displayed.
  • PO oral gavage
  • IV intravenous administration
  • BID twice daily dosing
  • BIW twice per week dosing
  • ICI immune checkpoint inhibitor (anti-mouse PD-1 antibody).
  • HC-5404-FU + Axitinib and anti-PDl + Axitinib (front line - SOC) combinations provide comparable antitumor activity in syngeneic RCC model RENCA (VHLwt).
  • Triplet combination (HC-5404 + Axitinib + anti-PDl mAb) provides enhanced efficacy over front line SOC Treatments were well-tolerated with no significant body weight loss observed in any of the groups (FIG. 58 and FIG. 59).
  • Example 19 HC-5404 and VEGFR-TKI Combination Induces Favorable Changes in Tumor- Infiltrating Myeloid Cells
  • This example investigated the effect of HC-5404 in combination with axitinib on RENCA RCC model following procedures analogous to those described above. Addition of HC-5404 to axitinib increased stimulatory macrophage phenotype and reduced axitinib -induced MDSC infiltration in mouse RENCA tumors.
  • mice with established subcutaneous syngeneic RENCA RCC tumors were treated with vehicle (0.5% methylcellulose in water, BID, PO), HC-5404 (30 mg/kg, BID, PO), axitinib (30 mg/kg, BID, PO), or the combination of HC-5404 and Axitinib. Following 13 days of treatment, animals were euthanized, and tumors were evaluated by multicolor flow cytometry.
  • HC-5404 + Axitinib combination alters populations of macrophages and MDSCs in tumor (RENCA RCC model). Increased Ml macrophage polarization (MHCII+CD206-) compared to both monotherapies. Reduced monocytic MDSC frequency compared to Axitinib monotherapy HC-5404 + Axitinib combination increases activation markers on Ml macrophages in tumor. Increased CD86 compared to either monotherapy Increased MHCII compared to either monotherapy (FIG. 60, FIG. 61).
  • This example investigated the effect of HC-5404 in combination with axitinib on CD4 T- Cells in tumor-draining lymph nodes following procedures analogous to those described above. Addition of HC-5404 to axitinib increased CD4 T-cell activation and effector phenotype in the draining lymph node while increasing TNFa and reducing exhaustion marker expression on CD4 T-cells in RENCA RCC tumors.
  • mice with established subcutaneous syngeneic RENCA RCC tumors were treated with vehicle (0.5% methylcellulose in water, BID, PO), HC-5404 (30 mg/kg, BID, PO), axitinib (30 mg/kg, BID, PO), or the combination of both HC-5404 and axitinib.
  • vehicle 0.5% methylcellulose in water
  • BID, PO HC-5404
  • axitinib 30 mg/kg, BID, PO
  • TDLN tumor-draining LN
  • HC-5404 + Axitinib combination increases activated phenotype of CD4 T-cells in tumordraining lymph node. Increased early activation marker CD69. Increased effector memory phenotype (CD62L-CD44+) was observed. HC-5404 + Axitinib combination reduces exhausted phenotype on CD4 T-cells in tumor, increased TNFa and decreased exhaustion markers (EOMES, TIM-3, LAG-3) (FIG. 62).
  • Example 21 HC-5404 and VEGFR-TKI Combination Reshapes Immunosuppressive TME [00587] This example investigated the effect of HC-5404 in combination with axitinib on myeloid cells in tumor microenvironment following procedures analogous to those described above.
  • Combination treatment of HC-5404 and axitinib decreased M2 markers and increased Ml markers on macrophages in RENCA RCC tumors.
  • mice bearing subcutaneous RENCA RCC tumors were treated for 7 days with vehicle (0.5% methylcellulose in water, BID, PO), HC-5404 (30 mg/kg, BID, PO), axitinib (30 mg/kg, BID, PO), or the combination of both HC-5404 or axitinib.
  • vehicle 0.5% methylcellulose in water
  • BID, PO vehicle
  • HC-5404 (30 mg/kg, BID, PO)
  • axitinib (30 mg/kg, BID, PO)
  • tumor sections were formalin-fixed, paraffin-embedded, and analyzed via fluorescence microscopy for the M2 marker CD206 (FIG. 64, left), and the Ml markers CD86 (FIG. 64, middle) and PD-L1 (FIG. 64, right) on macrophages.
  • HC-5404 + Axitinib combination increases activated phenotype myeloid cells in tumor microenvironment. There was a decrease in CD206 (M2 marker) and increase in CD86 and PD-L1 (Ml markers) in RENCA RCC model (IHC) (FIG. 63, FIG. 64).
  • This example investigated the effect of HC-5404 in combination with axitinib and aPD-1 on RENCA RCC model following procedures analogous to those described above.
  • HC-5404 + axitinib + aPD-1 triple combination showed trend of increased Ml macrophages and decreased M2 macrophages compared to dual combinations.
  • mice with established subcutaneous syngeneic RENCA RCC tumors were treated with vehicle (0.5% methylcellulose in water, BID, PO), HC-5404 (30 mg/kg, BID, PO), axitinib (30 mg/kg, BID, PO), anti-PD-1 Ab (10 mg/kg, BIW), or combinations of these treatments. Following 13 days of treatment, animals were euthanized, and tumors were evaluated by multicolor flow cytometry.
  • Example 23 HC-5404, VEGFR-TKI, and Immune Checkpoint Inhibitor Combination Increases T-Cell Infiltration and Cytokine Expression in TME
  • This example investigated the effect of HC-5404, axitinib, aPD-1, and HC-5404 in combination with axitinib and aPD-1 on T-Cell infiltration following procedures analogous to those described above.
  • HC-5404 + axitinib + aPD-1 triple combination increased CD8 T-cell tumor infiltration and expression of stimulatory cytokines.
  • mice with established subcutaneous syngeneic RENCA RCC tumors were treated with vehicle (0.5% methylcellulose in water, BID, PO), HC-5404 (30 mg/kg, BID, PO), axitinib (30 mg/kg, BID, PO), anti-PD-1 Ab (10 mg/kg, BIW) or combinations of these treatments. Following 13 days of treatment, animals were euthanized, and tumors were evaluated by multicolor flow cytometry.
  • T-cell infiltration and activation was observed with HC-5404 + axitinib + ICI triple combination treatment. Increased frequency and number of CD8 T-cells were observed, as well as increased expression of inflammatory cytokines on CD8 T-cells, including IFNy and TNFa. These increases were greater than any monotherapy or duo combination (FIG. 67).
  • Example 24 HC-5404 and Immune Checkpoint Inhibitor Combination Improves Efficacy and Immune Activation in Tumor Microenvironment
  • This example investigated the effect of HC-5404, aPD-1, and HC-5404 in combination with aPD-1 on myeloid cells in tumor microenvironment and T-cell activation following procedures analogous to those described above using MB49 syngeneic model.
  • HC-5404 + anti-PD- 1 combination showed improved efficacy and immune stimulation in syngeneic MB49 model.
  • Mice with established syngeneic MB49 tumors were randomized 6 days post tumor inoculation into groups that received either vehicle (0.5% methylcellulose in water, BID, PO), HC-5404 (10 or 30 mg/kg, BID, PO), anti-PD-1 Ab (10 mg/kg, BIW), or both HC-5404 and anti-PD-1.
  • HC-5404 + aPD-1 Combination efficacy of HC-5404 + aPD-1 was observed in multiple studies.
  • Myeloid activation increased type 1 interferon receptor (IFNAR1) expression on macrophages and MDSCs.
  • T-cell activation increased cytotoxic T and NK infiltration into tumor tissue and increased DC frequency and T-cell activation in draining lymph node (FIG. 68, FIG. 69).
  • Example 26 HC-5404 Sensitizes Antiangiogenics Across Multiple Indications
  • HC-5404 combines with anti angiogenic agents in tumor models of endometrial cancer, gastric cancer and pancreatic neuroendocrine cancers.
  • HC-5404 synergized with lenvatinib to induce an average 34% tumor regression in MFE280 MSS model of endometrial cancer (FIG. 74).
  • HC-5404 at 30 mg/kg, PO, BID showed only a trend for tumor growth inhibition with a TGI% value of -37%, while lenvatinib significantly inhibited tumor growth with a TGI% value of -85%.
  • the combination treatment resulted in average tumor regression of -34% (P>0.05 vs. Lenvatinib).
  • Example 28 ASNS and PSAT1 Induced by HC-5404 and Antiangiogenic Agent Human Endometrial Cancer Model
  • MFE280 tumors sampled after 7 days of treatment HC-5404 30 mpk BID; Lenvatinib 10 mpk QD.
  • HC-5404 as single-agent or in combination with Lenvatinib induces ASNS in endometrial cancer tumor model MFE280 (FIG. 79) and PSAT1 in endometrial cancer tumor model MFE280 (FIG. 80).
  • PD modulation consistent across RCC and endometrial (MSS) cancer model.
  • Example 29 HC-5404 Induces ATF4 Targets In Gastric PDX Model
  • This example investigated the effect of HC-5404 in combination with DC-101 in gastric PDX model following procedures analogous to those described above.
  • HC-5404 induces proteins involved in amino acid metabolism in gastric PDX (GA0114-R19P6).
  • HC-5404 induces ATF4 target genes, including ASNS, PSAT1 and other enzymes involved in amino acid metabolism (FIG. 81, FIG. 82).
  • ASNS ASNS
  • PSAT1 PSAT1
  • other enzymes involved in amino acid metabolism FIG. 81, FIG. 82.
  • Example 30 HC-5404 and ICIs
  • HC-5404 shows combination benefit in bladder cancer and melanoma with immune checkpoint inhibitor, anti-PDl, -50% increased TGI benefit over ICI alone. Combination with docetaxel in neuroendocrine (castration-resistant) prostate cancer leads to 45% TGI benefit. Addition of HC-5404 with gemcitabine in PDAC leads to a delayed recurrence compared to gemcitabine alone (FIG. 83, FIG. 84, FIG. 85).
  • Example 31 HC-5404 and VEGFR-TKI Combination Decreases Pro-angiogenic cytokines [00611]
  • This example investigated the effect of HC-5404 in combination with VEGFR-TKIs in modulating pro-angiogenic cytokines in plasma and in tumor tissue following procedures analogous to those described above.
  • HC-5404 and cabozantinib decrease pro-angiogenic cytokines in plasma and in tumor tissue.
  • VEGF-A Vascular endothelial growth factor-A
  • HGF Hepatocyte growth factor
  • Interleukin-8 was decreased by the combination of HC-5404 and cabozantinib in mouse plasma (FIG. 88).
  • Lymphatic vessel endothelial hyaluronan receptor- 1 (LYVE-1) was decreased by HC-5404 in a dose-dependent manner as either monotherapy or in combination with cabozantinib (FIG. 89).
  • Syndecan was decreased by HC-5404 as monotherapy or in combination with cabozantinib (FIG. 90).
  • Example 32 Tumors that Progress on Axitinib Retain Sensitivity To HC-5404 Combination Treatment
  • This example investigated the effect of HC-5404 in combination with VEGFR-TKIs in modulating tumor progression following procedures analogous to those described above. Tumors that progress on axitinib were sensitive to the addition of HC-5404 to the treatment regimen.
  • Mice harboring 786-0 xenografts were treated for 14 days with axitinib (30 mg/kg; PO; BID), following which the mice with tumors that progressed (approximately doubled in size) were rerandomized into four groups as indicated. After enabling 786-0 xenografts to progress on axitinib for two weeks, HC-5404 was added to the regimen, resulting in 20% tumor regression and inhibition of several vascular markers associated with immature and mature blood vessels (FIG.
  • HC-5404 and axitinib both decreased the proportion of smooth muscle actin (SMA) as monotherapy, which was further diminished by the combination treatment (FIG. 92).
  • Proportion of cells expressing Meca32 was decreased in the combination group relative to vehicle.
  • Meca32 is a marker of vascular endothelial cells (FIG. 92).
  • Proportion of cells expressing the vascular marker CD31 is decreased by axitinib, an effect that is enhanced by addition of HC-5404 in the combination group (FIG. 92).
  • Tumors randomized into the combination group have decreased proportion of cells expressing the pericyte markers NG2 and MCAM (FIG. 93).
  • Example 33 HC-5404 Combines with Antiangiogenic Therapies To Inhibit Tumor Growth [00617] This example investigated the effect of HC-5404 in combination with VEGFR-TKIs in inhibiting tumor growth following procedures analogous to those described above. Combination of HC-5404 with anti-VEGFR2 antibody DC 101 (mouse surrogate for ramucirumab) shows robust combinatorial benefit in RCC models.
  • HC-5404 combined with antiangiogenic monoclonal antibody selectively targeted mouse VEGFR-2.
  • DC 101 antiangiogenic monoclonal antibody
  • 786-0 and A498 RCC tumor models were treated with HC-5404 (30 mg/kg; PO; BID) and DC-101 (15 mg/kg; IP; BIW) for 28 days (FIG. 94, FIG. 95). No treatment- related BWL is observed in any of the groups.
  • Example 34 HC-5404 Shows Combination Benefit with VEGRD-TKIs in RENCA RCC Model
  • This example investigated the effect of HC-5404 in combination with VEGFR-TKIs in inhibiting tumor growth using RENCA RCC tumor model, following procedures analogous to those described above.
  • HC-5404 + Axitinib shows modest increase in efficacy compared to Axitinib alone PD and immunophenotyping samples were collected on day 13, after 4h treatment (FIG. 96).
  • Example 35 Efficacy of HC-5404 with and without VEGFR-TKIs in RCC PDX Models - A Single Animal Trial
  • This example investigated the effect of HC-5404 in combination with VEGFR-TKIs in inhibiting tumor growth using 12 RCC PDX tumor models, following procedures analogous to those described above.
  • HC-5404 and axitinib showed combination benefits in RXF-393 and RXF-2282 models (FIG. 97, FIG. 98). No combination benefits were observed in RXF-2178 and RXF-2502 RCC PDX models (FIG. 99, FIG. 100). Treatments were well-tolerated as determined by BW measurement. No treatment related BW loss was observed.
  • HC-5404 and axitinib showed combination benefits in SMTCA75, RXF-616, RXF-2304 and RXF-488 models (FIG. 101, FIG. 102, FIG. 103, FIG. 104), resulting in tumor stasis or regressions.
  • HC-5404 as a monotherapy exhibited antitumor efficacy in SMTCA75 and RXF-2304 models. Treatments were well-tolerated as determined by BW measurement. No treatment related BW loss was observed.
  • HC-5404 and axitinib showed combination benefits in RXF-2667 and RXF-2783 models (FIG. 105, FIG. 107), resulting in tumor regressions, while no combination benefit was observed in RXF-1220 and RXF-631 models (FIG. 106, FIG. 108).
  • HC-5404 as a monotherapy exhibited antitumor efficacy in RXF-2667 and RXF-2540 models. Treatments were well-tolerated as determined by BW measurement. No treatment related BW loss was observed.

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Abstract

Provided herein are methods of treating a cancer (e.g., a solid tumor) in a subject in need thereof. The present disclosure also provides methods of activating the immune system of a subject in need thereof (e.g., a subject having a cancer). The methods described herein generally comprise administering to the subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and an immunotherapy (e.g., an anti-PD-1 antibody) and/or an antiangiogenic agent (e.g., VEGFR-TKI).

Description

PERK INHIBITOR HC-5404 IN COMBINATION WITH ANTI-PD-1 ANTIBODY AND/OR AN ANTIANGIOGENETIC AGENT FOR USE IN THE TREATMENT OF CANCER
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of, and priority to, United States Provisional Application Serial Nos. 63/443,344, filed on February 3, 2023; 63/455,911, filed on March 30, 2023; and 63/413,116, filed on October 4, 2022, the contents of each of which are incorporated by reference herein in their entirety.
BACKGROUND
[0002] Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020 (World Health Organization). Targeted therapy and immunotherapy have expanded the horizons for treatment of cancers by improving prognosis drastically. However, tumor recurrence, drug resistance, and drug intolerance continue to be major challenges in the management of cancer (Wang et al., “Drug resistance and combating drug resistance in cancer,” Cancer Drug Resistance, 2019, 2(2): 141-160; Chakraborty et al., “The difficulties in cancer treatment” Ecancermedicalscience, 2012, 6:edl6). Cancer shows extensive proliferation of tumor cells which requires well-functioning protein machinery (Waczak et al, 2019). The endoplasmic reticulum (ER) plays a crucial role in regulating protein folding, synthesis, and maturation required for tumor cells, maintaining tumor homeostasis (Yoo et al, 2017). The accumulation of unfolded proteins in the ER lumen activates three main pathways, 1) PERK, 2) inositol -requiring enzyme-1 alpha (IREla), and 3) activating transcription factor 6 (ATF6) — together known as the unfolded protein response (UPR) — which allows cells to correct and survive this stress (Walter and Ron, 2011; Urra et al, 2016). Evidence suggests that in various types of cancer, UPR activation is a mechanism that allows tumor cells to adapt to demands on the ER and oxidative conditions imposed by conditions inherent to their biology such as an enhanced translational load caused by oncogenes and loss of tumor suppressor genes, and by environmental conditions common in tumor/metastasis microenvironments such as hypoxia among other signals (Blais et al, 2004; Chevet et al, 2015; Tameire et al, 2015; Hart et al, 2012; Martin-Perez et al, 2014; Rajasekhar and Holland, 2004;
Rajasekhar et al, 2003; Rojo et al, 2007; Sequeira et al 2009). Oncogene-activated pathways increase ER client protein load by activating mTOR signaling and translation initiation (Tameire et al, 2015; Hart et al, 2012; Ozcan et al, 2008). Other studies further show that UPR pathways contribute to adaptation to hypoxia and microenvironmental stress (Blais et al, 2004; Bi et al, 2005; Chen et al, 2014; Romero-Ramirez et al, 2009; Rouschop et al, 2010; Schewe et al, 2008; Ye et al, 2010), suggesting that UPR activation can allow for adaptation to a changing milieu. [0003] As a member of the UPR pathways, PERK is involved in pro-tumorigenesis processes, and this role makes it an attractive target for anticancer therapy. More specifically, PERK phosphorylates the eukaryotic translation initiation factor 2 alpha (eIF2a), nuclear factor (erythroid- derived 2)-like-2 (Nrf2), and forkhead box protein 01 (FOXO) proteins. Phosphorylation of eIF2a inhibits general protein synthesis and lowers the protein load, while inducing the targeted translation of uORF containing mRNAs such as ATF4. ATF4 then triggers a gene expression program consisting of antioxidant, chaperones, and autophagy genes among others aimed at restoring protein homeostasis and inducing cell survival in the context of ER stress. Cancer cells are, by definition, characterized by cellular activities and microenvironmental factors that contribute to the activation of the UPR (including oncogenic protein synthesis, hypoxic environments, dissemination and lodging into a different microenvironment, etc.) and, in addition, the action of many therapeutic agents further contribute to the activation of the UPR in the cells that are not eliminated by the therapy. The UPR pathways, including PERK, have not been a commonly investigated pathway / target for cancer therapy.
[0004] Thus, there remains an unmet need to develop new therapeutic strategies that utilize modulation of the PERK pathway for the treatment of a variety of cancers (e.g., solid tumors).
SUMMARY
[0005] In one aspect, provided herein are methods of activating the immune system of a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I)
Figure imgf000004_0001
or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD- 1 antibody.
[0006] In another aspect, provided herein are methods of activating the immune system of a subject having cancer, comprising administering to the subject a therapeutically effective amount of a compound of formula (I)
Figure imgf000005_0001
or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD- 1 antibody.
[0007] In certain embodiments, the method inhibits the activity of myeloid-derived suppressor cells (MDSCs). In certain embodiments, the method increases the infiltration of T cells and NK cells into a tumor.
[0008] In certain embodiments, the tumor is selected from the group consisting of bladder cancer, squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer.
[0009] In certain embodiments, the method increases the frequency of dendritic cells in a draining lymph node.
[0010] In another aspect, provided herein are methods of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I)
Figure imgf000005_0002
or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD- 1 antibody.
[0011] In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is selected from the group consisting of bladder cancer, squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer.
[0012] In certain embodiments, the cancer is a liquid tumor. In certain embodiments, the cancer is selected from the group consisting of Classical Hodgkin’s lymphoma, primary thymic mediastinal lymphoma, multiple myeloma, and B cell malignancies (e.g., non -Hodgkin lymphomas or chronic lymphocytic leukemia).
[0013] In certain embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, and cemiplimab. In certain embodiments, the anti-PD-1 antibody is pembrolizumab. In certain embodiments, the anti-PD-1 antibody is nivolumab.
[0014] In certain embodiments, the therapeutically effective amount of the anti-PD-1 antibody is administered once daily. In certain embodiments, the therapeutically effective amount of the anti- PD-1 antibody is administered twice daily.
[0015] In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered simultaneously. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered sequentially.
[0016] In certain embodiments, the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount of about 22 mg to about 451 mg twice daily. In certain embodiments, the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount of about 177 mg to about 900 mg twice daily.
[0017] In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered in the form of a capsule.
[0018] In certain embodiments, the method increases the tumor cell expression of type 1 interferon receptor (INF ARI) in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody. In certain embodiments, the method increases the peripheral blood monocyte surface expression of INF ARI in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody. In certain embodiments, the method increases tumor cell expression of calreticulin in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody. In certain embodiments, the method increases polymorphonuclear myeloid-derived suppressor cell (MDSC) and/or tumor associated macrophage (TAM) expression of INF ARI in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody. In certain embodiments, the method increases TAM expression of PD-L1 in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody. In certain embodiments, the method increases the infiltration of CD8 T cells and/or NK cells in a tumor in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody. In certain embodiments, the method increases expression of CD69 on T cells in a tumor draining lymph node in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody. In certain embodiments, the method reduces suppressive activity of MDSCs in the subject. In certain embodiments, the method increases dendritic cell frequency in a tumor draining lymph node in the subject. In certain embodiments, the method increases Ki67, Granzyme B (GzmB), and/or memory phenotypic markers on CD4 T-cells in the subject.
[0019] In certain embodiments, the compound of formula (I) is administered as a pharmaceutically acceptable salt. In certain embodiments, the pharmaceutically acceptable salt is a hemifumarate salt.
[0020] In another aspect, provided herein are methods of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I):
Figure imgf000007_0001
or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent. [0021] In another aspect, provided herein are methods of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I):
Figure imgf000008_0001
or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent, wherein the subject has been treated with an anti-cancer therapy.
[0022] In some embodiments, the subject is resistant or has acquired resistance to the anti-cancer therapy.
[0023] In some embodiments, the anti-cancer therapy is selected from the group consisting of: administration of an immunotherapeutic agent, a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, an anti angiogenic agent, or a combination thereof; radiation therapy; surgery; and combinations thereof.
[0024] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti angiogenic agent are administered simultaneously.
[0025] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti angiogenic agent are administered sequentially.
[0026] In some embodiments, the therapeutically effective amount of the anti angiogenic agent is administered to the subject daily.
[0027] In some embodiments, the therapeutically effective amount of the anti angiogenic agent is administered to the subject once daily.
[0028] In some embodiments, the therapeutically effective amount of the anti angiogenic agent is administered to the subject twice daily.
[0029] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti angiogenic agent reduces p-PERK levels in the subject relative to administering the therapeutically effective amount of the anti angiogenic agent alone. [0030] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti angiogenic agent increases ATF4 levels in the subject relative to administering the therapeutically effective amount of the anti angiogenic agent alone.
[0031] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti angiogenic agent modulates the activation of unfolded protein response (UPR) relative to administering the therapeutically effective amount of the anti angiogenic agent alone.
[0032] In another aspect, provided herein are methods of treating a cancer in a subject receiving a therapeutically effective amount of an anti angiogenic agent therapy, comprising administering to the subject a therapeutically effective amount of a compound of formula (I):
Figure imgf000009_0001
or a pharmaceutically acceptable salt thereof.
[0033] In some embodiments, the subject is resistant or has acquired resistance to the anti angiogenic agent therapy.
[0034] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, reduces p-PERK levels in the subject relative to administering the therapeutically effective amount of the anti angiogenic agent alone.
[0035] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, increases ATF4 levels in the subject relative to administering the therapeutically effective amount of the anti angiogenic agent alone.
[0036] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, modulates the activation of unfolded protein response (UPR) relative to administering the therapeutically effective amount of the anti angiogenic agent alone.
[0037] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, decreases tumor vasculature in the subject relative to administering the therapeutically effective amount of the anti angiogenic agent alone. [0038] In some embodiments, the anti angiogenic agent is a vascular endothelial growth factor receptor (VEGFR) modulator.
[0039] In some embodiments, the anti angiogenic agent is a vascular endothelial growth factor (VEGF) targeting antibody.
[0040] In some embodiments, the anti angiogenic agent is a tyrosine kinase inhibitor (TKI).
[0041] In some embodiments, the anti angiogenic agent is a VEGF receptor tyrosine kinase inhibitor (VEGFR- TKI).
[0042] In some embodiments, the anti angiogenic agent is selected from the group consisting of: apatinib, axitinib, brivanib, cabozantinib, canertinib, cediranib, dasatinib, erlotinib, gefitinib, leflunomide, lenvatinib, motesanib, nilotinib, nintedanib, pazopanib, regorafenib, semaxinib, sorafenib, sunitinib, tivozanib, vandetanib, vatalanib, XL0192, bevacizumab, ramucirumab, and pharmaceutically acceptable salts or biosimilars thereof.
[0043] In some embodiments, the anti angiogenic agent is selected from the group consisting of: bevacizumab, ramucirumab, and biosimilars thereof.
[0044] In some embodiments, the anti angiogenic agent is selected from the group consisting of: apatinib, axitinib, brivanib, cabozantinib, canertinib, cediranib, dasatinib, erlotinib, gefitinib, leflunomide, lenvatinib, motesanib, nilotinib, nintedanib, pazopanib, regorafenib, semaxinib, sorafenib, sunitinib, tivozanib, vandetanib, vatalanib, XL0192, and pharmaceutically acceptable salts thereof.
[0045] In some embodiments, the cancer is a metastatic cancer or a locally advanced cancer.
[0046] In some embodiments, the cancer is a solid tumor.
[0047] In some embodiments, the cancer is selected from the group consisting of: a bladder cancer, a breast cancer, a carcinoma, a cervical cancer, a colorectal cancer, a gastric cancer, a hepatocellular cancer, a kidney cancer, a lung cancer, a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer, and a thyroid cancer.
[0048] In some embodiments, the cancer is selected from the group consisting of: a breast cancer, a gastric cancer, a kidney cancer, and a lung cancer.
[0049] In some embodiments, the breast cancer is triple negative breast cancer or metastatic breast cancer; the carcinoma is carcinoma of unknown primary (CUP), endometrial carcinoma, head and neck squamous cell carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma; the gastric cancer is adenocarcinoma or gastrointestinal stromal tumor; the kidney cancer is a renal cell carcinoma (RCC); or the lung cancer is small cell lung cancer (SCLC) or nonsmall cell lung cancer.
[0050] In some embodiments, the breast cancer is triple negative breast cancer or metastatic breast cancer.
[0051] In some embodiments, the carcinoma is carcinoma of unknown primary (CUP), endometrial carcinoma, head and neck squamous cell carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma.
[0052] In some embodiments, the gastric cancer is adenocarcinoma or gastrointestinal stromal tumor.
[0053] In some embodiments, the kidney cancer is a renal cell carcinoma (RCC).
[0054] In some embodiments, the lung cancer is small cell lung cancer (SCLC) or non-small cell lung cancer.
[0055] In some embodiments, the renal cell carcinoma is clear cell renal cell carcinoma (ccRCC).
[0056] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[0057] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[0058] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, daily.
[0059] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, once daily.
[0060] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, twice daily.
[0061] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[0062] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[0063] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, daily.
[0064] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, once daily.
[0065] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, twice daily.
[0066] In some embodiments, the subject is in a fasting state.
[0067] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered with food.
[0068] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered about 30 minutes to about 1 hour after food.
[0069] In some embodiments, the therapeutically effective amount of the compound of formula (I) is administered as a pharmaceutically acceptable salt.
[0070] In some embodiments, the pharmaceutically acceptable salt is a hemifumarate salt. [0071] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an immunotherapy.
[0072] In an aspect, provided herein are methods of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I):
Figure imgf000013_0001
or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent, and a therapeutically effective amount of an immunotherapy.
[0073] In another aspect, provided herein are methods of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I):
Figure imgf000013_0002
or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent, and a therapeutically effective amount of an immunotherapy, wherein the subject has been treated with an anti -cancer therapy.
[0074] In some embodiments, the subject is resistant or has acquired resistance to the anti-cancer therapy.
[0075] In some embodiments, the anti-cancer therapy is selected from the group consisting of: administration of an immunotherapeutic agent, a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, an anti angiogenic agent, or a combination thereof; radiation therapy; surgery; and combinations thereof.
[0076] In another aspect, provided herein are methods of treating a cancer in a subject receiving a therapeutically effective amount of an anti angiogenic agent therapy, comprising administering to the subject a therapeutically effective amount of a compound of formula (I):
Figure imgf000014_0001
or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy.
[0077] In some embodiments, the immunotherapy is an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody.
[0078] In some embodiments, the immunotherapy is a therapeutically effective amount of an anti- PD-1 antibody.
[0079] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, and cemiplimab.
[0080] In some embodiments, the anti-PD-1 antibody is pembrolizumab.
[0081] In some embodiments, the anti-PD-1 antibody is nivolumab.
[0082] In some embodiments, the therapeutically effective amount of the immunotherapy is administered daily.
[0083] In some embodiments, the therapeutically effective amount of the immunotherapy is administered once daily.
[0084] In some embodiments, the therapeutically effective amount of the immunotherapy is administered twice daily.
[0085] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the immunotherapy are administered simultaneously.
[0086] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of an immunotherapy are administered sequentially.
[0087] In some embodiments, the therapeutically effective amount of the anti angiogenic agent and the therapeutically effective amount of an immunotherapy are administered simultaneously.
[0088] In some embodiments, the therapeutically effective amount of the anti angiogenic agent and the therapeutically effective amount of an immunotherapy are administered sequentially. BRIEF DESCRIPTION OF THE FIGURES
[0089] FIG. 1A is a graph showing the effects on tumor volume of single agent 10 mg/kg HC- 5404 twice daily or 30 mg/kg HC-5404 twice daily compared to vehicle in a renal cell carcinoma model.
[0090] FIG. IB is a graph showing the effects on tumor volume of single agent 30 mg/kg HC- 5404 twice daily compared to vehicle in a gastric cancer model.
[0091] FIG. 2A is a scheme showing a study design for the MB49 studies described in Example 2.
[0092] FIG. 2B is a graph showing the tumor volume post-MB49 cell inoculation in MB49 tumor-bearing mice administered with vehicle orally twice daily, 3 mg/kg HC-5404-FU orally twice daily, 10 mg/kg HC-5404-FU orally twice daily, 30 mg/kg HC-5404-FU orally twice daily, 5 mg/kg anti-PD-1 antibody intraperitoneally biweekly, 3 mg/kg HC-5404-FU orally twice daily + 5 mg/kg anti-PD-1 antibody intraperitoneally biweekly, 10 mg/kg HC-5404-FU orally twice daily + 5 mg/kg anti-PD-1 antibody intraperitoneally biweekly, or 30 mg/kg HC-5404-FU orally twice daily + 5 mg/kg anti-PD-1 antibody intraperitoneally biweekly.
[0093] FIG. 2C is a graph showing the tumor volume post-MB49 cell inoculation in MB49 tumor-bearing mice administered with vehicle, 30 mg/kg HC-5404-FU orally twice daily, 5 mg/kg anti-PD-1 antibody intraperitoneally biweekly, or 30 mg/kg HC-5404-FU orally twice daily + 5 mg/kg anti-PD-1 antibody intraperitoneally biweekly.
[0094] FIG. 2D is a graph showing the tumor volume of individual subjects and number of responders post-cell inoculation in the vehicle treatment group described in FIG. 2C.
[0095] FIG. 2E is a graph showing the tumor volume of individual subjects and number of responders post-cell inoculation in the 30 mg/kg HC-5404-FU treatment group described in FIG.
2C
[0096] FIG. 2F is a graph showing the tumor volume of individual subjects and number of responders post-cell inoculation in the anti-PD-1 antibody treatment group described in FIG. 2C.
[0097] FIG. 2G is a graph showing the tumor volume of individual subjects and number of responders post-cell inoculation in the 30 mg/kg HC-5404-FU + anti-PD-1 antibody treatment group described in FIG. 2C.
[0098] FIG. 3A is a graph showing the IFNAR1 expression on Ml macrophages from the treatment groups described in FIG. 2B 14 days post-inoculation (7 days post treatment), as determined by flow cytometry. [0099] FIG. 3B is a graph showing the IFNAR1 expression on M2 macrophages from the treatment groups described in FIG. 2B 14 days post-inoculation (7 days post treatment), as determined by flow cytometry.
[00100] FIG. 3C is a graph showing the IFNAR1 expression on polymorphonuclear MDSCs from the treatment groups described in FIG. 2B 14 days post-inoculation (7 days post treatment), as determined by flow cytometry.
[00101] FIG. 3D is a graph showing the IFNAR1 expression on tumor cells from the treatment groups described in FIG. 2B 14 days post-inoculation (7 days post treatment), as determined by flow cytometry.
[00102] FIG. 3E is a graph showing the PD-L1 expression on macrophages from the treatment groups described in FIG. 2B 14 days post-inoculation (7 days post treatment), as determined by flow cytometry.
[00103] FIG. 4A is a diagram showing proteins involved in immunogenic cell death in cancer therapy.
[00104] FIG. 4B is a graph showing the surface calreticulin expression on tumor cells from the treatment groups described in FIG. 2B 14 days post-inoculation (7 days post treatment), as determined by flow cytometry.
[00105] FIG. 4C is a comparison of images of 786-0 cells from a xenograft model treated with HC-5404 or vehicle showing HMGB1 in the cytoplasm detected by immunohistochemistry.
[00106] FIG. 4D is a graph showing HMGB1 cytoplasmic expression induced by 30 mg/kg HC- 5404 treatment compared to vehicle in 786-0 xenograft model on day 15 post treatment.
[00107] FIG. 5A is a graph showing the frequency of tumor-infiltrated CD8 T cells in the treatment groups described in FIG. 2B on day 7 post treatment, as determined by flow cytometry.
[00108] FIG. 5B is a graph showing the frequency of tumor-infiltrated CD8 NK cells in the treatment groups described in FIG. 2B on day 7 post treatment, as determined by flow cytometry.
[00109] FIG. 5C is a graph showing the frequency of CD69 expression on CD4 T cells from lymph nodes of the treatment groups described in FIG. 2B on day 7 post treatment, as determined by flow cytometry.
[00110] FIG. 5D is a graph showing the frequency of CD69 expression on CD8 T cells from lymph nodes of the treatment groups described in FIG. 2B on day 7 post treatment, as determined by flow cytometry. [00111] FIG. 6A is a graph showing the Ki67 expression on CD4 T-cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
[00112] FIG. 6B is a graph showing the EOMES expression on CD4 T-cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
[00113] FIG. 6C is a graph showing the Granzyme B expression on CD4 T-cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
[00114] FIG. 6D is a graph showing the effector memory populations of CD4 T-cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
[00115] FIG. 6E is a graph showing the TCF1 expression on CD4 T-cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
[00116] FIG. 7A is a graph showing the total dendritic cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
[00117] FIG. 7B is a graph showing the XCR1+ cDCl cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
[00118] FIG. 7C is a graph showing the cDC2 cells in lymph nodes of the treatment groups described in FIG. 2B on day 14 post treatment, as determined by flow cytometry.
[00119] FIG. 8 is a comparison of graphs showing the expression of IFNAR1 on monocytes from whole blood of the vehicle, 10 mg/kg HC-5404, and 30 mg/kg HC-5404 treatment groups described in FIG. 2B on days 7, 11, and 14 post treatment, as determined by flow cytometry.
[00120] FIG. 9 is a heat map showing the expression of myeloid (Ml- and M2-related), lymphoid (T-cell related), and interferon stimulated genes (ISG) in the vehicle, 10 mg/kg HC-5404, and 30 mg/kg HC-5404 treatment groups described in FIG. 2B on day 7 post treatment.
[00121] FIG. 10A shows the results of a T cell suppression assay performed using human CD3 T cells co-cultured with human MDSCs and treated with vehicle, 500 nM HC-5404, or 750 nM HC- 5404 (1 : 1 MDSC:T cell ratio).
[00122] FIG. 10B is a scheme showing the preparation of differentiated human MDSCs from cord blood. [00123] FIG. 10C is a histogram showing the results of the T cell suppression assay described in
FIG. 10A
[00124] FIG. 11A shows the results of a T cell suppression assay performed using mouse CD8 T cells co-cultured with MDSCs and treated with RPMI medium, LSLG, or LSLG + 500 nM HC- 5404 (8: 1 MDSGT cell ratio).
[00125] FIG. 11B is a scheme showing the preparation of mouse MDSC from bone marrow of non-tumor bearing mouse.
[00126] FIG. 11C is a set of histograms showing the results of the suppression assay described in FIG. 11A and a similar assay performed for mouse CD4 T cells.
[00127] FIG. 12 is a graph showing the FRET -based biochemical assays used to evaluate HC-5404 inhibition of GCN2, an ISR kinase, across concentration series ranging from 0.2 nM to 10 pM.
[00128] FIG. 13 is a graph showing the FRET -based biochemical assays used to evaluate HC-5404 inhibition of PERK, an ISR kinase, across concentration series ranging from 0.2 nM to 10 pM.
[00129] FIG. 14 is a graph showing the FRET -based biochemical assays used to evaluate HC-5404 inhibition of HRI, an ISR kinase, across concentration series ranging from 0.2 nM to 10 pM.
[00130] FIG. 15 is a graph showing the FRET -based biochemical assays used to evaluate HC-5404 inhibition of GCN2, an ISR kinase, across concentration series ranging from 0.2 nM to 10 pM.
[00131] FIG. 16 is a table demonstrating that HC-5404 is selective for PERK relative to four closely -related ISR kinases.
[00132] FIG. 17 is a Biochemical TreeSpot kinome panel assay demonstrating the selectivity of HC-5404 (100 nM, 1000 nM, 10,000 nM) against over 400 kinases. PERK is not included in the panel, and no interactions were observed when HC-5404 assayed at 100 nM.
[00133] FIG. 18 shows free drug levels of HC-5404 in plasma following single oral administration. Plasma sampled across a 24 h period and quantified by LC-MS/MS.
[00134] FIG. 19 shows PK/PD relationship of plasma exposure following oral administration of HC-5404 at 30 mg/kg and pPERK levels in mouse pancreas.
[00135] FIG. 20 shows PK/PD relationship of plasma exposure following oral administration of HC-5404 at 100 mg/kg and pPERK levels in mouse pancreas.
[00136] FIG. 21 shows PD effect of HC-5404 on pPERK in 786-0 tumors sampled 1, 4, 8, 12 h post last dose following 15 days of BID dosing. [00137] FIG. 22 is a graph showing the tumor volumes for 786-0 xenografts treated with HC-5404 at multiple dose levels and treatment regimens for 28 days. Values represent mean tumor volume ± SEM.
[00138] FIG. 23 is a graph showing the tumor volumes for mice harboring subcutaneous CAPAN- 2 pancreatic tumor xenografts treated with HC-5404 at 30 mg/kg and 100 mg/kg PO, BID for 48 days. Values represent mean tumor volume ± SEM.
[00139] FIG. 24 Mouse pancreas sections following three weeks of treatment with HC-5404 at either 30 mg/kg, PO, BID or 100 mg/kg PO, BID. The pancreas on the right was given a two-week washout period following treatment with 100 mg/kg PO BID, to demonstrate reversibility of the effect.
[00140] FIG. 25 is a graph demonstrating the pPERK/PERK protein ratio from 786-0 xenografts treated with sunitinib (40 mg/kg; PO; QD) for one or two weeks.
[00141] FIG. 26 A, D, G) 786-0 tumor xenografts treated with cabozantinib (15, 30, 45 mg/kg; PO; QD), lenvatinib (5 and 10 mg/kg; PO; QD), or axitinib (15 and 30 mg/kg; PO; BID) for 21 days. B, E, H) pPERK relative to total PERK were evaluated from tumor samples taken at the end of study, following 21 days of treatment. ANOVA statistical analysis compared differences between protein abundance. Bars highlight significant differences between groups, including p- values. C, F, I) Mouse body weights measured twice weekly across the course of treatment.
[00142] FIG. 27 shows a SimpleWestem protein analysis of pPERK and PERK in 786-0 xenografts treated with VEGFR-TKIs in panel B, sampled after seven days of treatment.
[00143] FIG. 28 is a graph demonstrating the tumor volume of 786-0 tumor xenografts treated with HC-5404 (30 mg/kg; BID), sunitinib (40 mg/kg; QD), lenvatinib (10 mg/kg; BID), axitinib (30 mg/kg; BID), or cabozantinib (30 mg/kg; QD) and combinations as indicated for 28 days.
[00144] FIG. 29 demonstrates that HC-5404 induces accumulation of ASNS, CBS, and CTH.
[00145] FIG. 30 shows pPERK and total PERK abundance evaluated by SimpleWestem in RCC tumor xenografts treated for seven days with HC-5404 (30 mg/kg; PO; BID), sunitinib (20 or 40 mg/kg as indicated; PO; QD), or a combination thereof.
[00146] FIG. 31 shows A498 RCC tumor xenografts treated with HC-5404 (30 mg/kg; PO; BID) and sunitinib (20 mg/kg; PO; QD) for 30 days.
[00147] FIG. 32 shows Caki-1 RCC tumor xenografts treated with HC-5404 (30 mg/kg; PO; BID) and sunitinib (20 mg/kg; PO; QD) for 30 days. [00148] FIG. 33 is a growth curve of a representative RCC PDX model across 28 days of treatment with either axitinib (30 mg/kg; PO; BID), HC-5404 (30 mg/kg; PO; BID), or a combination thereof.
[00149] FIG. 34 is a growth curve of a representative RCC PDX model across 28 days of treatment with either axitinib (30 mg/kg; PO; BID), HC-5404 (30 mg/kg; PO; BID), or a combination thereof.
[00150] FIG. 35 is a waterfall plot illustrating relative change in tumor volume of 15 diverse RCC PDX models following 28 days of treatment with either axitinib (30 mg/kg; PO; BID), HC-5404 (30 mg/kg; PO; BID), or a combination thereof. Models ranked on % change tumor volume in combination group. Progressive Disease (PD) = >30% increase from baseline; Stable Disease (SD) = <30% increase from baseline and <50% regression; Partial Response (PR) = >50% regression. Models that are VHL wild-type are indicated by an asterisk.
[00151] FIG. 36 shows the sensitivity across three RCC xenograft models of HC-5404 administered at 10 and 30 mg/kg, PO, BID for 28 days. 786-0 and A498 are VHL mutant models; Caki-1 VHL wild type.
[00152] FIG. 37 shows RCC tumor models treated with HC-5404 (30 mg/kg; PO; BID) and DC- 101 (15 mg/kg; IP; BIW) for 28 days. Shown here are mean tumor volumes ± SEM. B, D, F) Ratio of pPERK to total PERK across four treatment groups in three RCC models, evaluated by SimpleWestern protein analysis system. Note: low signal to noise ratio in Caki-1 model.
[00153] FIG. 38 shows IHC images of 786-0 xenograft sections stained with antibodies specific to Meca32, CD31, and SMA. Scale bar=150 pm.
[00154] FIG. 39 shows quantification of IHC staining of xenograft sections. Graphs indicate proportion of cells stained positive for Meca32+, positive for CD31 in absence of SMA (%CD31+SMA-), or cells that stain positive for both CD31 and SMA (%CD31+SMA+). One-way ANOVA statistical analysis evaluated differences between treatment groups; significant differences between treatment vs vehicle indicated by bars and p-values.
[00155] FIG. 40 shows 786-0 tumor growth across the study period. Xenografts that progressed in presence of axitinib (30 mg/kg; PO; BID) for 14 days were rerandomized and transferred to indicated treatment groups for an additional 28 days.
[00156] FIG. 41 shows IHC images of xenograft sections stained with antibodies specific for CD31 and SMA.
[00157] FIG. 42 shows quantification of proportion of cells that stained positive for SMA, CD31, or Meca32. One-way ANOVA was used to evaluate the statistical significance between groups. P- value indicated for significant differences between treatments and vehicle. Quantification of immunohistochemistry (IHC) staining of FFPE sections from 786-0 RCC tumor xenografts treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID), or combinations thereof. Quantification of % positive cells stained for CD31, a marker of the vascular endothelium.
[00158] FIG. 43 shows quantification of IHC staining of tumor sections using antibodies specific for the pericyte markers NG2 and MCAM. One-way ANOVA was used to evaluate the statistical significance between groups. P-value indicated for significant differences between treatment groups versus either baseline or vehicle. Quantification of immunohistochemistry (IHC) staining of FFPE sections from 786-0 RCC tumor xenografts treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID), or combinations thereof. Quantification of % positive cells stained with antibodies specific for NG2 and MCAM, two markers of pericyte cells.
[00159] FIG. 44 shows images and quantification of IHC staining of tumor sections using antibodies specific for the pericyte markers NG2 and MCAM. One-way ANOVA was used to evaluate the statistical significance between groups. P-value indicated for significant differences between treatment groups versus either baseline or vehicle.
[00160] FIG. 45 shows a tumor growth curve and pPERK/PERK levels relative to total PERK in 786-0 tumor xenografts treated with cabozantinib (15, 30, 45 mg/kg; PO; QD) for 21 days. ANOVA statistical analysis compared differences between protein abundance. Bars highlight significant differences between groups, including p-values.
[00161] FIG. 46 shows a tumor growth curve and pPERK/PERK levels relative to total PERK in 786-0 tumor xenografts treated with lenvatinib (5 and 10 mg/kg; PO; QD) for 21 days. ANOVA statistical analysis compared differences between protein abundance. Bars highlight significant differences between groups, including p-values.
[00162] FIG. 47 shows a tumor growth curve and pPERK/PERK levels relative to total PERK in 786-0 tumor xenografts treated with axitinib (15 and 30 mg/kg; PO; BID) for 21 days. ANOVA statistical analysis compared differences between protein abundance. Bars highlight significant differences between groups, including p-values.
[00163] FIG. 48 shows a tumor growth curve for 786-0 tumor xenografts treated with once daily oral administration of lenvatinib (10 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof. [00164] FIG. 49 shows a tumor growth curve for 786-0 tumor xenografts treated with twice daily oral administration of axitinib (30 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof.
[00165] FIG. 50 shows a tumor growth curve for A498 tumor xenografts treated with once daily oral administration of sunitinib (20 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof.
[00166] FIG. 51 shows a tumor growth curve for A498 tumor xenografts treated with twice weekly intraperitoneal injection of DC-101 (15 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof.
[00167] FIG. 52 shows a tumor growth curve for 786-0 tumor xenografts treated with twice daily oral administration of HC-5404 at 3, 10, or 30 mg/kg as a single agent or in combination with once daily oral dosing of cabozantinib at 30 mg/kg. HC-5404 at 10 or 30 mg/kg in combination with cabozantinib resulted in tumor regression -66%.
[00168] FIG. 53 shows a body mass curve for 786-0 tumor xenografts treated with twice daily oral administration of HC-5404 at 3, 10, or 30 mg/kg as a single agent or in combination with once daily oral dosing of cabozantinib at 30 mg/kg.
[00169] FIG. 54 shows images of IHC staining of CD31+SMA- cells treated with cabozantinib in combination with HC-5404.
[00170] FIG. 55 shows quantification of IHC staining of CD31+SMA- cells treated with cabozantinib in combination with HC-5404.
[00171] FIG. 56 shows images of IHC staining of pericyte cells treated with cabozantinib in combination with HC-5404.
[00172] FIG. 57 shows quantification of IHC staining of pericyte cells treated with cabozantinib in combination with HC-5404. Quantification of IHC staining of 786-0 RCC tumor xenografts treated with HC-5404 at 3, 10, or 30 mg/kg (PO; BID) as a single agent or in combination with cabozantinib (30 mg/kg; PO; QD).
[00173] FIG. 58 shows a tumor growth curve for RENCA (VHLwt) RCC tumor xenografts treated with HC-5404, axitinib, anti-PD-1, or a combination thereof.
[00174] FIG. 59 shows a body mass curve for RENCA (VHLwt) RCC tumor xenografts treated with HC-5404, axitinib, anti-PD-1, or a combination thereof. [00175] FIG. 60 shows graphs demonstrating the effect of HC-5404 in combination with axitinib on RENCA RCC model.
[00176] FIG. 61 shows graphs demonstrating the effect of HC-5404, axitinib, or HC-5404 in combination with axitinib on RENCA RCC model. Top left: Ml macrophages (CD1 lb+, F480+, MHCII+, CD206-) were quantified as a percentage of total F480+ macrophages. Top right: Monocytic MDSCs (CD1 lb+, Gr-1+, F480-, Ly6C+, Ly6G-) were quantified as a percentage of total CD45+ cells. Bottom left and right: The expression of the costimulatory and activation markers CD86 and MHCII on Ml macrophages was measured based on geometric mean fluorescence intensity (MFI). Graphs represent individual animals +/- SEM for each treatment group. Significance indicators are based on ordinary one-way ANOVA and Tukey’s multiple comparisons test.
[00177] FIG. 62 shows graphs demonstrating the effect of HC-5404, axitinib, or HC-5404 in combination with axitinib on CD4 T-Cells in tumor-draining lymph nodes. Top left: CD4 T-cells that expressed the activation marker CD69 were quantified as a % positive of total CD4 T-cells. Top right: Effector memory CD4 T-cells (CD4+, CD44+, CD62L-) in TDLN were quantified as a percentage of total CD4-cells. Middle left: TNFa+ CD4 T-cells were compared across treatment groups. Middle right; bottom left; bottom right: Multiple exhaustion markers were measured as % positive of total CD4 T-cells. Graphs represent individual animals +/- SD for each treatment group. Significance indicators are based on ordinary one-way ANOVA and Tukey’s multiple comparisons test.
[00178] FIG. 63 demonstrates the effect of HC-5404, axitinib, and HC-5404 in combination with axitinib on myeloid cells in tumor microenvironment in RENCA RCC tumors.
[00179] FIG. 64 demonstrates the effect of HC-5404, axitinib, and HC-5404 in combination with axitinib on myeloid cells in tumor microenvironment in RENCA RCC tumors. Fluorescence microscopy for the following markers on macrophages are shown: M2 marker CD206 (top left), and the Ml markers CD86 (top right) and PD-L1 (bottom). Values indicate the percentage of macrophages that express each marker. Dots represent individual animals, and error bars indicate the SD of each group. Significance indicators are based on ordinary one-way ANOVA and Tukey’s multiple comparisons test.
[00180] FIG. 65 demonstrates the effect of HC-5404, axitinib, aPD-1, or a combination of HC- 5404 with axitinib and aPD-1 on macrophages in RENCA RCC model. Macrophages with an Ml- like phenotype (MHCII+, CD206-) (left) or an M2 -like phenotype (MHCII-, CD206+) (right) were quantified as a percentage of total F480+ macrophages. Individual animals +/- SEM are displayed. Significance is based on a One-way ANOVA with Dunnett’s multiple comparisons.
[00181] FIG. 66 demonstrates the effect of HC-5404 in combination with axitinib and aPD-1 on Renca RCC model.
[00182] FIG. 67 demonstrates the effect of HC-5404, axitinib, aPD-1, and HC-5404 in combination with axitinib and aPD-1 on T-cell infiltration. Top left, top right: Frequency (% of total CD45+ cells) and cells/gram of CD8 T-cells in tumor tissue are displayed for different treatment groups. Bottom left, bottom right: Expression of the stimulatory cytokines ZFNy and TNFa was measured in CD8 T-cells via intracellular flow cytometry. Expression is displayed as a % positive of CD8 T-cells. Individual animals +/- SD are displayed. Significance is based on a One-way ANOVA with Dunnett’s multiple comparisons.
[00183] FIG. 68 demonstrates the effect of HC-5404 in combination with aPD-1 on myeloid cells in tumor microenvironment and T-cell activation. Tumor volume was measured over time and is displayed for each group (mean +/- SEM).
[00184] FIG. 69 demonstrates the effect of HC-5404 in combination with aPD-1 on myeloid cells in tumor microenvironment and T-cell activation in syngeneic MB49 model. Top (Ml Macs, M2 Macs, PMN-MDSCs): Expression of the type 1 interferon receptor (IFNAR1) was evaluated on Ml - and M2-like macrophages and MDSCs in tumor tissue via flow cytometry following 7 days of treatment. Expression levels are displayed as the geometric mean fluorescence intensity (MFI) for IFNAR1 on each cell type. Middle left (CD8 T-cells, NK cells): Tumor infiltrating CD8 T-cells and NK cells were evaluated via flow cytometry following 7 days of treatment. Cells/g of tissue were calculated for each group and are displayed. Middle right (In Draining LN: Increased DC): Dendritic cell (DC) frequency was measured in tumor-draining lymph nodes (TDLN) following 14 days of treatment. Values are expressed as a % of total CD45+ cells. Bottom (T-cell Activation: Ki67, GzmB, TCF1, CD69): CD4 or CD8 T-cells in TDLN were evaluated for proliferation and activation markers, including Ki67, Granzyme B, TCF1, and CD69. Expression levels are depicted as either % positive cells or MFI for each marker as indicated. Individual animals +/- SD are displayed. Significance is based on a One-way ANOVA with Dunnett’s multiple comparisons.
[00185] FIG. 70 shows the efficacy of HC-5404 on RT4 bladder cells.
[00186] FIG. 71 shows the efficacy of HC-5404 on HCT116 CRC cells.
[00187] FIG. 72 shows the efficacy of HC-5404 on A549 NSCLC cells.
[00188] FIG. 73 shows the efficacy of HC-5404 on A375 melanoma cells. [00189] FIG. 74 shows a tumor growth curve for MFE280 MSS model of endometrial cancer treated with twice daily oral administration of HC-5404 (30 mg/kg), once daily oral administration of lenvatinib (10 mg/kg), or a combination thereof.
[00190] FIG. 75 shows a tumor growth curve for GAO 114 PDX model of gastric cancer treated with HC-5404 and DC-101.
[00191] FIG. 76 shows a tumor growth curve for TM01125 PDX model of pancreatic cancer treated with HC-5404 and sunitinib.
[00192] FIG. 77 shows a tumor growth curve for MFE280 model of human endometrial cancer model treated with twice daily oral administration of HC-5404, once daily oral administration of lenvatinib (10 mg/kg), or a combination thereof.
[00193] FIG. 78 shows a body mass curve for MFE280 model of human endometrial cancer model treated with twice daily oral administration of HC-5404, once daily oral administration of lenvatinib (10 mg/kg), or a combination thereof.
[00194] FIG. 79 demonstrates the effect of HC-5404 in combination with lenvatinib on MFE280 human endometrial cancer model. IHC Images and quantification of FFPE sections from MFE280 tumors treated with HC-5404 (39 mg/kg; PO; BID), lenvatinib (10 mg/kg; PO; QD), or a combination thereof for 7 days. Tumors were sampled at 2 h following the morning dose.
[00195] FIG. 80 demonstrates the effect of HC-5404 in combination with envatinib on MFE280 human endometrial cancer model. IHC Images and quantification of FFPE sections from MFE280 tumors treated with HC-5404 (39 mg/kg; PO; BID), lenvatinib (10 mg/kg; PO; QD), or a combination thereof for 7 days. Tumors were sampled at 2 h following the morning dose.
[00196] FIG. 81 demonstrates the effect of HC-5404 in combination with DC-101 in gastric PDX model. SimpleWestem protein analysis (JESS) of pPERK, PERK, and five proteins involved in amino acid metabolism: asparagine synthetase (ASNS), phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), phosphoserine phosphatase (PSPH), serine hy droxymethyltransf erase 1 (SHMT1). Gastric cancer patient-derived xenograft (PDX) model GA0114-R19P6 treated with HC-5404 (30 mg/kg; PO; BID), DC101 (20 mg/kg; IP; BIW), or a combination thereof for seven days and sampled 1 h after final morning dose. These proteins can serve a biomarkers of HC-5404 activity in vivo.
[00197] FIG. 82 demonstrates the effect of HC-5404 in combination with DC-101 in gastric PDX model. Quantification of SimpleWestem signal represented in panel FIG. 81. [00198] FIG. 83 is a tumor growth curve for CAPAN2 PDAC model treated with HC-5404 in combination with gemcitabine.
[00199] FIG. 84 is a tumor growth curve for MB49 bladder cancer model treated with HC-5404 at 3, 10, 30 mg/kg PO BID in combination with anti-PDl (IP; BIW) antibody for 14 days.
[00200] FIG. 85 is a tumor growth curve for NCI-H660 NEPC model treated with HC-5404 in combination with docetaxel.
[00201] FIG. 86 shows plasma analysis from mice bearing 786-0 tumors, treated with HC-5404 at 3, 10, or 30 mg/kg (PO; BID) as a single agent or in combination with cabozantinib (30 mg/kg; PO; QD). Vascular endothelial growth factor-A (VEGF-A) was decreased in plasma by HC-5404 as a single agent in a dose-dependent fashion.
[00202] FIG. 87 shows plasma analysis from mice bearing 786-0 tumors, treated with HC-5404 at 3, 10, or 30 mg/kg (PO; BID) as a single agent or in combination with cabozantinib (30 mg/kg; PO; QD). Hepatocyte growth factor (HGF) was decreased by the combination of HC-5404 and cabozantinib in mouse plasma.
[00203] FIG. 88 shows plasma analysis from mice bearing 786-0 tumors, treated with HC-5404 at 3, 10, or 30 mg/kg (PO; BID) as a single agent or in combination with cabozantinib (30 mg/kg; PO; QD). Interleukin-8 (IL-8) was decreased by the combination of HC-5404 and cabozantinib in mouse plasma.
[00204] FIG. 89 shows plasma analysis from mice bearing 786-0 tumors, treated with HC-5404 at 3, 10, or 30 mg/kg (PO; BID) as a single agent or in combination with cabozantinib (30 mg/kg; PO; QD). Lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) was decreased by HC-5404 in a dose-dependent manner as either monotherapy or in combination with cabozantinib.
[00205] FIG. 90 shows plasma analysis from mice bearing 786-0 tumors, treated with HC-5404 at 3, 10, or 30 mg/kg (PO; BID) as a single agent or in combination with cabozantinib (30 mg/kg; PO; QD). Syndecan was decreased by HC-5404 as monotherapy or in combination with cabozantinib.
[00206] FIG. 91 is a tumor growth curve for mice harboring 786-0 xenografts that were treated for 14 days with axitinib (30 mg/kg; PO; BID), following which the mice with tumors that progressed (approximately doubled in size) were rerandomized into four groups. After enabling 786-0 xenografts to progress on axitinib for two weeks, HC-5404 was added to the regimen. Data presented are mean ± SEM. [00207] FIG. 92 shows quantification of IHC images from FFPE tumor sections sampled 7 days after treating mice harboring 786-0 xenografts for 14 days with axitinib (30 mg/kg; PO; BID), randomizing the mice with tumors that progressed (approximately doubled in size), and then treating with HC-5404. Top left: HC-5404 and axitinib both decreased the proportion of smooth muscle actin (SMA) as monotherapy, which was further diminished by the combination treatment. Top right: Proportion of cells expressing Meca32 was decreased in the combination group relative to vehicle. Meca32 is a marker of vascular endothelial cells. Bottom: Proportion of cells expressing the vascular marker CD31 is decreased by axitinib, an effect that is enhanced by addition of HC-5404 in the combination group.
[00208] FIG. 93 shows quantification of IHC images from FFPE tumor sections sampled 7 days after treating mice harboring 786-0 xenografts for 14 days with axitinib (30 mg/kg; PO; BID), randomizing the mice with tumors that progressed (approximately doubled in size), and then treating with HC-5404. Tumors randomized into the combination group have decreased proportion of cells expressing the pericyte markers NG2 and MCAM.
[00209] FIG. 94 is a tumor growth curve for 786-0 RCC tumor models treated with HC-5404 (30 mg/kg; PO; BID) and DC-101 (15 mg/kg; IP; BIW) for 28 days. Mean tumor volumes ± SEM.
[00210] FIG. 95 is a tumor growth curve for A498 RCC tumor models treated with HC-5404 (30 mg/kg; PO; BID) and DC-101 (15 mg/kg; IP; BIW) for 28 days. Mean tumor volumes ± SEM.
[00211] FIG. 96 is a tumor growth curve for RENCA RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID), or a combination thereof.
[00212] FIG. 97 is a tumor growth curve for RXF-393 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
[00213] FIG. 98 is a tumor growth curve for RXF-2282 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
[00214] FIG. 99 is a tumor growth curve for RXF-2178 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
[00215] FIG. 100 is a tumor growth curve for RXF-2502 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
[00216] FIG. 101 is a tumor growth curve for SMTCA75 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof. [00217] FIG. 102 is a tumor growth curve for RXF-616 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
[00218] FIG. 103 is a tumor growth curve for RXF-2304 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
[00219] FIG. 104 is a tumor growth curve for RXF-488 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
[00220] FIG. 105 is a tumor growth curve for RXF-2667 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
[00221] FIG. 106 is a tumor growth curve for RXF-1220 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
[00222] FIG. 107 is a tumor growth curve for RXF-2783 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
[00223] FIG. 108 is a tumor growth curve for RXF-631 RCC tumor model treated with HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
DETAILED DESCRIPTION
[00224] As generally described herein, the present disclosure provides methods of treating a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof. The present disclosure also provides methods of activating the immune system of a subject in need thereof (e.g., a subject having cancer). The methods described herein generally comprise administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy (e.g., an anti-PD-1 antibody).
[00225] The present disclosure also provides methods of treating a cancer (e.g., a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer), and a thyroid cancer) in a subject in need thereof. The methods described herein generally comprise administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent. Also disclosed herein are methods comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent, and a therapeutically effective amount of an immunotherapy.
Definitions
[00226] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.
[00227] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.
[00228] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
[00229] In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.
[00230] Further, it should be understood that elements and/or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present invention and/or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.
[00231] The articles “a” and “an” are used in this disclosure to refer to one or more than one (i.e., to at least one) of the grammatical object of the article, unless the context is inappropriate. By way of example, “an element” means one element or more than one element.
[00232] The term “and/or” is used in this disclosure to mean either “and” or “or” unless indicated otherwise.
[00233] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and/or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.
[00234] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.
[00235] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred from the context.
[00236] At various places in the present specification, variables or parameters are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual subcombination of the members of such groups and ranges. For example, an integer in the range of 0 to 40 is specifically intended to individually disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40, and an integer in the range of 1 to 20 is specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.
[00237] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.
[00238] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.
[00239] As used herein, “pharmaceutical composition” or “pharmaceutical formulation” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.
[00240] “Pharmaceutically acceptable” means approved or approvable by a regulatory agency of the federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly, in humans.
[00241] As used herein, “pharmaceutically acceptable salt” refers to any salt of an acidic or a basic group that may be present in a compound of the present invention (e.g., a compound of formula (I)), which salt is compatible with pharmaceutical administration.
[00242] As is known to those of skill in the art, “salts” of compounds may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene- -sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acid. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid addition salts.
[00243] Examples of bases include, but are not limited to, alkali metal (e.g., sodium and potassium) hydroxides, alkaline earth metal (e.g., magnesium and calcium) hydroxides, ammonia, and compounds of formula NW4+, wherein W is Ci-4 alkyl, and the like.
[00244] Examples of salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemifumarate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na+, K+, Ca2+, NH4+, and NW4+ (where W can be a Ci-4 alkyl group), and the like.
[00245] For therapeutic use, salts of the compounds of the present invention (e.g., a compound of formula (I)) are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.
[00246] As used herein, “pharmaceutically acceptable excipient” refers to a substance that aids the administration of an active agent to and/or absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, such as a phosphate buffered saline solution, emulsions (e.g., such as an oil/water or water/oil emulsions), lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer’s solution), alcohols, oils, gelatins, carbohydrates, fatty acid esters, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention. For examples of excipients, see Martin, Remington’s Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).
[00247] A “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and/or non-human animals, e.g., mammals such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and/or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is an adult human. In certain embodiments, the subject is a non-human animal.
[00248] As used herein, “solid dosage form” means a pharmaceutical dose(s) in solid form, e.g., tablets, capsules, granules, powders, sachets, reconstitutable powders, dry powder inhalers and chewables.
[00249] As used herein, “administering” means oral administration, administration as a suppository, topical contact, intravenous administration, parenteral administration, intraperitoneal administration, intramuscular administration, intralesional administration, intrathecal administration, intracranial administration, intranasal administration or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “co-administer” it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies (e.g., anti-cancer agent, chemotherapeutic, or immunotherapy). The compound of formula (I), or a pharmaceutically acceptable salt thereof, can be administered alone or can be co-administered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compound individually or in combination (more than one compound or agent). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation).
[00250] As used herein, “fasting state” means at least 1 hour before food or at least 2 hours after food is consumed by a subject.
[00251] The terms “disease,” “disorder,” and “condition” are used interchangeably herein.
[00252] As used herein, and unless otherwise specified, the terms “treat,” “treating,” and “treatment” contemplate an action that occurs while a subject is suffering from the specified disease, disorder or condition, which reduces the severity of the disease, disorder or condition, or retards or slows the progression of the disease, disorder or condition (e.g., “therapeutic treatment”).
[00253] In general, an “effective amount” or a “therapeutically effective amount” of a compound (e.g., a compound of formula (I), or a pharmaceutically acceptable salt thereof) refers to an amount sufficient to elicit the desired biological response, e.g., to treat a cancer or activate the immune system in a subject in need thereof. As will be appreciated by those of ordinary skill in this art, the therapeutically effective amount of a compound of the disclosure may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, weight, health, and condition of the subject.
[00254] As used herein, the term “activating” refers to stimulation of an immune cell (e.g., a T cell, NK cell, B cell, dendritic cell, or macrophage) that results in cellular proliferation, maturation, cytokine production and/or induction of regulatory or effector functions. Methods of evaluating immune cell activation or function are well known in the art and include, but are not limited to, proliferation assays such as BRDU and thymidine incorporation, cytotoxicity assays such as chromium release, cytokine secretion assays such as intracellular cytokine staining ELISPOT and ELISA, and expression assays of activation markers such as CD25 and CD69 using flow cytometry and multimer (e.g., tetramer) assays.
[00255] As used herein, the term “myeloid derived suppressor cell” or “MDSC” refers to a collection of immature myeloid cells that modulate the activity of a variety of effector cells and antigen-presenting cells, such as T cells, NK cells, dendritic cells, and macrophages, among others. Myeloid derived suppressor cells are distinguished by their gene expression profile, and express all or a subset of proteins and small molecules selected from the group consisting of B7-1 (CD80), B7- H1 (PD-L1 ), CCR2, CDld, CDldl , CD2, CD31 (PECAM-1), CD43, CD44, complement component C5aRl , F4/80 (EMR1), Fey RIII (CD16), Fey RII (CD32), Fey RIIA (CD32a), Fey RUB (CD32b), Fey RIIB/C (CD32b/c), Fey RIIC (CD32c), Fey RIIIA (CD16A), Fey RIIIB (CD16b), galectin-3, GP130, Gr-1 (Ly-6G), ICAM-1 (CD54), IL-1RI, IL-4Ra, IL- 6Ra, integrin aL (CD49d), integrin aL (CD1 la), integrin aM (CD1 lb), M-CSFR, MGL1 (CD301a), MGL1/2 (CD301a/b), MGL2 (CD301b), nitric oxide, PSGL-1 (CD162), L-selectin (CD62L), siglec-3 (CD33), transferrin receptor (TfR), VEGFR1 (Fit- 1 ), and VEGFR2 (KDR or Flk-1). Particularly, MDSCs do not express proteins selected from the group consisting of B7-2 (CD86), B7-H4, CDl lc, CD14, CD21 , CD23 (FCERII), CD34, CD35, CD40 (TNFRSF5), CD117 (c-kit), HLA-DR, and Sca-1 (Ly6).
[00256] As used herein, the phrase “tumor-infiltrating lymphocytes” refers to white blood cells (e.g., T cells and/or NK cells) of a subject afflicted with a cancer (e.g., a cancer described herein), that are resident in a tumor or otherwise have left the circulation (blood or lymphatic fluid) and have migrated into a tumor.
[00257] As used herein, the term “immunotherapy” refers to a treatment of disease by inducing, enhancing, or suppressing an immune response. Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress an immune response are classified as suppression immunotherapies.
[00258] As used herein, the phrase “immune checkpoint inhibitor” refers to an agent that blocks certain proteins made by some types of immune system cells, such as T cells, and some cancer cells. These proteins help keep immune responses in check and can prevent T cells from killing cancer cells. When these proteins are blocked, immune system function is restored and the immune system is released enabling T cells to kill cancer cells. [00259] As used herein, the term “anti angiogenic agent” refers an agent that modulates (e.g., inhibits) angiogenesis, vasculogenesis, or vascular permeability. Anti angiogenic agents include agents that block the angiogenic activity of the angiogenic factor or its receptor.
[00260] As used herein, the term “antibody” refers to a functional component of serum and is often referred to either as a collection of molecules (antibodies or immunoglobulins) or as one molecule (the antibody molecule or immunoglobulin molecule). An antibody is capable of binding to or reacting with a specific antigenic determinant (the antigen or the antigenic epitope), which in turn may lead to induction of immunological effector mechanisms. An individual antibody is usually regarded as monospecific, and a composition of antibodies may be monoclonal (i.e., consisting of identical antibody molecules) or polyclonal (i.e., consisting of two or more different antibodies reacting with the same or different epitopes on the same antigen or on distinct, different antigens). Each antibody has a unique structure that enables it to bind specifically to its corresponding antigen, and all natural antibodies have the same overall basic structure of two identical light chains and two identical heavy chains. Antibodies are also known collectively as immunoglobulins. An antibody may be of human or non-human (for example, rodent such as murine, dog, camel, etc.) origin (e.g., may have a sequence originally developed in a human or non-human cell or organism), or may be or comprise a chimeric, humanized, reshaped, or reformatted antibody based, e.g., on a such a human or non-human antibody (or, in some embodiments, on an antigen-binding portion thereof).
[00261] As used herein, “biosimilar” is used to describe a biological agent that is highly similar to a reference biological agent. The term is generally used to describe a subsequent version of a biological agent (e.g., from a different source) and is unlikely to be exactly the same as the reference biological agent.
[00262] As used herein, the term “antagonist” refers to an agent that (i) decreases or suppresses one or more effects of another agent; and/or (ii) decreases or suppresses one or more biological events. In some embodiments, an antagonist may reduce the level and/or activity of one or more agents that it targets. In some embodiments, an antagonist may be a receptor antagonist, e.g., a receptor ligand or drug that blocks or dampens a biological response by binding to and blocking a receptor rather than activating it like an agonist.
[00263] As used herein, the term “agonist” refers to an agent that (i) increases or induces one or more effects of another agent; and/or (ii) increases or induces one or more biological events. In some embodiments, an agonist may increase the level and/or activity of one or more agents that it targets. PERK Inhibitors
[00264] The compound of formula (I), as depicted below, is a selective PERK inhibitor, and is also known as (A)-2-amino-5-(4-(2-(3,5-difluorophenyl)-2-hydroxyacetamido)-2-methylphenyl)-N- i sopropy Ini cotinami de :
Figure imgf000036_0001
[00265] The compound of formula (I) may also be referred to as HC-5404 throughout the present disclosure. The hemifumarate salt of the compound of formula (I) may be referred to as HC-5404- FU throughout the present disclosure. A method of chemically synthesizing the compound of formula (I) and its hemifumarate salt is described in Example 1.
[00266] In one aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00267] In another aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00268] In another aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD-1 antibody for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00269] In another aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD-1 antibody for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00270] In another aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
[00271] In another aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD-1 antibody for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
[00272] In various embodiments, provided herein is a method of administering a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00273] In various embodiments, provided herein is a method of administering a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00274] In various embodiments, provided herein is a method of administering a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and a therapeutically effective amount of an anti-PD-1 antibody for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00275] In various embodiments, provided herein is a method of administering a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and a therapeutically effective amount of an anti-PD-1 antibody for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00276] In various embodiments, provided herein is a method of administering a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
[00277] In various embodiments, provided herein is a method of administering a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and a therapeutically effective amount of an anti-PD-1 antibody for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
[00278] In certain embodiments, the immunotherapy is a PD-1 or PD-L1 inhibitor. In certain embodiment, the immunotherapy is an anti-PD-1 antibody. [00279] In another aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer (e.g., malignant melanoma)), and a thyroid cancer) in a subject in need thereof.
[00280] In another aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer), and a thyroid cancer) in a subject in need thereof.
[00281] In another aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer (e.g., malignant melanoma)), and a thyroid cancer) in a subject in need thereof, wherein the subject has been treated with an anti-cancer therapy.
[00282] In another aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer), and a thyroid cancer) in a subject in need thereof, wherein the subject has been treated with an anti -cancer therapy.
[00283] In another aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer (e.g., malignant melanoma)), and a thyroid cancer) in a subject receiving a therapeutically effective amount of an anti angiogenic agent therapy. [00284] In another aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer), and a thyroid cancer) in a subject receiving a therapeutically effective amount of an anti angiogenic agent therapy.
[00285] In various embodiment, provided herein the method further comprises administering to the subject a therapeutically effective amount of an immunotherapy (e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody).
[00286] In one aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)), and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti- CTLA-4 antibody) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer (e.g., malignant melanoma)), and a thyroid cancer) in a subject in need thereof.
[00287] In one aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)), and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti- CTLA-4 antibody) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer), and a thyroid cancer) in a subject in need thereof.
[00288] In another aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)), and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti- CTLA-4 antibody) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer (e.g., malignant melanoma)), and a thyroid cancer) in a subject in need thereof, wherein the subject has been treated with an anti-cancer therapy.
[00289] In another aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)), and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti- CTLA-4 antibody) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer), and a thyroid cancer) in a subject in need thereof, wherein the subject has been treated with an anti-cancer therapy.
[00290] In another aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer (e.g., malignant melanoma)), and a thyroid cancer) in a subject receiving a therapeutically effective amount of an anti angiogenic agent therapy.
[00291] In another aspect, provided herein is a method of administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer), and a thyroid cancer) in a subject receiving a therapeutically effective amount of an anti angiogenic agent therapy.
[00292] In certain embodiments, the pharmaceutically acceptable salt of the compound of formula (I) is a fumarate salt. In certain embodiments, the pharmaceutically acceptable salt of the compound of formula (I) is a hemifumarate salt.
Antiangiogenic Agents
[00293] Antiangiogenic agents are limited by primary and secondary resistance mechanisms. As part of their mechanism, VEGFR-TKIs induce hypoxia and nutrient-deprivation that drives ER stress. Tumors evade deleterious ER stress by activating the PERK branch of the integrated stress response, which arrests global translation and restores protein homeostasis.
[00294] In some embodiments, an antiangiogenic agent is an antibody or a biosimilar thereof. In some embodiments, an antiangiogenic agent is a small molecule or a pharmaceutically acceptable salt thereof. In some embodiments, an antiangiogenic agent is a vascular endothelial growth factor receptor (VEGFR) modulator. In some embodiments, an antiangiogenic agent is a vascular endothelial growth factor (VEGF) targeting antibody. In some embodiments, an antiangiogenic agent is a tyrosine kinase inhibitor (TKI). In some embodiments, an antiangiogenic agent is a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI).
[00295] Examples of antiangiogenic agents include, but are not limited to, apatinib, axitinib, brivanib, cabozantinib, canertinib, cediranib, dasatinib, erlotinib, gefitinib, leflunomide, 42envatinib, motesanib, nilotinib, nintedanib, pazopanib, regorafenib, semaxinib, sorafenib, sunitinib, tivozanib, vandetanib, vatalanib, XL0192, bevacizumab, ramucirumab, and pharmaceutically acceptable salts or biosimilars thereof.
Pharmaceutical Compositions
[00296] Provided herein are pharmaceutical compositions generally comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.
[00297] In one aspect, provided herein is a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00298] In another aspect, provided herein is a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00299] In another aspect, provided herein is a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an anti-PD-1 antibody for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00300] In another aspect, provided herein is a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an anti-PD-1 antibody for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00301] In another aspect, provided herein is a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
[00302] In another aspect, provided herein is a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an anti-PD-1 antibody for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
[00303] In another aspect, provided herein is a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor), for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, and urothelial carcinoma) in a subject in need thereof.
[00304] In another aspect, provided herein is a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor), for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, and urothelial carcinoma) in a subject in need thereof.
[00305] In another aspect, provided herein is a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients and a therapeutically effective amount of an anti-PD-1 antibody, for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00306] In another aspect, provided herein is a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients and a therapeutically effective amount of an anti-PD-1 antibody, for the treatment of a cancer (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00307] In another aspect, provided herein is a method of administering a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor) for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
[00308] In another aspect, provided herein is a method of administering a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients, and a therapeutically effective amount of an anti-PD-1 antibody for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
[00309] In another aspect, provided herein is a pharmaceutical composition for treating a cancer (e.g., a cancer described herein), wherein the pharmaceutical composition comprises a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and wherein the pharmaceutical composition is to be administered with a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor).
[00310] In another aspect, provided herein is a pharmaceutical composition for treating a cancer (e.g., a cancer described herein), wherein the pharmaceutical composition comprises a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and wherein the pharmaceutical composition is to be administered with a therapeutically effective amount of an anti-PD-1 antibody. [00311] In certain embodiments, the immunotherapy is an inhibitor of PD-1 or PD-L1. In certain embodiments, the immunotherapy is an anti-PD-1 antibody.
[00312] In various embodiments, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.
[00313] In various embodiments, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients.
[00314] In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 22 mg to about 226 mg, about 35 mg to about 226 mg, about 45 mg to about 226 mg, about 55 mg to about 226 mg, about 65 mg to about 226 mg, about 75 mg to about 226 mg, about 85 mg to about 226 mg, about 95 mg to about 226 mg, about 105 mg to about 226 mg, about 115 mg to about 226 mg, about 125 mg to 226 200 mg, about 135 mg to about 226 mg, about 145 mg to about 226 mg, about 155 mg to about 226 mg, about 165 mg to about 226 mg, about 175 mg to about 226 mg, about 185 mg to about 226 mg, about 195 mg to about 226 mg, about 22 mg to about 190 mg, about 22 mg to about 180 mg, about 22 mg to about 170 mg, about 22 mg to about 160 mg, about 22 mg to about 150 mg, about 22 mg to about 140 mg, about 22 mg to about 130 mg, about 22 mg to about 120 mg, about 22 mg to about 110 mg, about 22 mg to about 100 mg, about 22 mg to about 90 mg, about 22 mg to about 80 mg, about 22 mg to about 70 mg, about 22 mg to about 60 mg, about 22 mg to about 50 mg, about 22 mg to about 40 mg, about 22 mg to about 30 mg, about 35 mg to about 185 mg, about 45 mg to about 175 mg, about 55 mg to about 165 mg, about 65 mg to about 155 mg, about 75 mg to about 145 mg, about 85 mg to about 135 mg, about 95 mg to about 125 mg, or about 105 mg to about 115 mg.
[00315] In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 177 mg to about 900 mg, about 250 mg to about 900 mg, about 300 mg to about 900 mg, about 350 mg to about 900 mg, about 400 mg to about 900 mg, about 450 mg to about 900 mg, about 500 mg to about 900 mg, about 550 mg to about 900 mg, about 600 mg to about 900 mg, about 650 mg to about 900 mg, about 700 mg to about 900 mg, about 750 mg to about 900 mg, about 800 mg to about 900 mg, about 850 mg to about 900 mg, about 177 mg to about 850 mg, about 177 mg to about 800 mg, about 177 mg to about 750 mg, about 177 mg to about 700 mg, about 177 mg to about 650 mg, about 177 mg to about 600 mg, about 177 mg to about 550 mg, about 177 mg to about 500 mg, about 177 mg to about 450 mg, about 177 mg to about 400 mg, about 177 mg to about 350 mg, about 177 mg to about 300 mg, about 177 mg to about 250 mg, about 250 mg to about 850 mg, about 300 mg to about 800 mg, about 350 mg to about 750 mg, about 400 mg to about 700 mg, about 450 mg to about 650 mg, about 500 mg to about 600 mg, or about 550 mg to about 600 mg.
[00316] In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 22 mg to about 451 mg, about 50 mg to about 451 mg, about 75 mg to about 451 mg, about 100 mg to about 451 mg, about 125 mg to about 451 mg, about 150 mg to about 451 mg, about 175 mg to about 451 mg, about 200 mg to about 451 mg, about 225 mg to about 451 mg, about 250 mg to about 451 mg, about 275 mg to about 451 mg, about 300 mg to about 451 mg, about 325 mg to about 451 mg, about 350 mg to about 451 mg, about 375 mg to about 451 mg, about 22 mg to about 375 mg, about 22 mg to about 350 mg, about 22 mg to about 325 mg, about 22 mg to about 300 mg, about 22 mg to about 275 mg, about 22 mg to about 250 mg, about 22 mg to about 225 mg, about 22 mg to about 200 mg, about 22 mg to about 175 mg, about 22 mg to about 150 mg, about 22 mg to about 125 mg, about 22 mg to about 100 mg, about 22 mg to about 75 mg, about 22 mg to about 50 mg, about 50 mg to about 375 mg, about 75 mg to about 350 mg, about 100 mg to about 325 mg, about 125 mg to about 300 mg, about 150 mg to about 275 mg, about 175 mg to about 250 mg, about 200 mg to about 225 mg, about 50 mg to about 100 mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250 mg, about 250 mg to about 300 mg, about 300 mg to about 350 mg, or about 350 mg to about 451 mg.
[00317] In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 22 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about 650 mg, about 660 mg, about
670 mg, about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about 750 mg, about 760 mg, about 770 mg, about 780 mg, about 790 mg, about 800 mg, about 810 mg, about 820 mg, about 830 mg, about 840 mg, about 850 mg, about 860 mg, about
870 mg, about 880 mg, about 890 mg, or about 900 mg.
[00318] In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 22 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 50 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 75 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 100 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 125 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 150 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 175 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 200 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 250 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 300 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 350 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 400 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 450 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 500 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 550 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 600 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 650 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 700 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 750 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 800 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 850 mg. In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 900 mg.
[00319] In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 22 mg to about 226 mg, about 35 mg to about 226 mg, about 45 mg to about 226 mg, about 55 mg to about 226 mg, about 65 mg to about 226 mg, about 75 mg to about 226 mg, about 85 mg to about 226 mg, about 95 mg to about 226 mg, about 105 mg to about 226 mg, about 115 mg to about 226 mg, about 125 mg to about 226 mg, about 135 mg to about 226 mg, about 145 mg to about 226 mg, about 155 mg to about 226 mg, about 165 mg to about 226 mg, about 175 mg to about 226 mg, about 185 mg to about 226 mg, about 195 mg to about 226 mg, about 22 mg to about 190 mg, about 22 mg to about 180 mg, about 22 mg to about 170 mg, about 22 mg to about 160 mg, about 22 mg to about 150 mg, about 22 mg to about 140 mg, about 22 mg to about 130 mg, about 22 mg to about 120 mg, about 22 mg to about 110 mg, about 22 mg to about 100 mg, about 22 mg to about 90 mg, about 22 mg to about 80 mg, about 22 mg to about 70 mg, about 22 mg to about 60 mg, about 22 mg to about 50 mg, about 22 mg to about 40 mg, about 22 mg to about 30 mg, about 35 mg to about 185 mg, about 45 mg to about 175 mg, about 55 mg to about 165 mg, about 65 mg to about 155 mg, about 75 mg to about 145 mg, about 85 mg to about 135 mg, about 95 mg to about 125 mg, or about 105 mg to about 115 mg.
[00320] In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 177 mg to about 900 mg, about 250 mg to about 900 mg, about 300 mg to about 900 mg, about 350 mg to about 900 mg, about 400 mg to about 900 mg, about 450 mg to about 900 mg, about 500 mg to about 900 mg, about 550 mg to about 900 mg, about 600 mg to about 900 mg, about 650 mg to about 900 mg, about 700 mg to about 900 mg, about 750 mg to about 900 mg, about 800 mg to about 900 mg, about 850 mg to about 900 mg, about 177 mg to about 850 mg, about 177 mg to about 800 mg, about 177 mg to about 750 mg, about 177 mg to about 700 mg, about 177 mg to about 650 mg, about 177 mg to about 600 mg, about 177 mg to about 550 mg, about 177 mg to about 500 mg, about 177 mg to about 450 mg, about 177 mg to about 400 mg, about 177 mg to about 350 mg, about 177 mg to about 300 mg, about 177 mg to about 250 mg, about 250 mg to about 850 mg, about 300 mg to about 800 mg, about 350 mg to about 750 mg, about 400 mg to about 700 mg, about 450 mg to about 650 mg, about 500 mg to about 600 mg, or about 550 mg to about 600 mg.
[00321] In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 22 mg to about 451 mg, about 50 mg to about 451 mg, about 75 mg to about 451 mg, about 100 mg to about 451 mg, about 125 mg to about 451 mg, about 150 mg to about 451 mg, about 175 mg to about 451 mg, about 200 mg to about 451 mg, about 225 mg to about 451 mg, about 250 mg to about 451 mg, about 275 mg to about 451 mg, about 300 mg to about 451 mg, about 325 mg to about 451 mg, about 350 mg to about 451 mg, about 375 mg to about 451 mg, about 22 mg to about 375 mg, about 22 mg to about 350 mg, about 22 mg to about 325 mg, about 22 mg to about 300 mg, about 22 mg to about 275 mg, about 22 mg to about 250 mg, about 22 mg to about 225 mg, about 22 mg to about 200 mg, about 22 mg to about 175 mg, about 22 mg to about 150 mg, about 22 mg to about 125 mg, about 22 mg to about 100 mg, about 22 mg to about 75 mg, about 22 mg to about 50 mg, about 50 mg to about 375 mg, about 75 mg to about 350 mg, about 100 mg to about 325 mg, about 125 mg to about 300 mg, about 150 mg to about 275 mg, about 175 mg to about 250 mg, about 200 mg to about 225 mg, about 50 mg to about 100 mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250 mg, about 250 mg to about 300 mg, about 300 mg to about 350 mg, or about 350 mg to about 451 mg.
[00322] In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 22 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about 650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about 750 mg, about 760 mg, about 770 mg, about 780 mg, about 790 mg, about 800 mg, about 810 mg, about 820 mg, about 830 mg, about 840 mg, about 850 mg, about 860 mg, about 870 mg, about 880 mg, about 890 mg, or about 900 mg.
[00323] In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 22 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 50 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 75 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 100 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 125 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 150 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 175 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 200 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 250 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 300 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 350 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 400 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 450 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 500 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 550 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 600 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 650 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 700 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 750 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 800 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 850 mg. In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in a pharmaceutical composition described herein is about 900 mg.
[00324] In various embodiments, provided herein are pharmaceutical compositions comprising:
(i) about 22 mg to about 451 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof,; and
(ii) one or more pharmaceutically acceptable excipients.
[00325] In various embodiments, provided herein are pharmaceutical compositions comprising:
(i) about 177 mg to about 900 mg of a pharmaceutically acceptable salt of a compound of formula (I); and
(ii) one or more pharmaceutically acceptable excipients.
[00326] In another aspect, provided herein are pharmaceutical compositions comprising about 22 mg to about 177 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00327] In another aspect, provided herein are pharmaceutical compositions comprising about 22 mg to about 177 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00328] In another aspect, provided herein are pharmaceutical compositions comprising about 177 mg to about 900 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00329] In another aspect, provided herein are pharmaceutical compositions comprising about 177 mg to about 900 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00330] In another aspect, provided herein are pharmaceutical compositions comprising about 22 mg to about 226 mg of a pharmaceutically acceptable salt of a compound of formula (I), and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, M cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00331] In another aspect, provided herein are pharmaceutical compositions comprising about 22 mg to about 226 mg of a pharmaceutically acceptable salt of a compound of formula (I), and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, M cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00332] In another aspect, provided herein are pharmaceutical compositions comprising about 177 mg to about 900 mg of a pharmaceutically acceptable salt of a compound of formula (I), and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, malignant melanoma, M cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00333] In another aspect, provided herein are pharmaceutical compositions comprising about 177 mg to about 900 mg of a pharmaceutically acceptable salt of a compound of formula (I), and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a solid tumor, which, in some embodiments, is selected from the group consisting of squamous cell head and neck cancer, M cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer) in a subject in need thereof.
[00334] In another aspect, provided herein are pharmaceutical compositions comprising about 22 mg to about 226 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
[00335] In another aspect, provided herein are pharmaceutical compositions comprising about 177 mg to about 900 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
[00336] In another aspect, provided herein are pharmaceutical compositions comprising about 22 mg to about 226 mg of a pharmaceutically acceptable salt of a compound of formula (I), and one or more pharmaceutically acceptable excipients, for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
[00337] In another aspect, provided herein are pharmaceutical compositions comprising about 177 mg to about 900 mg of a pharmaceutically acceptable salt of a compound of formula (I), and one or more pharmaceutically acceptable excipients, for activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
[00338] In various embodiments, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of a compound of formula (I), a therapeutically effective amount of an anti angiogenic agent, and one or more pharmaceutically acceptable excipients.
[00339] In certain embodiments, the amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition described herein is about 22 mg to about 798 mg, about 35 mg to about 798 mg, about 44 mg to about 798 mg, about 66 mg to about 798 mg, about 88 mg to about 798 mg, about 110 mg to about 798 mg, about 113 mg to about 798 mg, about 155 mg to about 798 mg, about 177 mg to about 798 mg, about 199 mg to about 798 mg, about 221 mg to 798 mg, about 244 mg to about 798 mg, about 266 mg to about 798 mg, about 310 mg to about 798 mg, about 355 mg to about 798 mg, about 399 mg to about 798 mg, about 443 mg to about 798 mg, about 488 mg to about 798 mg, about 532 mg to about 798 mg, about 576 mg to about 798 mg, about 621 mg to about 798 mg, about 665 mg to about 798 mg, about 709 mg to about 798 mg, about 753 mg to about 798 mg, about 22 mg to about to about 177 mg, about 44 mg to about 177 mg, about 44 mg to about 160 mg, about 44 mg to about 150 mg, about 44 mg to about 140 mg, about 44 mg to about 130 mg, about 44 mg to about 120 mg, about 44 mg to about 110 mg, about 44 mg to about 100 mg, about 44 mg to about 90 mg, about 44 mg to about 80 mg, about 44 mg to about 70 mg, about 44 mg to about 60 mg, or about 44 mg to about 50 mg.
[00340] In various embodiments, provided herein are pharmaceutical compositions comprising:
(i) about 22 mg to about 177 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and an anti angiogenic agent; and
(ii) one or more pharmaceutically acceptable excipients. [00341] In various embodiments, provided herein are pharmaceutical compositions comprising:
(i) about 44 mg to about 177 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and an anti angiogenic agent; and
(ii) one or more pharmaceutically acceptable excipients.
[00342] In various embodiments, provided herein are pharmaceutical compositions comprising:
(i) about 177 mg to about 798 mg of a pharmaceutically acceptable salt of a compound of formula (I), and an anti angiogenic agent; and
(ii) one or more pharmaceutically acceptable excipients.
[00343] In another aspect, provided herein are pharmaceutical compositions comprising about 22 mg to about 798 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer (e.g., malignant melanoma)), and a thyroid cancer) in a subject in need thereof.
[00344] In another aspect, provided herein are pharmaceutical compositions comprising about 22 mg to about 798 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer), and a thyroid cancer) in a subject in need thereof.
[00345] In another aspect, provided herein are pharmaceutical compositions comprising about 177 mg to about 798 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer (e.g., malignant melanoma)), and a thyroid cancer) in a subject in need thereof.
[00346] In another aspect, provided herein are pharmaceutical compositions comprising about 177 mg to about 798 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer), and a thyroid cancer) in a subject in need thereof.
[00347] In another aspect, provided herein are pharmaceutical compositions comprising about 44 mg to about 177 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer (e.g., malignant melanoma)), and a thyroid cancer) in a subject in need thereof.
[00348] In another aspect, provided herein are pharmaceutical compositions comprising about 44 mg to about 177 mg of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, for the treatment of a cancer described herein (e.g., a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer), and a thyroid cancer) in a subject in need thereof.
[00349] In certain embodiments, the pharmaceutically acceptable salt of the compound of formula (I) is a fumarate salt. In certain embodiments, the pharmaceutically acceptable salt of the compound of formula (I) is a hemifumarate salt.
[00350] The pharmaceutical compositions described herein can be administered by a variety of routes including, but not limited to, oral (enteral) administration, parenteral (by injection) administration, rectal administration, transdermal administration, intradermal administration, intrathecal administration, subcutaneous (SC) administration, intravenous (IV) administration, intramuscular (IM) administration, and intranasal administration. In certain embodiments, the pharmaceutical compositions described herein are administered orally.
[00351] The pharmaceutical compositions described herein may also be administered chronically (“chronic administration”). Chronic administration refers to administration of the compound or pharmaceutical composition thereof over an extended period of time, e.g., for example, over 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or may be continued indefinitely, for example, for the rest of the subject’s life. In certain embodiments, the chronic administration is intended to provide a constant level of the compound in the blood, e.g., within the therapeutic window over the extended period of time.
[00352] The pharmaceutical compositions described herein may be presented in unit dosage forms to facilitate accurate dosing. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.
[00353] In certain embodiments, the pharmaceutical compositions provided herein are administered to the patient as a solid dosage form. In certain embodiments, the solid dosage form is a capsule. [00354] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation. General considerations in the formulation and/or manufacture of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005.
Methods of Use and Treatment
[00355] Provided herein are methods of activating the immune system of a subject in need thereof. The methods generally comprise administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor).
[00356] Also provided herein are methods of activating the immune system of a subject in need thereof. The methods generally comprise administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, and a therapeutically effective amount of an anti-PD- 1 antibody.
[00357] Also provided herein are methods of activating the immune system of a subject having a cancer (e.g., a cancer described herein), comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor).
[00358] Also provided herein are methods of activating the immune system of a subject having a cancer (e.g., a cancer described herein), comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, and a therapeutically effective amount of an anti-PD- 1 antibody.
[00359] In some embodiments, the methods of activating the immune system described herein inhibit the activity of myeloid derived suppressor cells. In some embodiments, the methods of activating the immune system described herein increase the infiltration of T cells and NK cells in a tumor. In some embodiments, the methods of activating the immune system described herein increase the frequency of dendritic cells in a draining lymph node.
[00360] Also provided herein are methods of treating a cancer in a subject in need thereof. The methods generally comprise administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein, and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor). In certain embodiments, the cancer is a solid tumor.
[00361] Also provided herein are methods of treating a cancer in a subject in need thereof. The methods generally comprise administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein, and a therapeutically effective amount of an anti-PD-1 antibody. In certain embodiments, the cancer is a solid tumor.
[00362] The compound of formula (I), or a pharmaceutically acceptable salt thereof, in combination with an immunotherapy (e.g., an anti-PD-1 antibody) is contemplated to be useful in treating cancers including, but not limited to, pancreatic cancer; bladder cancer; colorectal cancer; breast cancer, including metastatic breast cancer; prostate cancer, including androgen-dependent and androgen-independent prostate cancer; kidney or renal cancer, including, e.g., metastatic renal cell carcinoma; hepatocellular cancer; lung cancer, including, e.g., NSCLC, bronchioloalveolar carcinoma (BAC), and adenocarcinoma of the lung; ovarian cancer, including, e.g., progressive epithelial or primary peritoneal cancer; cervical cancer; gastric cancer; esophageal cancer; head and neck cancer, including, e.g., squamous cell carcinoma of the head and neck; melanoma; neuroendocrine cancer, including metastatic neuroendocrine tumors; brain tumors, including, e.g., glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme, and adult anaplastic astrocytoma; bone cancer; and soft tissue sarcoma, hepatic carcinoma, rectal cancer, penile carcinoma, vulval cancer, thyroid cancer, salivary gland carcinoma, endometrial or uterine carcinoma, hepatoma, hepatocellular cancer, liver cancer, gastric or stomach cancer including gastrointestinal cancer, cancer of the peritoneum, squamous carcinoma of the lung, gastroesophageal cancer, biliary tract cancer, gall bladder cancer, colorectal/appendiceal cancer, and squamous cell cancer (e.g., epithelial squamous cell cancer).
[00363] In certain embodiments, the cancer is selected from the group consisting of bladder cancer, squamous cell head and neck cancer, malignant melanoma, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer.
[00364] In certain embodiments, the cancer is selected from the group consisting of bladder cancer, squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer.
[00365] In certain embodiments, the cancer is a liquid tumor. In certain embodiments, the cancer is selected from the group consisting of multiple myeloma, leukemia, and lymphoma. In certain embodiments, the leukemia is selected from the group consisting of chronic lymphocytic leukemia, chronic myelocytic leukemia, acute lymphoblastic leukemia, and acute myeloid leukemia (AML). In some embodiments, the lymphoma is selected from the group consisting of classical Hodgkin’s lymphoma, primary thymic mediastinal lymphoma, and non-Hodgkin’s lymphoma. In certain embodiments, the lymphoma is selected from classical Hodgkin’s lymphoma and primary thymic mediastinal lymphoma.
[00366] In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 22 mg to about 226 mg, about 35 mg to about 226 mg, about 45 mg to about 226 mg, about 55 mg to about 226 mg, about 65 mg to about 226 mg, about 75 mg to about 226 mg, about 85 mg to about 226 mg, about 95 mg to about 226 mg, about 105 mg to about 226 mg, about 115 mg to about 226 mg, about 125 mg to about 226 mg, about 135 mg to about 226 mg, about 145 mg to about 226 mg, about 155 mg to about 226 mg, about 165 mg to about 226 mg, about 175 mg to about 226 mg, about 185 mg to about 226 mg, about 195 mg to about 226 mg, about 22 mg to about 190 mg, about 22 mg to about 180 mg, about 22 mg to about 170 mg, about 22 mg to about
160 mg, about 22 mg to about 150 mg, about 22 mg to about 140 mg, about 22 mg to about 130 mg, about 22 mg to about 120 mg, about 22 mg to about 110 mg, about 22 mg to about 100 mg, about 22 mg to about 90 mg, about 22 mg to about 80 mg, about 22 mg to about 70 mg, about 22 mg to about 60 mg, about 22 mg to about 50 mg, about 22 mg to about 40 mg, about 25 mg to about 30 mg, about 35 mg to about 185 mg, about 45 mg to about 175 mg, about 55 mg to about 165 mg, about 65 mg to about 155 mg, about 75 mg to about 145 mg, about 85 mg to about 135 mg, about 95 mg to about 125 mg, or about 105 mg to about 115 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. [00367] In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 177 mg to about 900 mg, about 250 mg to about 900 mg, about 300 mg to about 900 mg, about 350 mg to about 900 mg, about 400 mg to about 900 mg, about 450 mg to about 900 mg, about 500 mg to about 900 mg, about 550 mg to about 900 mg, about 600 mg to about 900 mg, about 650 mg to about 900 mg, about 700 mg to about 900 mg, about 750 mg to about 900 mg, about 800 mg to about 900 mg, about 850 mg to about 900 mg, about 177 mg to about 850 mg, about 177 mg to about 800 mg, about 177 mg to about 750 mg, about 177 mg to about 700 mg, about 177 mg to about 650 mg, about 177 mg to about 600 mg, about 177 mg to about 550 mg, about 177 mg to about 500 mg, about 177 mg to about 450 mg, about 177 mg to about 400 mg, about 177 mg to about 350 mg, about 177 mg to about 300 mg, about 177 mg to about 250 mg, about 250 mg to about 850 mg, about 300 mg to about 800 mg, about 350 mg to about 750 mg, about 400 mg to about 700 mg, about 450 mg to about 650 mg, about 500 mg to about 600 mg, or about 550 mg to about 600 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[00368] In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 22 mg to about 451 mg, about 50 mg to about 451 mg, about 75 mg to about 451 mg, about 100 mg to about 451 mg, about 125 mg to about 451 mg, about 150 mg to about 451 mg, about 175 mg to about 451 mg, about 200 mg to about 451 mg, about 225 mg to about 451 mg, about 250 mg to about 451 mg, about 275 mg to about 451 mg, about 300 mg to about 451 mg, about 325 mg to about 451 mg, about 350 mg to about 451 mg, about 375 mg to about 451 mg, about 22 mg to about 375 mg, about 22 mg to about 350 mg, about 22 mg to about 325 mg, about 22 mg to about 300 mg, about 22 mg to about 275 mg, about 22 mg to about 250 mg, about 22 mg to about 225 mg, about 22 mg to about 200 mg, about 22 mg to about 175 mg, about 22 mg to about 150 mg, about 22 mg to about 125 mg, about 22 mg to about 100 mg, about 22 mg to about 75 mg, about 22 mg to about 50 mg, about 50 mg to about 375 mg, about 75 mg to about 350 mg, about 100 mg to about 325 mg, about 125 mg to about 300 mg, about 150 mg to about 275 mg, about 175 mg to about 250 mg, about 200 mg to about 225 mg, about 50 mg to about 100 mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250 mg, about 250 mg to about 300 mg, about 300 mg to about 350 mg, or about 350 mg to about 451 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. [00369] In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 22 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about 650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about 750 mg, about 760 mg, about 770 mg, about 780 mg, about 790 mg, about 800 mg, about 810 mg, about 820 mg, about 830 mg, about 840 mg, about 850 mg, about 860 mg, about 870 mg, about 880 mg, about 890 mg, or about 900 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[00370] In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 22 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 50 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 75 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 100 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 125 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 150 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 175 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 200 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 250 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 300 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 350 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 400 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 450 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 500 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 550 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 600 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 650 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 700 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 750 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 800 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 850 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 900 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[00371] In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 22 mg to about 226 mg, about 35 mg to about 226 mg, about 45 mg to about 226 mg, about 55 mg to about 226 mg, about 65 mg to about 226 mg, about 75 mg to about 226 mg, about 85 mg to about 226 mg, about 95 mg to about 226 mg, about 105 mg to about 226 mg, about 115 mg to about 226 mg, about 125 mg to about 226 mg, about 135 mg to about 226 mg, about 145 mg to about 226 mg, about 155 mg to about 226 mg, about 165 mg to about 226 mg, about 175 mg to about 226 mg, about 185 mg to about 226 mg, about 195 mg to about 226 mg, about 22 mg to about 190 mg, about 22 mg to about 180 mg, about 22 mg to about 170 mg, about 22 mg to about 160 mg, about 22 mg to about 150 mg, about 22 mg to about 140 mg, about 22 mg to about 130 mg, about 22 mg to about 120 mg, about 22 mg to about 110 mg, about 22 mg to about 100 mg, about 22 mg to about 90 mg, about 22 mg to about 80 mg, about 22 mg to about 70 mg, about 22 mg to about 60 mg, about 22 mg to about 50 mg, about 22 mg to about 40 mg, about 22 mg to about 30 mg, about 35 mg to about 185 mg, about 45 mg to about 175 mg, about 55 mg to about 165 mg, about 65 mg to about 155 mg, about 75 mg to about 145 mg, about 85 mg to about 135 mg, about 95 mg to about 125 mg, or about 105 mg to about 115 mg of the pharmaceutically acceptable salt of the compound of formula (I). [00372] In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 177 mg to about 900 mg, about 250 mg to about 900 mg, about 300 mg to about 900 mg, about 350 mg to about 900 mg, about 400 mg to about 900 mg, about 450 mg to about 900 mg, about 500 mg to about 900 mg, about 550 mg to about 900 mg, about 600 mg to about 900 mg, about 650 mg to about 900 mg, about 700 mg to about 900 mg, about 750 mg to about 900 mg, about 800 mg to about 900 mg, about 850 mg to about 900 mg, about 177 mg to about 850 mg, about 177 mg to about 800 mg, about 177 mg to about 750 mg, about 177 mg to about 700 mg, about 177 mg to about 650 mg, about 177 mg to about 600 mg, about 177 mg to about 550 mg, about 177 mg to about 500 mg, about 177 mg to about 450 mg, about 177 mg to about 400 mg, about 177 mg to about 350 mg, about 177 mg to about 300 mg, about 177 mg to about 250 mg, about 250 mg to about 850 mg, about 300 mg to about 800 mg, about 350 mg to about 750 mg, about 400 mg to about 700 mg, about 450 mg to about 650 mg, about 500 mg to about 600 mg, or about 550 mg to about 600 mg of the pharmaceutically acceptable salt of the compound of formula (I).
[00373] In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 22 mg to about 451 mg, about 50 mg to about 451 mg, about 75 mg to about 451 mg, about 100 mg to about 451 mg, about 125 mg to about 451 mg, about 150 mg to about 451 mg, about 175 mg to about 451 mg, about 200 mg to about 451 mg, about 225 mg to about 451 mg, about 250 mg to about 451 mg, about 275 mg to about 451 mg, about 300 mg to about 451 mg, about 325 mg to about 451 mg, about 350 mg to about 451 mg, about 375 mg to about 451 mg, about 22 mg to about 375 mg, about 22 mg to about 350 mg, about 22 mg to about 325 mg, about 22 mg to about 300 mg, about 22 mg to about 275 mg, about 22 mg to about 250 mg, about 22 mg to about 225 mg, about 22 mg to about 200 mg, about 22 mg to about 175 mg, about 22 mg to about 150 mg, about 22 mg to about 125 mg, about 22 mg to about 100 mg, about 22 mg to about 75 mg, about 22 mg to about 50 mg, about 50 mg to about 375 mg, about 75 mg to about 350 mg, about 100 mg to about 325 mg, about 125 mg to about 300 mg, about 150 mg to about 275 mg, about 175 mg to about 250 mg, about 200 mg to about 225 mg, about 50 mg to about 100 mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250 mg, about 250 mg to about 300 mg, about 300 mg to about 350 mg, or about 350 mg to about 451 mg of the pharmaceutically acceptable salt of the compound of formula (I). [00374] In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 22 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about 650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about 750 mg, about 760 mg, about 770 mg, about 780 mg, about 790 mg, about 800 mg, about 810 mg, about 820 mg, about 830 mg, about 840 mg, about 850 mg, about 860 mg, about 870 mg, about 880 mg, about 890 mg, or about 900 mg of the pharmaceutically acceptable salt of the compound of formula (I).
[00375] In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 22 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 50 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 75 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 100 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 125 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 150 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 175 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 200 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 250 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 300 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 350 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 400 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 450 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 500 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 550 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 600 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 650 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 700 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 750 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 800 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 850 mg of the pharmaceutically acceptable salt of the compound of formula (I). In some embodiments, administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) comprises administering to the subject about 900 mg of the pharmaceutically acceptable salt of the compound of formula (I).
[00376] In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject daily. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily.
[00377] In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive days. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for 21 consecutive days.
[00378] In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, or 36 consecutive days. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for at least 21 consecutive days.
[00379] In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, or 36 consecutive days. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for 21 consecutive days.
[00380] In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive days. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for at least 21 consecutive days.
[00381] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for 1 year. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for 2 years.
[00382] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for at least 1 year. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for at least 2 years.
[00383] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for 1 year. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for 2 years.
[00384] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for at least 1 year. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for at least 2 years.
[00385] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 22 mg to about 451 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[00386] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 451 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[00387] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 451 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, daily.
[00388] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 451 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, once daily.
[00389] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 451 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, twice daily.
[00390] In certain embodiments, the subject is in a fasting state. In certain embodiments, the subject is not in a fasting state. In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject the effective amount within about 30 minutes of completing a meal.
[00391] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 22 mg to about 451 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, within about 30 minutes of completing a meal.
[00392] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 177 mg to about 900 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[00393] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 177 mg to about 900 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[00394] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 177 mg to about 900 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, daily.
[00395] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 177 mg to about 900 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, once daily.
[00396] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 177 mg to about 900 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, twice daily.
[00397] In certain embodiments, the subject is in a fasting state. In certain embodiments, the subject is not in a fasting state. In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject the therapeutically effective amount within about 30 minutes of completing a meal.
[00398] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 177 mg to about 900 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, within about 30 minutes of completing a meal.
[00399] In some embodiments, the immunotherapy is selected from the group consisting of an immune checkpoint inhibitor, an adoptive cell therapy, and a monoclonal antibody. In some embodiments, the immunotherapy is selected from an immune checkpoint inhibitor and an adoptive cell therapy.
[00400] In certain embodiments, the immunotherapy is an adoptive cell therapy. In certain embodiments, the adoptive cell therapy is selected from the group consisting of tumor-infiltrating lymphocyte therapy, engineered T-cell receptor therapy, CAR T-cell therapy, and natural killer cell therapy.
[00401] In some embodiments, the immunotherapy is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 (programmed cell death protein 1) antagonist, a PD-L1 (programmed cell death protein ligand 1) antagonist, a PD-L2 antagonist, a CD27 (cluster of differentiation 27) antagonist, a CD28 antagonist, a CD70 antagonist, a CD80 antagonist, a CD86 antagonist, a CD137 antagonist, a CD276 antagonist, a KIRs (killer-cell immunoglobulin-like receptors) antagonist, a LAG3 (lymphocyte-activation gene 3) antagonist, a TNFRSF4 (tumor necrosis factor receptor superfamily, member 4) antagonist, a GITR (glucocorticoid-induced TNFR-related protein) antagonist, a GITRL (GITR ligand) antagonist, a 4-1BBL (4-1BB ligand) antagonist, a CTLA-4 (cytotoxic T lymphocyte associated antigen 4) antagonist, an A2AR (adenosine A2A receptor) antagonist, a VTCN1 (V-set domain-containing T-cell activation inhibitor 1) antagonist, a BTLA (B- and T-lymphocyte attenuator) antagonist, an IDO (Indoleamine 2,3 -dioxygenase) antagonist, a TIM-3 (T-cell immunoglobulin and mucin-domain containing-3) antagonist, a VISTA (V-domain Ig suppressor of T cell activation) antagonist, a KLRA antagonist, and a combination thereof.
[00402] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, lambrolizumab, BMS-936559, atezolizumab, AMP-224, AMP224, AUNP12, BGB108, MCLA134, MEDI0680, spartalizumab, cemiplimab, SHR1210, STIA110X, STIA1110, TSR042, MPDL3280A, MEDI- 4736, MSB0010718C, ALN-PDL, BGBA317, KD033, KY1003, STIA100X, STIA1010, STIA1011, STIA1012, STIA101, BMS-663513, and PF-05082566.
[00403] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the immunotherapy are administered simultaneously. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the immunotherapy are administered separately. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the immunotherapy are administered sequentially.
[00404] In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) and the therapeutically effective amount of the immunotherapy are administered simultaneously. In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) and the therapeutically effective amount of the immunotherapy are administered separately. In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) and the therapeutically effective amount of the immunotherapy are administered sequentially.
[00405] In some embodiments, the therapeutically effective amount of the immunotherapy is administered over a course of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. In some embodiments, the therapeutically effective amount of the immunotherapy is administered over a course of 1 year. In some embodiments, the therapeutically effective amount of the immunotherapy is administered over a course of 2 years.
[00406] In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti- PD-L1 antibody, or an anti-CTLA-4 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody.
[00407] In some embodiments, the anti-PD-1 antibody is a PD-1 antagonist. In some embodiments, the anti-PD-1 antibody is a PD-1 agonist. In some embodiments, the anti-PD-1 antibody is a PD-1 inhibitor.
[00408] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, spartalizumab, tislelizumab, or cemiplimab. In some embodiments, the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, and cemiplimab. In certain embodiments, the anti-PD-1 antibody is selected from pembrolizumab and nivolumab.
[00409] In certain embodiments, the anti-PD-1 antibody is pembrolizumab. In certain embodiments, the anti-PD-1 antibody is nivolumab. In certain embodiments, the anti-PD-1 antibody is cemiplimab.
[00410] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered simultaneously. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered separately. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered sequentially.
[00411] In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) and the therapeutically effective amount of the anti- PD-1 antibody are administered simultaneously. In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) and the therapeutically effective amount of the anti-PD-1 antibody are administered separately. In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I) and the therapeutically effective amount of the anti-PD-1 antibody are administered sequentially. [00412] In some embodiments, the therapeutically effective amount of the anti-PD-1 antibody is administered over a course of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. In some embodiments, the therapeutically effective amount of the anti-PD-1 antibody is administered over a course of 1 year. In some embodiments, the therapeutically effective amount of the anti-PD-1 antibody is administered over a course of 2 years.
[00413] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered over a course of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody is administered over a course of 1 year. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody is administered over a course of 2 years.
[00414] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered over a course of at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody is administered over a course of at least 1 year. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti- PD-1 antibody is administered over a course of at least 2 years.
[00415] In some embodiments, administering the therapeutically effective amount of the anti-PD-1 antibody comprises administering about 200 mg to about 400 mg of the anti-PD-1 antibody to the subject. In some embodiments, administering the therapeutically effective amount of the anti-PD-1 antibody comprises administering about 200 mg to about 400 mg of the anti-PD-1 antibody to the subject every about 2 to about 4 weeks. In some embodiments, administering the therapeutically effective amount of the anti-PD-1 antibody comprises administering about 200 mg to about 400 mg of the anti-PD-1 antibody to the subject every about 2 to about 4 weeks for about 6 months. In some embodiments, administering the therapeutically effective amount of the anti-PD-1 antibody comprises administering about 200 mg to about 400 mg of the anti-PD-1 antibody to the subject every about 2 to about 4 weeks for about 1 year. In some embodiments, administering the therapeutically effective amount of the anti-PD-1 antibody comprises administering about 200 mg to about 400 mg of the anti-PD-1 antibody to the subject every about 2 to about 4 weeks for about 2 years.
[00416] In some embodiments, the anti-PD-1 antibody is pembrolizumab. In certain embodiments, the pembrolizumab is administered in an amount of about 200 mg every 3 weeks. In certain embodiments, the pembrolizumab is administered in an amount of about 200 mg every 3 weeks for about 6 months. In certain embodiments, the pembrolizumab is administered in an amount of about 200 mg every 3 weeks for about 1 year. In certain embodiments, the pembrolizumab is administered in an amount of about 200 mg every 3 weeks for about 2 years. In certain embodiments, the pembrolizumab is administered in an amount of about 400 mg every 6 weeks. In certain embodiments, the pembrolizumab is administered in an amount of about 400 mg every 6 weeks for about 6 months. In certain embodiments, the pembrolizumab is administered in an amount of about 400 mg every 6 weeks for about 1 year. In certain embodiments, the pembrolizumab is administered in an amount of about 400 mg every 6 weeks for about 2 years.
[00417] In some embodiments, the anti-PD-1 antibody is nivolumab. In certain embodiments, the nivolumab is administered in an amount of about 240 mg every 2 weeks. In certain embodiments, the nivolumab is administered in an amount of about 240 mg every 2 weeks for about 6 months. In certain embodiments, the nivolumab is administered in an amount of about 240 mg every 2 weeks for about 1 year. In certain embodiments, the nivolumab is administered in an amount of about 240 mg every 2 weeks for about 2 years. In certain embodiments, the nivolumab is administered in an amount of about 480 mg every 4 weeks. In certain embodiments, the nivolumab is administered in an amount of about 480 mg every 4 weeks for about 6 months. In certain embodiments, the nivolumab is administered in an amount of about 480 mg every 4 weeks for about 1 year. In certain embodiments, the nivolumab is administered in an amount of about 480 mg every 4 weeks for about 2 years.
[00418] In some embodiments, the anti-PD-1 antibody is cemiplimab. In certain embodiments, the cemiplimab is administered in an amount of about 350 mg every 3 weeks. In certain embodiments, the cemiplimab is administered in an amount of about 350 mg every 3 weeks for about 6 months. In certain embodiments, the cemiplimab is administered in an amount of about 350 mg every 3 weeks for about 1 year. In certain embodiments, the cemiplimab is administered in an amount of about 350 mg every 3 weeks for about 2 years.
[00419] In some embodiments, provided herein is a method of treating a cancer (e.g., a cancer described herein) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, as described herein; or a pharmaceutical composition comprising the compound of formula (I), or a pharmaceutically acceptable salt thereof, as described herein; and a therapeutically effective amount of pembrolizumab.
[00420] In some embodiments, provided herein is a method of treating a cancer (e.g., a cancer described herein) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, as described herein; or a pharmaceutical composition comprising the compound of formula (I), or a pharmaceutically acceptable salt thereof, as described herein; and a therapeutically effective amount of nivolumab.
[00421] In some embodiments, provided herein is a method of treating a cancer (e.g., a cancer described herein) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, as described herein; or a pharmaceutical composition comprising the compound of formula (I), or a pharmaceutically acceptable salt thereof, as described herein; and a therapeutically effective amount of cemiplimab.
[00422] In certain embodiments, the subject has previously been administered at least one prior line of therapy. In certain embodiments, the subject has previously been administered fewer than five prior lines of therapy. In certain embodiments, the subject has previously been administered one, two, three, or 4 prior lines of therapy. In certain embodiments, the subject has not been administered a prior line of therapy.
[00423] In certain embodiments, the subject has previously been administered at least one and no more than 5 prior lines of therapy.
[00424] Prior lines of therapy include, but are not limited to, surgery, radiation therapy (e.g., external beam radiation therapy or internal radiation therapy), chemotherapy (e.g., alkylating agents, nitrosoureas, anti-metabolites, plant alkaloids and natural products, anti-tumor antibiotics, hormonal agents, and biological response modifiers), gene therapy, DNA therapy, viral therapy (e.g., oncolytic virus therapy), RNA therapy, adjuvant therapy, and immunotherapy (e.g., immune checkpoint inhibition, adoptive cell therapies, (e.g., tumor-infiltrating lymphocyte therapy, engineered T-cell receptor therapy, CAR T-cell therapy, natural killer cell therapy), or monoclonal antibodies).
[00425] In certain embodiments, the methods include administering a therapeutically effective amount of a pharmaceutically acceptable salt of the compound of formula (I). In certain embodiments, the pharmaceutically acceptable salt is a fumarate salt. In certain embodiments, the fumarate salt is a hemifumarate salt.
[00426] In certain embodiments, the methods described herein further comprise administering a therapeutically effective amount of a third therapeutic agent to the subject. In certain embodiments, the second therapeutic agent is selected from the group consisting of a checkpoint inhibitor, an EGFR inhibitor, an anti angiogenic agent, venetoclax, fluorouracil, and combinations thereof.
[00427] In certain embodiments, the third therapeutic agent is a checkpoint inhibitor. In certain embodiments, the third therapeutic agent is a PD-L1 inhibitor. In certain embodiments, the third therapeutic agent is selected from the group consisting of atezolizumab, avelumab, and durvalumab.
[00428] In certain embodiments, the third therapeutic agent is an EGFR inhibitor. In some embodiments, the EGFR inhibitor is selected from the group consisting of erlotinib, gefitinib, afatinib, and osimertinib.
[00429] In certain embodiments, the third therapeutic agent is an anti angiogenic agent. In certain embodiments, the anti angiogenic agent is a VEGFR inhibitor. In certain embodiments, the anti angiogenic agent is a VEGFR tyrosine kinase inhibitor.
[00430] In certain embodiments, the third therapeutic agent is a VEGFR inhibitor. In certain embodiments, the VEGFR inhibitor is selected from the group consisting of sunitinib, axitinib, lenvatinib, tivozanib, pazopanib, cabozantinib, and ramucirumab.
[00431] In another aspect, provided herein are methods of treating a cancer (e.g., a cancer described herein) in a subject in need thereof, comprising administering any one of the pharmaceutical compositions described herein and a therapeutically effective amount of an anti- PD-1 antibody (e.g., an anti-PD-1 antibody described herein) to the subject.
[00432] In another aspect, provided herein are methods of activating the immune system of a subject in need thereof, comprising administering any one of the pharmaceutical compositions described herein and a therapeutically effective amount of an anti-PD-1 antibody (e.g., an anti-PD- 1 antibody described herein) to the subject.
[00433] In another aspect, provided herein are methods of activating the immune system of a subject having a cancer (e.g., a cancer described herein), comprising administering any one of the pharmaceutical compositions described herein and a therapeutically effective amount of an anti- PD-1 antibody (e.g., an anti-PD-1 antibody described herein) to the subject.
[00434] In some embodiments, the methods described herein increase the tumor cell expression of type 1 interferon receptor (INF ARI) in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
[00435] In some embodiments, the methods described herein increase the peripheral blood monocyte surface expression of INF ARI in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
[00436] In some embodiments, the methods described herein increase tumor cell expression of calreticulin in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
[00437] In some embodiments, the methods described herein increase polymorphonuclear myeloid-derived suppressor cell (MDSC) and/or tumor associated macrophage (TAM) expression of INF ARI in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
[00438] In some embodiments, the methods described herein increase TAM expression of PD-L1 in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
[00439] In some embodiments, the methods described herein increase the infiltration of CD8 T cells and/or NK cells in a tumor in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody. [00440] In some embodiments, the methods described herein increase expression of CD69 on T cells in a tumor draining lymph node in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
[00441] In some embodiments, the methods described herein reduce suppressive activity of MDSCs in the subject.
[00442] In some embodiments, the methods described herein increase dendritic cell frequency in a tumor draining lymph node in the subject.
[00443] In some embodiments, the methods described herein increase Ki67, GzmB, and memory phenotypic markers on CD4 T-cells in the subject.
[00444] In some embodiments, the methods described herein increase the immuno-stimulatory phenotype of macrophages in the tumor microenvironment.
[00445] In some embodiments, the methods described herein decrease the immuno-inhibitory phenotype of macrophages in the tumor microenvironment.
[00446] In some embodiments, the methods described herein increase an Ml phenotype and/or function of macrophages, or decrease an M2 phenotype or function of macrophages.
[00447] In some embodiments, the methods described herein increase surface expression of activation markers (e.g., MHCII, HLA-DR, and/or CD86) on macrophages or other myeloid cells.
[00448] In some embodiments, the methods described herein increase the immuno-stimulatory phenotype of dendritic cells in the draining lymph node or tumor microenvironment.
[00449] In some embodiments, provided herein are a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy for use in treating a cancer (e.g., a cancer described herein).
[00450] In some embodiments, provided herein are a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD-1 antibody for use in treating a cancer (e.g., a cancer described herein).
[00451] In some embodiments, provided herein are a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy for use in activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein). [00452] In some embodiments, provided herein are a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD-1 antibody for use in activating the immune system of a subject in need thereof (e.g., a subject having a cancer described herein).
[00453] Also provided herein are methods of treating a cancer in a subject in need thereof. The methods generally comprise administering to the subject a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, and a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)). In certain embodiments, the cancer is a solid tumor.
[00454] Also provided herein are methods of treating a cancer in a subject in need thereof. The methods generally comprise administering to the subject a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein, a therapeutically effective amount of an anti angiogenic agent (e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)), and a therapeutically effective amount of an immunotherapy (e.g., an immune checkpoint inhibitor). In certain embodiments, the cancer is a solid tumor.
[00455] The compound of formula (I), or a pharmaceutically acceptable salt thereof, in combination with an anti angiogenic agent (e.g., e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)) is contemplated to be useful in treating cancers including, but not limited to, a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer (e.g., malignant melanoma)), and a thyroid cancer.
[00456] The compound of formula (I), or a pharmaceutically acceptable salt thereof, in combination with an anti angiogenic agent (e.g., e.g., a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI)) is contemplated to be useful in treating cancers including, but not limited to, a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer), and a thyroid cancer.
[00457] In certain embodiments, the cancer is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer (e.g., malignant melanoma)), and a thyroid cancer.
[00458] In certain embodiments, the cancer is selected from the group consisting of a bladder cancer, a breast cancer (e.g., triple negative breast cancer or metastatic breast cancer), a carcinoma (e.g., carcinoma of unknown primary (CUP), endometrial carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma), a cervical cancer, a colorectal cancer, a hepatocellular cancer, a gastric cancer (e.g., adenocarcinoma or gastrointestinal stromal tumor), a kidney cancer (e.g., a renal cell carcinoma (RCC) such as clear cell renal carcinoma (ccRCC)), a lung cancer (e.g., small cell lung cancer (SCLC) or non-small cell lung cancer), a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer), and a thyroid cancer.
[00459] In some embodiments, the cancer is malignant melanoma.
[00460] In some embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 22 mg to about 798 mg, about 35 mg to about 798 mg, about 44 mg to about 798 mg, about 66 mg to about 798 mg, about 88 mg to about 798 mg, about 110 mg to about 798 mg, about 113 mg to about 798 mg, about 155 mg to about 798 mg, about 177 mg to about 798 mg, about 199 mg to about 798 mg, about 221 mg to 798 mg, about 244 mg to about 798 mg, about 266 mg to about 798 mg, about 310 mg to about 798 mg, about 355 mg to about 798 mg, about 399 mg to about 798 mg, about 443 mg to about 798 mg, about 488 mg to about 798 mg, about 532 mg to about 798 mg, about 576 mg to about 798 mg, about 621 mg to about 798 mg, about 665 mg to about 798 mg, about 709 mg to about 798 mg, about 753 mg to about 798 mg, about 22 mg to about to about 177 mg, about 44 mg to about 177 mg, about 44 mg to about 160 mg, about 44 mg to about 150 mg, about 44 mg to about 140 mg, about 44 mg to about 130 mg, about 44 mg to about 120 mg, about 44 mg to about 110 mg, about 44 mg to about 100 mg, about 44 mg to about 90 mg, about 44 mg to about 80 mg, about 44 mg to about 70 mg, about 44 mg to about 60 mg, or about 44 mg to about 50 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[00461] In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject daily. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily.
[00462] In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive days. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for 21 consecutive days.
[00463] In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, or 36 consecutive days. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for at least 21 consecutive days.
[00464] In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, or 36 consecutive days. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for 21 consecutive days. [00465] In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive days. In certain embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for at least 21 consecutive days.
[00466] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for 1 year. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for 2 years.
[00467] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for at least 1 year. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for at least 2 years.
[00468] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for 1 year. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for 2 years.
[00469] In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject twice daily for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for at least 1 year. In some embodiments, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally to the subject once daily for at least 2 years.
[00470] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent (e.g., VEGFR-TKI) comprises administering to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[00471] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent (e.g., VEGFR-TKI) comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[00472] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent (e.g., VEGFR-TKI) comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, daily.
[00473] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent (e.g., VEGFR-TKI) comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, once daily. [00474] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an antiangiogenic agent (e.g., VEGFR-TKI) comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, twice daily.
[00475] In certain embodiments, the subject is in a fasting state. In certain embodiments, the subject is not in a fasting state. In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an antiangiogenic agent (e.g., VEGFR-TKI) comprises administering to the subject the therapeutically effective amount within about 30 minutes of completing a meal.
[00476] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an antiangiogenic agent (e.g., VEGFR-TKI) comprises administering to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, within about 30 minutes of completing a meal.
[00477] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an antiangiogenic agent (e.g., VEGFR-TKI) comprises administering to the subject about 177 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[00478] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an antiangiogenic agent (e.g., VEGFR-TKI) comprises administering orally to the subject about 177 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
[00479] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an antiangiogenic agent (e.g., VEGFR-TKI) comprises administering orally to the subject about 177 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, daily.
[00480] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent (e.g., VEGFR-TKI) comprises administering orally to the subject about 177 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, once daily.
[00481] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent (e.g., VEGFR-TKI) comprises administering orally to the subject about 177 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, twice daily.
[00482] In certain embodiments, the subject is in a fasting state. In certain embodiments, the subject is not in a fasting state. In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent (e.g., VEGFR-TKI) comprises administering to the subject the therapeutically effective amount within about 30 minutes of completing a meal.
[00483] In certain embodiments, administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent (e.g., VEGFR-TKI) comprises administering to the subject about 177 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, within about 30 minutes of completing a meal.
[00484] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis.
[00485] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis.
[00486] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis, daily. [00487] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis, once daily.
[00488] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis, twice daily.
[00489] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis.
[00490] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis.
[00491] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis, daily.
[00492] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis, once daily.
[00493] In some embodiments, administering the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free base equivalent weight basis, twice daily.
[00494] In certain embodiments, the subject is a human. In certain embodiments, the subject is an adult human. EXAMPLES
[00495] In order that the disclosure may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope.
Abbreviations BID twice daily
BIW twice weekly
DMEM Dulbecco’s Modified Eagle Medium
DMSO dimethyl sulfoxide dppf 1 , 1’ -bi s(diphenylphosphino)ferrocene
EtOH ethanol
FBS fetal bovine serum h hour(s)
HPLC high performance liquid chromatography
IMDM Iscove’ s modified Dulbecco’ s medium
IP intraperitoneal
IP A isopropyl alcohol
LN lymph node
LOQ limit of quantification mAb monoclonal antibody
MEM minimum essential medium
NEAA non-essential amino acids
NMR nuclear magnetic resonance spectroscopy
PBS phosphate buffered saline
PO oral
SEM standard error of mean
SFC supercritical fluid chromatography
TFA trifluoroacetic acid
TGI tumor growth inhibition Example 1: Synthesis of (7?)-2-amino-5-(4-(2-(3,5-difluorophenyl)-2-hydro yacetamido)-2- methylphenyl)-N-isopropylnicotinamide (Compound of Formula (I)) and hemifumarate salt
Step 1: Isolation of (2R)-2-(3,5-difluorophenyl)-2-hydroxy-acetic acid
Figure imgf000093_0001
[00496] Lipase PS Amano (see Mendiola, J. et al, Org. Process Res. Dev. 2012, 16, 1312-1316) was supported in diatomaceous earth prior to use by mixing 200 g of diatomaceous earth and 200 g of lipase PS Amano SD. H2O was added to cover the solid, and the mixture was stirred. H2O was removed in an oven at 4 mbar and 40 °C for 16 h. H2O was below 1% through Karl Fischer titration for water determination.
[00497] Supported lipase PS Amano SD (250 g) and vinyl acetate (312 mL; 3.36 mol) were added to a suspension of racemic 2-(3,5-difluorophenyl)-2-hydroxyacetic acid (125 g, 664 mmol) in methyl tert-butyl ether (2.50 L), and the mixture was stirred at 26 °C for 72 h. After this time, the solid was rinsed and filtered with methyl tert-butyl ether (1.50 L), and combined filtrates were concentrated under reduced pressure. The residue was slurried in di chloromethane (160 mL) at 23 °C for 4 h. The mixture was filtered, and the solid was washed with petroleum ether (150 mL), and dried to give the title compound (47.0 g, 36%).
Figure imgf000093_0002
NMR (de-DMSO) 5 5.11 (s, 1H), 6.20 (bs, 1H), 7.11-7.21 (m, 3H), 12.8 (bs, 1H). Absolute configuration was determined by vibrational circular dichroism (see Freedman T.B et al, Chirality, 2003 Nov., 15(9), 743-758). Chiral HPLC: Rt = 7.39 min (UV); Column: Chiralpak® AD 4.6 x 150mm 5pm; 5% EtOH in n-hexane (0.05% TFA) isocratic; Flow Rate: 1.5 mL/min, ee >98%.
Step 2: Synthesis of 2-Amino-5-(4-amino-2-methyl-phenyl)-N-isopropyl-pyridine-3-carboxamide
Figure imgf000093_0003
[00498] 3-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)aniline (93.6 g, 0.401 mol),
K2CO3 (H9g, 0.860 mol), and Pd(dppf)2C12 (10.6 g, 140 mmol) were added to a solution of 2- amino-5-bromo-N-isopropyl-pyridine-3-carboxamide (74.0 g, 0.287 mol) in dioxane (888 mL) and H2O (296 mL), and the mixture was heated at 55 °C overnight. The mixture was cooled to 23 °C, ethyl acetate (150 mL) was added, the resulting suspension was filtered through diatomaceous earth, and the solid was rinsed with ethyl acetate (50 mL). The combined filtrates were washed with H2O (30 mL) and saturated aqueous NaCl (300 mL), and concentrated under reduced pressure to give the title compound (78.0 g, 96%). ES/MS m/z 285.1 (M+H).
Step 3: Synthesis of (R)-2-amino-5-(4-(2-(3,5-dijluorophenyl)-2-hydroxyacetamido)-2- methylphenyl)-N-isopropylnicotinamide
Figure imgf000094_0001
[00499] A mixture of (2R)-2-(3,5-difluorophenyl)-2 -hydroxyacetic acid (29.0 g, 0.154 mol), 2- amino-5-(4-amino-2-methyl-phenyl)-N-isopropyl-pyridine-3-carboxamide (43.83 g, 0.154 mol), and N,N-diisopropylethylamine (39.8 g, 0.308 mol) in tetrahydrofuran (960 mL) was treated with (l-[bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) (87.9 g, 0.231 mol) at 0 °C for 30 min, and the mixture was warmed to 20 °C and stirred for 2 h. Ethyl acetate (50 mL) was added, and the mixture was filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by chromatography (eluent: 2: 1 petroleum ether / ethyl acetate) and then by Supercritical Fluid Chromatography, SFC (Column: Chiralpak® IC 30 x 250 mm 5 pm (Daicel); MeOH / CO2= 30:70 isocratic; Flow rate: 80 g/min; Back pressure: 100 Bar; Column temperature: 40°C) to give the title compound (27.5 g, 39%) as a white solid. ES/MS m/z 455.2 (M+H).
Step 4: Synthesis of (R)-2-amino-5-(4-(2-(3,5-difluorophenyl)-2-hydroxyacetamido)-2- methylphenyl)-N-isopropylnicotinamide hemifumarate salt
Figure imgf000095_0001
MW = 454.48 W = 51ZS2
[00500] To a 300 L reactor under N2 with agitation at 25°C was added IPA (174.3 kg, 40.0 V) and HC-5404 (5.48 kg, 0.012 moles, 1.0 eq). The resulting mixture was stirred for 3 h at 35°C and filtered through a 3M carbon filter (IPC: Pd content < LOQ {2 ppm}). The filtrate was combined with fumaric acid (0.72 kg, 0.55 eq) and was stirred for 9 h at 25°C to afford HC-5404-FU.
Example 2: Studies of HC-5404 + anti-PD-1 antibody (RMP1-14 mouse antibody) combination therapy in murine MB49 syngeneic bladder cancer model
[00501] C57BL/6 mice were subcutaneously inoculated with MB49 cells, and treatment started on day 8 post cell inoculation. A group of animals (n=10/group) received either vehicle, HC-5404 (PO, BID) (3 mg/kg, 10 mg/kg, or 30 mg/kg), 5 mg/kg anti-PD-1 antibody (aPD-1) (RMP1-14) (IP, BIW), or the combination of both. At various timepoints, animals were sacrificed, and flow cytometry was performed on blood or single cell suspensions from tumors or lymph nodes (n=6). While HC-5404 alone showed only a modest anti-tumor effect (32% tumors growth inhibition (TGI)), the addition of HC-5404 to aPD-1 provided combination antitumor benefits (75% TGI) and significantly improved the effects of aPD-1 alone (53% TGI) (FIGS. 2B, 2C, 2D, 2E, 2F, and 2G).
[00502] HC-5404 + aPD-1 treatment efficacy was correlated with increased expression of type 1 interferon receptor (IFNAR1) and increased surface calreticulin on tumor cells (FIG. 3D and 4B). Additionally, IFNAR1 expression was also significantly increased on polymorphonuclear myeloid- derived suppressor cells (PMN-MDSCs) and tumor-associated macrophages (TAM) (FIGS. 3A, 3B, and 3C). TAMs also showed increased expression of PD-L1 with combination treatment (FIG. 3E). Concomitant to the activation of myeloid cells, combination treatment increased the frequency of CD8 T-cells in the tumor (FIGS. 5A and 5B) along with increased expression of activation marker CD69 on T-cells in the tumor draining lymph node FIGS. 5C and 5D). Notably, the effect of HC-5404 on IFNAR1 was also detected on monocytes in peripheral blood, demonstrating surface expression of IFNAR1 as a potential biomarker for HC-5404 activity (FIG. 8). In vitro evaluation of human cord blood-derived and mouse bone marrow-derived MDSCs showed a reduced inhibition of T-cells in the presence of HC-5404 (FIGS. 10A, IOC, HA, and 11C). Collectively, these data demonstrate the efficacious and immuno-stimulatory effects of HC- 5404 co-administered with anti-PDl mAb and outline its potential application in ICI-treated cancers.
[00503] HC-5404 + aPD-1 treatment was also correlated with increased CD4 activation and memory T cell induction (demonstrated by Ki67 expression, EOMES expression, Granzyme B expression, TCF-1 expression, and/or effector memory population) (FIGS. 6A, 6B, 6C, 6D, and 6E), and expansion of DC populations in the lymph node (LN) of the treated mice (FIGS. 7A, 7B, and 7C).
Table 1 - Tumor growth inhibition % from two independent studies
Figure imgf000096_0001
Animal Characteristics
[00504] Female C57BL/6 mice (7-8 weeks of age animals, 20-25 grams, from Envigo) were used.
Cell Line
[00505] MB49 (lot # 18B016) mouse urothelial carcinoma cell line was purchased from Sigma - The European Collection of Authenticated Cell Cultures (EC ACC). Cells were free of human and animal pathogens as tested by h-IMPACT-I and IMPACT-III (IDEXX Bioresearch, Columbia, MO). Additionally, the genetic profile of the cells matched with the ECACC reference standard as tested by STR based DNA fingerprinting assay. Cells were maintained in DMEM with 10% FBS, and cultured up to 4 passages prior to implantation. Cells were harvested at -80% confluence, washed twice with DMEM without FBS and suspended in DMEM without FBS at 5 x 106 cells/mL for implantation. Establishing MB49 Model
[00506] Female Balb/c mice (7-8 week of age) were implanted into the right flank area subcutaneously under Isoflurane anesthesia with 0.5 x 106 MB49 cells/mouse in a total volume of 100 mL on Day 0. Animals were randomized into treatment groups when the average tumor volume reached ~ 50 mm3 on Day 5 post inoculation.
Measurement Parameters
[00507] Tumor volume was measured twice weekly.
[00508] Tumor volume was calculated using the formula [tumor volume (mm3) = JT/6 x length x width2) and plotted as means ± SEM. The %T/C (ratio between the tumor volume in the treated group and in the control group) was calculated by the formula 100 x AT/AC, if AT > 0 of the mean values. AT, mean tumor volume of the drug-treated group on the observation day of the study - mean tumor volume of the drug-treated group on initial day of dosing; AC, mean tumor volume of the control group on the observation day of the study - mean tumor volume of the control group on initial day of dosing. Regression was calculated using the formula = 100 x AT/Tinitial, if AT < 0. Animals with <14 mm3 tumor volume for three consecutive measurements were considered as complete responders and tumors with >50% regressions were partial responders.
[00509] Animals were euthanized at the end of the study using CO2 asphyxiation.
Statistical Analyses
[00510] Tumor volume data were analyzed for statistics using 2-way ANOVA followed by Dunnett multiple comparisons test (GraphPad Prism).
Flow cytometric analysis of myeloid and lymphoid cells in MB49
Tumor Tissue Preparation
[00511] Tumor tissue samples were obtained 7 days post treatment (14 days post inoculation). Single cells were prepared by enzymatic digestion of tumor tissues and subsequently analyzed by flow cytometry on a Cytek Aurora. Expression of IFNAR1 (A) and PD-L1 (B) on the myeloid populations and tumors were shown.
[00512] Surface calreticulin was analyzed by flow cytometry on MB49 tumor cells d7 post treatment. LN Preparation
[00513] Single cell suspensions were washed in PBS twice and then stained with the LIVE/DEAD fixable blue dead cell stain (Invitrogen cat # L23105) at 1 :3000 dilution for 30 minutes at 4 °C.
Cells were washed in FACS buffer (PBS with 2.5% FBS and 0.1% sodium azide) once, Fc receptor blocked for 5-10 minutes and then stained with the Ab cocktail in Super Bright Complete Staining Buffer (Invitrogen cat # SB-4401) for 30 min at 4 °C and protected from light. The labeled cells were then fixed with Fix/Perm buffer (Tonbo Biosciences) for 30 minutes at 4 °C, washed three times with Perm buffer (Tonbo Biosciences) before staining with intracellular staining Abs for 30 minutes at 4 °C. The stained cells were then washed once with Perm buffer and twice with FACS buffer sequentially. Stained cells were analyzed by flow cytometry on the Cytek Aurora (Cytek Biosciences) platform and data were analyzed by Flow Jo (BD).
[00514] LN cells from day 14 post treatment samples were analyzed by flow cytometry.
Expression of Ki67, EOMES, and Granzyme B on CD4+ T cell, effector memory (EM) population and TCF-1 expression on activated T cells was shown. Effector memory (EM) CD4 cells are defined as CD62L" CD44+ CD4+. Total DC is identified as CD1 lc+ MHCII+ CD1 lbdimA, cDCl is identified as XCR1+DC, and cDC2 is identified as XCR1" CD24+DC.
Peripheral Blood Cells
[00515] Whole blood was collected on the indicated date post treatment in a MB49 model.
IFNAR1 expression was determined by flow cytometry and gated on CD 14+ monocytes
Staining panels:
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000099_0002
Blood cell IFNAR panel
Figure imgf000100_0001
HMGB1 IHC staining
[00516] HC-5404 has demonstrated single agent and combinatorial efficacy in multiple solid tumor xenograft models.
[00517] 786-0 tumor cells were maintained in vitro with MEM medium supplemented with 10% heat inactivated fetal bovine serum and 0.01 mM NEAA, at 37 °C in an atmosphere of 5% CO2. Each mouse was inoculated subcutaneously at the right front region with 786-0 tumor cells (5x106) in 0.1 ml of PBS (1 : 1 matrigel) for tumor development. The randomization for efficacy study began when the mean tumor size reached approximately 200-300 mm3, preferably to initiate with 250 mm3.
[00518] Animals were treated with HC-5404 at 30mpk, BID for 28 days. Animals were euthanized and tumors collected and fixed in 10% Neutral buffered formalin and paraffin embedded for IHC analysis.
[00519] Paraffin sections of tumor samples were stained on slides for HMGB1 using the Abeam antibody (cat# ab 18256) and chromogenic DAB staining on Bond Rx (Leica Biosystems). The slides were digitized by Leica AT2 slide scanner with 40x objective. The scanned images were analyzed using custom-written macro in ImageJ/FIJI (NIH) to measure nuclear and non-nuclear signal (FIGS. 4C and 4D).
RNA purification
[00520] Mouse tumor, spleen and lung tissues were snap frozen and stored in -800 °C freezer.
The frozen tissue was soaked in lOx volume of the RNAlater®- ICE (ThermoFisher) at -200 °C for >16 hr. Then the tissue was transferred into M tube (Miltenyi Biotech) containing RET lysis buffer (Qiagen) and homogenized by gentleMACS Dissociators with program of RNA_02 (Miltenyi Biotech). Total RNA was isolated from homogenized tissue using the RNeasy Plus Mini Kit (Qiagen). RNA concentration was determined by Epoch Microplate Spectrophotometer (Biotek). QuantiGene® Plex assay
[00521] A custom-made 77-plex mouse Immune QuantiGene Assay kit (ThermoFisher Scientific) using a bead-based multiplex assay were used to measure the expression of 69 immune genes, 4 stress genes plus 4 reference genes with Luminex xMAP 200 (ThermoFisher Scientific) according to the manufacturer’s instructions.
Data analysis
[00522] The limit of detection of each gene analyzed was determined by subtracting the average intensity of the negative control wells from each unknown sample. The target gene expression values were normalized relative to geomean of 4 housekeeping genes. Fold changes of each treatment groups relative to the mean of the controls were log 2 transformed and presented as Heatmap (FIG. 9).
[00523] Data were analyzed using one-way ANOVA with multiple comparison of a post hoc (Tukey) test by Prism software (version 9.1.0) and were expressed as mean ± SEM. A p-value of < 0.05 was considered statistically significant.
Human and mouse MDSC study
Human MDSC
Reagents
Lymphoprep (StemCell tech cat # 07851)
Easy Sep Human Cord Blood CD34 Positive selection Kit II (StemCell Tech cat # 17896)
IMDM medium (Gibco cat# 12440-046)
RPMI (Corning cat# 10-040-CV)
FBS (Gibco cat# 10438-026)
Penicillin-streptomycin (Gibco cat# 15140122)
L-glutamax (Gibco cat# 35050-061)
Human GM-CSF (R&D systems cat# 215-GM)
Human G-CSF (R&D systems cat# 214-CS) Human SCF (R&D systems cat# 7466-SC)
EasySep Human T cell isolation kit (StemCell Tech cat# 17951)
Dynabeads Human T-activator CD3/CD28 (Gibco cat# 1113 ID)
Cell Trace Violet (CTV) Cell Proliferation kit (Invitrogen cat# C34557)
IL-2 (R&D systems cat# 202-IL)
[00524] Human MDSC Preparation: Fresh cord blood was obtained from NDRI (Protocol ID # RCHA4 01). Mononuclear cells were enriched from cord blood by Lymphoprep density gradient centrifugation, followed by CD34+ cell isolation using EasySep Human Cord Blood CD34 Positive selection Kit II, and the enriched CD34+ cells were cultured in a 48-well plate (BD Falcon, Bedford, MA) at 1 x 105 cells/ well/ ml of IMDM complete medium (IMDM supplemented with 10% FBS, lx penicillin-streptomycin and 2 mM L-glutamax). 100 ng/ml human GM-CSF, 100 ng/ml human G-CSF and 50 ng/ml human SCF were added to the IMDM complete medium and cells were expanded for 13-14 days in a 37 °C, 5% CO2 humidified incubator and medium was refreshed on day 7. MDSC differentiation was induced by culturing the expanded CD34+ cells in a 24 well plate at 0.1-0.15 x 106 cells/ well in 2 ml of IMDM complete medium supplemented with 100 ng/ml human GM-CSF, 50 ng/mL G-CSF and 30 ng/ml IL-6 for additional 6-7 days with medium changed on day 3. For HC-5404 treatment, the indicated dose of HC-5404 was added to the cell culture on day 0 of the MDSC differentiation phase, medium was refreshed on day 3 and HC-5404 was added afresh. MDSCs were harvested on day 6 or 7 and used in the T cell suppression assay.
[00525] Human T cell Suppression Assay. PBMCs were enriched from Trima Cone obtained from Innovative Blood Resources (Protocol ID # 120160627) by Ficoll density gradient centrifugation. CD3 T cells were further enriched from PBMC using EasySep Human T cell isolation kit. CD3 T cells were labeled with Cell Trace Violet (CTV) according to Vendor’s protocol. CTV-labeled T cells were resuspended in RPMI medium supplemented with 10% FBS, lx penicillin-streptomycin and 2 mM L-glutamax and added at 50,000 cells per well in a 96 well round-bottom plate. Dynabeads Human T-activator CD3/CD28 were washed twice with PBS and then added to T cells at 1 : 1 ratio. HC-5404 treated and un-treated MDSC were harvested from the plates, washed three times with RPMI medium and then added to the wells at various ratio of MDSC: T (4: 1; 2: 1; 1 : 1). IL-2 (30u/ml) was added to each well and the 96 well plate was incubated in a 37 0C, 5% CO2 humidified incubator to 3-5 days. T cell proliferation was analyzed by flow cytometry. Cells were washed 2-3 times in PBS, and then stained with fluorescent- conjugated anti-CD3 Ab for 30 minutes at 4 °C and protected from light. The labeled cells were analyzed on a BD Fortessa cytometer and data was analyzed by FlowJo. Statistical Analyses was performed using Prism software Prism (GraphPad Software). Data were analyzed using one-way ANOVA with multiple comparison corrected with Dunnett test. *P < .05; **P < .01; ***p < .005, ****P < .0001 (FIGS. 10A, 10B, and 10C).
Mouse MDSC
Reagents
RPMI (Corning cat# 10-040-CV)
FBS (Gibco cat# 10438-026) penicillin-streptomycin (Gibco
L-glutamax (Gibco cat# 35050-061)
HEPES (Gibco cat# 15630-080)
Sodium pyruvate (Gibco cat#l 1360-070)
2-ME (Gibco cat# 21985-023)
MEM NEAA (Gibco cat# 11140-050)
Glucose free RPMI (Gibco cat # 118790-020)
Glucose (Gibco cat# A24940-01)
Mouse GMCSF (PeproTech cat#315-03)
Mouse IL-6 (PeproTech cat# 216-16)
EasySep Mouse T cell isolation kit (StemCell Tech cat# 19851 A)
Dynabeads Mouse T-activator CD3/CD28 (Gibco cat# 114543D)
[00526] Mouse MDSC Preparation: Single cell suspensions from bone marrow were prepared from the tibia and femur bone of C57BL/6 mice. Cells were cultured in RPMI complete medium (RPMI medium supplemented with 10% FBS, lOmM HEPES, 1 mM Sodium pyruvate, 50uM 2- ME, 1 mM MEM NEAA, lx penicillin-streptomycin and 2 mM L-glutamax) at 2.5 x 106 cells per 10 ml in a 100 mm2 tissue culture dish for 4 days in a 37 0 °C, 5% CO2 humidified incubator. Cytokines GM-CSF (10 ng/ml) and IL-6 (10 ng/mL) were added to the cell culture on day 0. For low serum low glucose (LSLG) medium treatment, cultured cells were first harvested from the plate and washed twice with RPMI medium to rinse off residual serum and glucose, and then cultured in 5 ml of LSLG medium (glucose-free RPMI supplemented with 1% FBS, Ig/L glucose, lx penicillin-streptomycin and 2 mM L-glutamax) in a 15 ml conical tube for 6 hours with or without HC-5404 addition. Cells were harvested and used in the T cell suppression assay.
[00527] Mouse T cell suppression assay: Spleens were harvested from C57BL/6 mice. Single cell suspensions were prepared and CD3 T cells were isolated using EasySep Mouse T cell isolation kit. CD3 T cells were labeled with Cell Trace Violet (CTV) according to Vendor’s protocol. CTV-labeled T cells were resuspended in RPMI medium supplemented with 10% FBS, lx penicillin-streptomycin and 2 mM L-glutamax and added at 100,000 cells per well in a 96 well round-bottom plate. Dynabeads mouse T-activator CD3/CD28 were washed twice with PBS and then added to T cells at 1 : 1 ratio. LSLG +/- HC5404- treated and un-treated MDSC were harvested from the plates, washed three times with RPMI medium and then added to the wells at various ratio of MDSC: T (8: 1, 4: 1; 2: 1). IL-2 (30 ug/ml) was added to each well and the 96 well plate was incubated in a 37 °C, 5% CO2 humidified incubator to 3-5 days. T cell proliferation was analyzed by flow cytometry. Cells were washed 2-3 times in PBS, and then stained with fluorescent- conjugated anti-CD4 and anti-CD8 Ab for 30 minutes at 4 °C and protected from light. The labeled cells were analyzed on a BD Fortessa cytometer and data was analyzed by FlowJo. Statistical Analyses was performed using Prism software Prism (GraphPad Software). Data were analyzed using one-way ANOVA with multiple comparison corrected with Dunnett test. *P < .05; **P < .01; ***p < .005, ****P < .0001 (FIGS. 11A, 11B, and 11C).
Example 3: Cell Based Assays
[00528] Cell based assays HEK293 cells were plated at 1 x 106 cells per well and treated with HC- 5404 30 minutes prior to addition of tunicamycin (1 pM) to induce ER stress. Cells were harvested 4 hours following tunicamycin addition and lysed for western blotting in RIPA buffer supplemented with protease and phosphatase inhibitors. Total protein concentrations were determined by BCA and 30 mg of protein were loaded on to SDS-PAGE gels, transferred to nitrocellulose and probed with a primary antibody against phosphorylated T-982 of human PERK or total ATF4 followed by incubation with an infrared labelled secondary antibody. Blots were imaged on a LiCor imager and normalized to total protein in the wells using Revert™ stain. Band intensities were quantified and normalized to DMSO treated samples. Normalized band intensities were plotted as percent inhibition against 10-point; 3-fold dilution series of HC-5404. IC50 values were calculated using 4-parameter logistical fitting in XLFit.
Example 4: Biochemical Assays
[00529] The potency of HC-5404 against four ISR kinases was evaluated in cell-free FRET-based biochemical assays, following manufacturer’s instructions (Carna).
Example 5: Protein Analysis
[00530] Samples were put in 500 pL of Novex lysis buffer consisting of 2X Novex Tris-Glycine SDS sample buffer (ThermoFisher LC2676), IX Phosphatase Inhibitor Cocktail III (Sigma P0044), IX PhosphataseArrest I (Gbioscience 113P-D), IX 2-mercaptoethanol (Gibco 21985023), IX Benzonase (EMD Millipore Corp 70746-3), and IX Protease inhibitor cocktail tablet (Roche/Sigma 11836170001). Samples were homogenized with the Polytron and heated for 10 min at 95 °C. They were then centrifuged at max speed for 10 min and the supernatant was transferred. Protein concentrations of samples were measured using the Pierce 660 nm Protein assay kit (Thermo Scientific 22660) according to the manufacturer’s instructions.
[00531] Western Blot analysis was performed on the SimpleWestern high-throughput protein analysis platform (ProteinSimple 004-650) according to manufacturer’s protocol using a 12-230 kDA Separation Module (ProteinSimple SM-W004) and Total Protein Detection Module (ProteinSimple DM-TP01). Antibodies used were: (Add info for other antibodies used) pPERK (Lilly; 1 :50) and PERK (Cell Signaling Technology 5683; 1 :200). All protein analytes were normalized to total protein loaded on capillary.
Example 6: Immunohistochemistry and Image Analysis
[00532] Formalin-fixed paraffin-embedded samples were sectioned at 5 pm thickness and mounted on Superfrost Plus microscope slides (Fisher Scientific). All immunohistochemical (IHC) staining was performed on Bond Rx auto-stainer (Leica Biosystems) using the conventional TSA- amplified detection system. Primary antibodies used were as follows: a-CD31 (Abeam #ab 182981), a-smooth muscle actin (SMA; Abeam #abl24964), a-Meca32 (Novus Biologicals #NB 100-77668). TSA-conjugated Alexa488 and Alexa647 fluorophores from Invitrogen (#B40953 and #B40958) were used at 1 :500 dilution. Slides were counterstained with DAPI and cover slipped using Mowiol antifade mounting media (Sigma #D2522).
[00533] Slides were imaged with Aperio Versa 200 (Leica Biosystems) whole-slide scanner using 10x/0.32NA objective, and the data were analyzed using custom-written macros in ImageJ/FUI (NIH). Briefly, 150 pm of outer edges of the tumor sections were eliminated from the analysis. Gaussian-blurred DAPI channel was used to measure the area of the tissue. Scans of 104envat- staining were processed with median filter to remove noise, then positive areas were measured with appropriate threshold levels. % positive area for each marker was calculated. CD31-SMA double positive areas were measured via image calculation of the binary images. One-way ANOVA and pair-wise comparisons were performed on the data using Prism GraphPad.
Example 7: In Vivo Mouse Tumor Studies
Animals
[00534] Animals were allowed to acclimate for 7 days upon arrival in animal facility. Mice were housed in a 12-hour light/dark cycle facility under pathogen-free conditions with standard laboratory chow and water ad libitum.
Xenograft Models
[00535] Xenograft RCC models were generated as follows: Female NOD.SCID mice (6-8 weeks of age) were injected with A498 RCC cells (5xl06 cells/mouse), female Balb/c Nude mice (6-8 weeks old) were implanted with 786-0 RCC cells (5xl06 cells/mouse) or Caki-1 RCC cells (6xl06 cells/mouse), female Athymic Nude-Foxnlnu mice (7-8 weeks of age) were implanted with MFE280 endometrial cells (10xl06 cells/mouse). Cells were implanted into the flank of animals.
PDX models
[00536] For patient derived xenograft model generation, small pieces of neuroendocrine pancreatic cancers were implanted into female NSG mice (6-8 weeks of age), fragments of Huprime gastric cancers were implanted into female Balb/c nude mice and for a single animal trial in RCC PDX models, tumor fragments from selected RCC PDX models were implanted into mice unilaterally.
Treatments
[00537] Once tumor tumors reached volume of typically 150-250 mm3, animals were randomized by tumor volume into the treatment groups of 8 or 10 animals per group. In the single animal trial with RCC PDX models, only one animal per group was enrolled. The treatment groups typically included the vehicle group (10 pl/g body weight, orally (PO), twice per day (BID)), HC-5404 (3, 10 or 30 mg/kg, PO, BID), cabozantinib (30 mg/kg, PO, daily (QD)), axitinib (30 mg/kg, PO, BID), 104envatinib (10 mg/kg, PO, BID), sunitinib (20 mg/kg, PO, QD) or DC101 (15 mg/kg, 15 mg/kg or 20 mg/kg, intraperitoneally (IP), twice per week (BIW)).
[00538] Body weight and tumor volume were measured twice weekly. Tumor volume was calculated using the formula [tumor volume (mm3) = JT/6 x length x width2] and plotted as means + SEM. The percentage of tumor growth inhibition was calculated at the end of the study using the formula: 100 - [1 - (AT/AC)], if AT > 0, AT, mean tumor volume of the drug-treated group on the observation day of the study - mean tumor volume of the drug-treated group on initial day of dosing; AC, mean tumor volume of the control group on the observation day of the study - mean tumor volume of the control group on initial day of dosing. Regression was calculated using the formula = 100 x AT/Tinitial, if AT < 0. Animals with >50% regressions were partial responders. Animals with tumor volume of 0 (no measurable tumor) were considered as complete responders, and animals with <50% tumor regression and 30% growth over the baseline were considered as stable disease. Animals with tumors >30% increase over the baseline were considered progressive disease. The % change in body weight was calculated by the formula (body weight on observation day - body weight on initial day)/body weight on initial day x 100%.
Multiple In Vivo Studies
[00539] At the start of the study, 116 mice harboring 786-0 xenografts (-200 mm3) began treatment with axitinib as a single agent (30 mg/kg, PO; BID). After 14 days of treatments, 77 mice bearing the largest tumors were rerandomized into four PD arms (n=8), four efficacy arms (n=10), and 5 tumors sampled on day of rerandomization as a baseline. The remaining animals were removed from study. The four treatment groups were vehicle, HC-5404 (30 mg/kg, PO; BID), axitinib (30 mg/kg, PO; BID), or the combination thereof, which continued for 28 days.
Example 8: HC-5404 is a Potent and Selective PERK Inhibitor
[00540] PERK is one of four closely related kinases of the integrated stress response (ISR) that phosphorylate eIF2a in response to cellular stress. Both tumorigenesis and treatment with many anticancer agents have been shown to activate ISR, and hypoxic stress is associated with activation of PERK signaling as an adaptive response. To understand the specific role of PERK in RCC, the aminopyridine PERK inhibitor HC-5404 was investigated.
[00541] Cell-free biochemical assays were used to evaluate the selectivity of HC-5404 for PERK, relative to the ISR kinases GCN2, HRI, and PKR. In these assays, recombinant target kinases were incubated with ATP and an eIF2a-based fluorogenic substrate, and kinase activity was evaluated in presence of HC-5404 across a concentration series. These experiments revealed an IC50 for PERK of 1 nM, representing a >2000-fold biochemical selectivity over GCN2 (IC50 = 2.17 pM), HRI (IC50 = 2.96 pM) and PKR (IC50 > 10 pM) (FIG. 12, FIG. 13, FIG. 14, FIG. 15, FIG. 16).
[00542] The selectivity of HC-5404 against the broader kinome was then evaluated using a KINOMEScan biochemical panel assay that measured binding against 468 unique kinases, including 403 wild-type human kinases and 59 mutant isoforms. Notably, the kinome panel does not include PERK (also called EIF2AK3). HC-5404 was evaluated at 100 nM, 1000 nM, and 10,000 nM, which revealed a high level of selectivity across the kinome (FIG. 17). There were no interactions observed when HC-5404 was assayed at 100 nM, whereas BLK and the atypical kinase RIOK2 were inhibited at 1000 nM and only 9 kinases inhibited >65% at 10,000 nM HC-5404.
[00543] The cell -based activity of HC-5404 was evaluated using HEK293 treated with tunicamycin (Tm; 1 pM) to induce ER stress. Tm treatment resulted in PERK autophosphorylation at Thr982 (pPERK) and increased ATF4 levels, both of which were inhibited by HC-5404 in a concentration-dependent manner (IC50 = 23 nM and 115 nM, respectively; FIG. 12, FIG. 13, FIG.
14, FIG. 15, FIG. 16)
[00544] Taken together, these results demonstrate that HC-5404 is a potent, selective, and cell active PERK kinase inhibitor.
Example 9: In vivo Characterization of HC-5404
[00545] Initial in vivo studies aimed at understanding the pharmacokinetic and pharmacodynamic (PK/PD) relationship and optimizing the dosing regimen were conducted. HC-5404 was quantified from mouse plasma following a single oral administration (PO) at doses ranging from 3 to 100 mg/kg. Dose-proportional increases in exposure were observed up to 100 mg/kg, with Cmax before 1 h and average half-life of 2.22 h. Mouse plasma protein binding (PPB%) was determined to be 95.6%, which enabled us to calculate the free drug plasma exposure across time (FIG. 18). Oral dosing at 30 mg/kg resulted in free drug exposure of 355 nM at Cmax, well below the 1000 nM concentration assayed in the kinome scan that revealed minimal secondary binding.
[00546] The PK/PD relationship was evaluated in mouse pancreas, as the pancreas has elevated pPERK activity that makes it suitable for PD analysis. Treatment with HC-5404 resulted in a timedependent and dose-dependent inhibition of p-PERK in pancreas tissue. When administered at 30 mg/kg, HC-5404 reached 3668 ng/mL (355 nM, unbound) in plasma at 1 h, as shown in table below. At these concentrations, pPERK inhibition was approximately 90% (FIG. 19). As HC- 5404 is cleared from plasma, pPERK gradually increased until basal levels were restored by 12 h after dosing.
Figure imgf000108_0001
[00547] Single oral administration at 100 mg/kg achieves a proportional increase in exposure, reaching 16583 ng/mL (1605 nM, unbound) at Cmax (FIG. 20). At this higher dose level, HC-5404 exposure remains above 3200 ng/mL (311 nM, unbound) in plasma up to 12 h after dosing, resulting in pPERK inhibition (<20% activity) for the first 12 h following treatment.
[00548] The ability of HC-5404 to suppress tumor growth was evaluated in the 786-0 xenograft model of RCC. The 786-0 tumor model is homozygous for a frameshift mutation following Glyl04 in von Hippel -Lindau protein (VHLmut), resulting in HIF stabilization and activation of UPR, including elevated pPERK. Similar to the pancreas, administration of 30 mg/kg HC-5404 induced -90% pPERK inhibition in 786-0 tumors at 1 h post dose, which returned to baseline by 8 h (FIG. 21).
[00549] To evaluate the single-agent activity of HC-5404 and explore the effect of fractioning the dose between QD and BID dosing, mice harboring 786-0 xenografts were treated with 3, 10, or 30 mg/kg PO, BID dosing alongside 6, 20, or 60 mg/kg PO, QD dosing to evaluate the effect of the dose regimen on tumor growth. Although the differences between BID and QD dosing were not statistically significant, the general trend indicated better antitumor effects with BID dosing. Of the doses tested, 30 mg/kg BID induced the greatest effect at 48% tumor growth inhibition (FIG. 22). Based on this result, 30 mg/kg BID dosing was used for subsequent in vivo tumor studies.
[00550] The dose levels of HC-5404 was further explored using Capan-2 pancreatic tumor xenografts, a model previously reported to be sensitive to PERK inhibition, were treated with 30 and 100 mg/kg HC-5404 BID, which resulted in TGI of 50% and 43%, respectively (FIG. 23). No difference between the dose levels was observed, suggesting that increasing doses above 30 mg/kg, when administered twice per day, was not likely to improve the single agent activity of HC-5404. As PERK inhibition has been associated with pancreatic toxicity, pancreas sections were evaluated from mice treated with 30 mg/kg or 100 mg/kg HC-5404. This revealed histological changes in the pancreas only in the 100 mg/kg dose, which was reversible with a two- week washout period (FIG. 24). Together, these findings further supported the decision to dose at 30 mg/kg HC-5404 in subsequent in vivo studies.
Example 10: HC-5404 Sensitizes RCC Tumor Models to VEGFR-TKIs
[00551] Receptor tyrosine kinase inhibitors that target the VEGF receptor (VEGFR-TKIs) are anti angiogenic agents that disrupt tumor vascular development as a critical component of their mechanism of action, driving tumor hypoxia and nutrient deprivation. Hypoxia and glucose deprivation are known drivers of ER stress and PERK pathway activation in tumors. [00552] The effects of three second-generation VEGFR-TKIs on tumor growth and PERK activation were evaluated. Multiple dose levels of cabozantinib (15, 30 and 60 mg/kg), lenvatinib (5 and 10 mg/kg), or axitinib (15 and 30 mg/kg) were orally administered to mice harboring 786-0 xenografts for 21 days, after which the tumors were resected and analyzed for pPERK levels. All three VEGFR-TKIs induced dose-dependent increases in pPERK that reflected the level of tumor growth inhibition (FIG. 26). The doses used were well tolerated, as no treatment-associated body weight loss was observed (FIG. 26). Sunitinib was evaluated in 786-0 xenografts and also demonstrated increased pPERK. This effect increased over time (FIG. 27). Based on these experiments, dose regimens were selected for follow-up combination studies.
[00553] Next, studies evaluating the effects of the addition of HC-5404 were conducted. Axitinib, cabozantinib, lenvatinib and sunitinib were each tested in combination with HC-5404 in studies with 786-0 RCC xenografts. All studies included a PD arm that was sampled after 7 days of dosing to evaluate the effect of treatments on angiogenesis and pPERK levels. Consistent with initial dosedetermining studies, the PD arms of the experiments revealed that all VEGFR-TKIs induced pPERK accumulation, which was inhibited by the addition of HC-5404 (FIG. 27).
[00554] In the efficacy arms of each tumor growth study, HC-5404 and the second generation VEGFR-TKIs (axitinib, lenvatinib, and cabozantinib) resulted in modest effects on tumor volume. In contrast, when the VEGFR-TKIs were combined with HC-5404, tumors regressed relative to baseline (FIG. 28). At the dose level tested, sunitinib was a more effective inhibitor of 786-0 tumors compared to the second generation of VEGFR-TKIs. Sunitinib as a single agent was an effective inhibitor of 786-0 growth, yet combination with HC-5404 still enhanced the level of TGI, underscoring the mechanistic interplay between the VEGFR-TKIs and PERK inhibitors, and highlighting the therapeutic benefit that might be achieved by combining VEGFR-TKIs with HC- 5404. HC-5404 sensitizes RCC tumor models to anti angiogenic VEGFR-TKIs and enhance their antitumor effect.
[00555] The effect of HC-5404 was further validated in tumors by assessing the response of markers of in vivo PERK inhibition. Previous studies have highlighted a surprising induction of downstream targets of ATF4, including asparagine synthetase (ASNS) and phosphoserine aminotransferase (PSAT1). Other canonical targets of ATF4, including CBS and CTH, were investigated. Treatment with HC-5404 induced a dose-dependent accumulation of ASNS, CBS and CTH in 786-0 tumors (FIG. 29). Example 11: Sensitivity to VEGFR-TKIs and HC-5404 Combination is Independent of VHL Mutation Status
[00556] RCC is frequently characterized by mutations in and epigenetic silencing of VHL, which results in dysregulated HIF expression that drives a proangiogenic tumor environment associated with increased VEGF expression and activated UPR. VHL mutation status, pPERK expression, and sensitivity to VEGFR-TKIs may be linked. Three RCC tumor models were investigated: 786-0 and A498 (Glyl44 frameshift) were selected as representative VHL mutant models, and Caki-1 model was chosen as a VHL wild-type model.
[00557] The models were initially characterized to assess basal levels of pPERK levels. Consistent with the HIF pathway activation resulting from VHL mutation driving ER stress, 786-0 and A498 tumors had elevated levels of pPERK in the vehicle group (FIG. 30). In contrast, pPERK was barely detectible in Caki-1. As expected, treatment with HC-5404 strongly inhibited pPERK expression across the three models (FIG. 30).
[00558] The presence of basal PERK pathway engagement may predict sensitivity to PERK inhibition, and sensitivity to HC-5404 was investigated in three RCC models. Sensitivity to HC- 5404 varied across the three xenograft models in a manner that reflected their basal levels of pPERK activity and VHL status. HC-5404 induced TGI of approximately 52% and 41% in 786-0 and A498, respectively, whereas HC-5404 had minimal effect on the growth of the Caki-1 model (FIG. 36)
[00559] To evaluate differences in sensitivity to PERK inhibition and VEGFR-TKI treatments, A498 and Caki-1 models were treated with HC-5404, sunitinib, or a combination thereof for a 28- day period. The level of tumor response across the three models was reflective of the genetic background: the VHL mutant A498 was sensitive to HC-5404 as a single agent, although the TGI only reached -40% (FIG. 31). Similarly, the A498 model was sensitive to sunitinib with robust single agent activity that resulted in 80% TGI. When sunitinib and HC-5404 were combined in the VHL mutant model, tumor regression of 22% was observed in A498 (FIG. 31). In contrast, Caki-1 was far less sensitive to either HC-5404 or sunitinib as monotherapy, and although an effect of the combination treatments was observed, the TGI only reached 47% in the combination group (FIG.
32).
[00560] The observation that Cakil was less responsive to HC-5404 and sunitinib treatment suggested that VHL mutation status could be used as a selection criterion that predicts sensitivity to treatment. Accordingly, 15 diverse RCC patient-derived xenografts (PDXs) were evaluated in a single-animal study that directly compared models with wild-type and mutant VHL. Animals were treated to axitinib, HC-5404, or a combination thereof for 28 days, and scored for the magnitude of combination benefit observed. All five VHLwt models that were included in the study responded to the combination treatment with significant improvement over monotherapy (representative growth charts in FIG. 33 and FIG. 34). The models were ranked based on change in tumor volume relative to starting baseline and scored for the number of partial responses (PRs). In this case, PR was considered as a regression in volume greater than 50% compared to starting baseline. While axitinib as a monotherapy resulted in 1/15 PRs, the combination of HC-5404 with axitinib resulted in 7/15 PRs (FIG. 35). Importantly, the VHLwt models retained responsiveness to the combination treatment, although three of the models appeared to have decreased sensitivity to axitinib. From this study, it was concluded that HC-5404 improves tumor response to VEGFR-TKI across diverse tumor contexts, and this effect is independent of VHL mutation status.
Example 12: HC-5404 Demonstrates Combination Benefit with Other Agents Targeting Angiogenesis without Induction of Tumor pPERK
[00561] While the common target associated with anti angiogenic activity is inhibition of the VEGF axis, the secondary targets of the VEGFR-TKIs may improve antitumor activity. To test whether inhibition of the VEGF/VEGFR axis is sufficient to drive the combination benefit with HC-5404 or whether secondary effects were necessary, a mouse monoclonal antibody that selectively targets VEGFR-2 was investigated.
[00562] DC-101 is a surrogate mAb to human anti-VEGFR2 mAb ramucirumab, that is approved for use in multiple cancer types. DC-101 was tested to determine whether treatment with HC-5404 could enhance the activity of DC-101 across three models of RCC (786-0, A498, and Caki-1). Similar to the observations of HC-5404 with multi-kinase-targeted TKIs, the combination treatment significantly improved the effects of each monotherapy in 786-0 and A498 xenografts with TGI values of 82% and 78%, respectively (FIG. 37). In contrast, cotreatment of DC-101 and HC-5404 did not exhibit significant combination benefits resulting in a TGI value of only 24% in Caki-1 following 28 days of treatment (FIG. 37). The combination treatments did not result in tumor regression in any of the models tested.
[00563] The effect of DC-101 on PERK activation was evaluated in tumor samples resected after 4 days of treatment, and protein lysates analyzed to determine pPERK levels. DC-101 did not affect pPERK levels in any of the three RCC models tested (FIG. 37). In 786-0 and A498 models, pPERK was unchanged compared to the vehicle group, while HC-5404 inhibited the pPERK signal, as expected. pPERK was variable across the Cakil samples, as the data had low signal -to- noise and not affected by either DC-101 or HC-5404 treatments. Example 13: PERK Enhances the Antiangiogenic Effects of VEGFR-TKIs
[00564] PERK is an adaptive stress response that links hypoxia and nutrient deprivation with proangiogenic signaling. The effect of HC-5404 on tumor vasculature when administered in combination with antiangiogenic VEGFR-TKIs was investigated. H4C staining of tumor crosssections confirmed that axitinib decreased the proportion of Meca32 (also called PLVAP) expressing vascular endothelial cells, and that this effect was enhanced by the addition of HC-5404 (FIG. 38, FIG. 39). Notably, HC-5404 alone did not affect this vascular endothelium marker, highlighting a context-dependent effect of HC-5404 on tumor angiogenesis.
[00565] As immature blood vessels mature, they develop a mural layer of cells that express smooth muscle actin (SMA), which encircles and stabilizes the maturing vessel. Co-staining for both CD31 and SMA was conducted to determine whether treatments were affecting mature or immature tumor blood vessels. Similar to effects observed with the endothelial marker Meca32, axitinib as a single agent decreased the proportion of immature CD31+SMA- vessels, which was enhanced in the combination treatment group (FIG. 39). In contrast, axitinib did not affect the number of mature CD31+SMA+ cells at the dose and time points analyzed, consistent with the predominant role that VEGF axis plays in the production of new vasculature through sprouting angiogenesis. HC-5404 as a single agent did not affect the expression of any vascular markers, whereas the combination treatments resulted in a significant decrease in mature CD31+SMA+ blood vessel cells (FIG. 39).
Example 14: PERK is a Vulnerability for RCC Xenografts That Have Progressed on VEGFR-TKIs
[00566] Despite initial responses to VEGFR-TKI therapy, tumor regrowth necessitates subsequent rounds of alternate therapies to improve patient outcomes. This effect was modeled by challenging 786-0 xenografts to progress on axitinib, and then observing whether adding HC-5404 to axitinib treatment could maintain the combination benefits in this setting.
[00567] A period of growth in presence of VEGFR-TKI could drive heightened sensitivity to the PERK inhibitor. Tumors that were transferred to the vehicle group experienced an increase in growth rate, suggesting that although the tumors had advanced on axitinib, some sensitivity to treatment remained. Groups that were transferred to single agent axitinib or HC-5404 progressed at approximately the same rate as prior to rerandomization. By the end of study, single agent activity of HC-5404 or axitinib inhibited tumor growth by 47% or 38%, relative to vehicle (FIG. 40). When HC-5404 was added to the axitinib regimen to establish a combination group, xenografts regressed in volume following a brief period of continued growth, resulting in average tumor regression of
Ill 20% relative to baseline (FIG. 40). Thus, tumors that have previously progressed on axitinib retain sensitivity to HC-5404 when administered in combination with the VEGFR-TKI.
[00568] Analysis of tumor vasculature revealed that the combination treatments inhibited both immature and mature blood vessels, including pericyte cells. Consistent with earlier findings, axitinib decreased the number of Meca32+ and CD31+ cells in xenograft sections, which was enhanced by the addition of HC-5404 in the combination group (FIG. 41, FIG. 42).
[00569] The pericytes form a layer of mural cells that surround blood vessels, and are important for vascular morphogenesis and branching. Two pericyte markers (NG2 and MCAM) were used to evaluate the effect of axitinib and HC-5404 on this layer. The proportion of cells that stained positive for NG2+ decreased only in the combination group relative to vehicle, highlighting the impact of the combination groups on this pericyte marker. MCAM+ cells were decreased by axitinib, and this effect was enhanced by the addition of HC-5404 (FIG. 43, FIG. 44). In both cases, the proportion of NG2+ and MCAM+ cells were decreased relative to the baseline samples, suggesting that following rerandomization the tumors in the combination group underwent a loss of mature vascular cells, rather than simply inhibition of neovascular growth. HC-5404 and axitinib combination inhibits markers of tumor vasculature endothelium and pericyte cells.
Example 15: Adaptive Stress Drives Resistance to VEGFR Inhibitors
[00570] Anti angiogenic therapies enhance tumor hypoxia. Tumor cells adapt to hypoxia via activation of adaptive stress pathways, notably PERK, which leads to cell survival.
[00571] 786-0 RCC tumor xenografts were treated with cabozantinib (15, 30, 45 mg/kg; PO; QD), lenvatinib (5 and 10 mg/kg; PO; QD), and axitinib (15 and 30 mg/kg; PO; BID) for 21 days, following analogous procedures as described in the examples above. In vivo characterization of VEGFR-TKIs are shown in the tumor growth curves and pPERK/PERK levels as shown in FIG. 45, FIG. 46, and FIG. 47
Example 16: HC-5404 Potentiates Antiangiogenic Agents in 786-0 RCC Xenograft Models [00572] This example investigated the inhibition of PERK-mediated stress adaptation using HC- 5404. HC-5404 combines with VEGRF-TKIs to inhibit tumor growth in 786-0 xenograft models of RCC.
[00573] 786-0 tumor xenografts were treated with once daily oral administration of lenvatinib (10 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof and twice daily oral administration of axitinib (30 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof following analogous procedures as described in the examples above. Tumor growth curves are shown in FIG. 48 and FIG. 49. A498 tumor xenografts were treated with once daily oral administration of sunitinib (20 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof and twice weekly intraperitoneal injection of DC-101 (15 mg/kg), twice daily oral administration of HC-5404 (30 mg/kg), or a combination thereof following analogous procedures as described in the examples above. Tumor growth curves are shown in FIG. 50 and FIG. 51.
[00574] 786-0 tumor xenografts treated with twice daily oral administration of HC-5404 at 3, 10, or 30 mg/kg as a single agent or in combination with once daily oral dosing of cabozantinib at 30 mg/kg following analogous procedures as described in the examples above. Tumor growth and body mass curves are shown in FIG. 52 and FIG. 53. HC-5404 at 3, 10 and 30 mg/kg, PO, BID showed a trend for dose-dependent antitumor efficacy with the maximum efficacy at 30 mg/kg with a TGI% value of -61% while cabozantinib monotherapy resulted in tumor stasis with 2/8 PRs in this model. The combination treatment with 10 and 30 mg/kg of HC-5404 significantly improved the effects of cabozantinib alone and exhibited similar efficacy resulting in average tumor regression of -66% with 8/8 PRs in each group. No treatment-related body weight loss was observed. Treatment with HC-5404 or cabozantinib, either as monotherapy or in combination, did not affect mouse body weight, suggesting the treatments are well tolerated.
Example 17: Cabozantinib Inhibits Formation of New Tumor Vasculature and Disrupts Pericytes
[00575] This example investigated the inhibition the effect of cabozantinib on the formation of new tumor vasculature and on pericytes following procedures analogous to those described above.
[00576] Cabozantinib induced near complete loss of immature vasculature (CD31+SMA- cells). HC-5404 decreased mature CD31+SMA+ vascular cells as a trend only in presence of cabozantinib. New vascular formation is highly dependent on VEGF signaling. Images of IHC staining are shown in FIG. 54, and FIG. 55 shows quantification of the IHC staining.
[00577] Pericyte is a stabilizing cell layer only associated with mature blood vessels. HC-5404 and cabozantinib combination decreased pericyte markers NG2 and MCAM. Statistical comparisons only shown for significant differences between vehicle and treatment groups. MCAM trend was observed for Cabo+HC-5404 groups. Images of IHC staining are shown in FIG. 56, and FIG. 57 shows quantification of the IHC staining.
Example 18: Triplet Combination Induces Anti-Tumor Immune Response
[00578] This example investigated the effect of treating tumors with a combination of HC-5404, an anti angiogenic agent, and an immunotherapy following procedures analogous to those described above. HC-5404 showed improved efficacy in combination with axitinib and anti-PD-1 antibody in RENCA tumor-bearing BALB/c mice.
[00579] Mice were inoculated subcutaneously with syngeneic RENCA RCC tumor cells. The indicated treatments were initiated on Day 6 post cell inoculation following randomization for equivalent starting tumor size. Tumor volume (FIG. 58) and body weight (FIG. 59) were monitored over time and are displayed. PO = oral gavage; IV = intravenous administration; BID = twice daily dosing; BIW = twice per week dosing; ICI = immune checkpoint inhibitor (anti-mouse PD-1 antibody).
[00580] HC-5404-FU + Axitinib and anti-PDl + Axitinib (front line - SOC) combinations provide comparable antitumor activity in syngeneic RCC model RENCA (VHLwt). Triplet combination (HC-5404 + Axitinib + anti-PDl mAb) provides enhanced efficacy over front line SOC Treatments were well-tolerated with no significant body weight loss observed in any of the groups (FIG. 58 and FIG. 59).
Example 19: HC-5404 and VEGFR-TKI Combination Induces Favorable Changes in Tumor- Infiltrating Myeloid Cells
[00581] This example investigated the effect of HC-5404 in combination with axitinib on RENCA RCC model following procedures analogous to those described above. Addition of HC-5404 to axitinib increased stimulatory macrophage phenotype and reduced axitinib -induced MDSC infiltration in mouse RENCA tumors.
[00582] Mice with established subcutaneous syngeneic RENCA RCC tumors were treated with vehicle (0.5% methylcellulose in water, BID, PO), HC-5404 (30 mg/kg, BID, PO), axitinib (30 mg/kg, BID, PO), or the combination of HC-5404 and Axitinib. Following 13 days of treatment, animals were euthanized, and tumors were evaluated by multicolor flow cytometry.
[00583] HC-5404 + Axitinib combination alters populations of macrophages and MDSCs in tumor (RENCA RCC model). Increased Ml macrophage polarization (MHCII+CD206-) compared to both monotherapies. Reduced monocytic MDSC frequency compared to Axitinib monotherapy HC-5404 + Axitinib combination increases activation markers on Ml macrophages in tumor. Increased CD86 compared to either monotherapy Increased MHCII compared to either monotherapy (FIG. 60, FIG. 61). Example 20: HC-5404 and VEGFR-TKI Combination Increases Activation and Reduces Exhausted Phenotype of T-Cells
[00584] This example investigated the effect of HC-5404 in combination with axitinib on CD4 T- Cells in tumor-draining lymph nodes following procedures analogous to those described above. Addition of HC-5404 to axitinib increased CD4 T-cell activation and effector phenotype in the draining lymph node while increasing TNFa and reducing exhaustion marker expression on CD4 T-cells in RENCA RCC tumors.
[00585] Mice with established subcutaneous syngeneic RENCA RCC tumors were treated with vehicle (0.5% methylcellulose in water, BID, PO), HC-5404 (30 mg/kg, BID, PO), axitinib (30 mg/kg, BID, PO), or the combination of both HC-5404 and axitinib. Following 13 days of treatment, animals were euthanized, and tumors and tumor-draining LN (TDLN) were evaluated by multicolor flow cytometry.
[00586] HC-5404 + Axitinib combination increases activated phenotype of CD4 T-cells in tumordraining lymph node. Increased early activation marker CD69. Increased effector memory phenotype (CD62L-CD44+) was observed. HC-5404 + Axitinib combination reduces exhausted phenotype on CD4 T-cells in tumor, increased TNFa and decreased exhaustion markers (EOMES, TIM-3, LAG-3) (FIG. 62).
Example 21: HC-5404 and VEGFR-TKI Combination Reshapes Immunosuppressive TME [00587] This example investigated the effect of HC-5404 in combination with axitinib on myeloid cells in tumor microenvironment following procedures analogous to those described above.
Combination treatment of HC-5404 and axitinib decreased M2 markers and increased Ml markers on macrophages in RENCA RCC tumors.
[00588] Mice bearing subcutaneous RENCA RCC tumors were treated for 7 days with vehicle (0.5% methylcellulose in water, BID, PO), HC-5404 (30 mg/kg, BID, PO), axitinib (30 mg/kg, BID, PO), or the combination of both HC-5404 or axitinib. Following 7 days of treatment, tumor sections were formalin-fixed, paraffin-embedded, and analyzed via fluorescence microscopy for the M2 marker CD206 (FIG. 64, left), and the Ml markers CD86 (FIG. 64, middle) and PD-L1 (FIG. 64, right) on macrophages.
[00589] HC-5404 + Axitinib combination increases activated phenotype myeloid cells in tumor microenvironment. There was a decrease in CD206 (M2 marker) and increase in CD86 and PD-L1 (Ml markers) in RENCA RCC model (IHC) (FIG. 63, FIG. 64). Example 22: HC-5404, VEGFR-TKI, and Immune Checkpoint Inhibitor Combination Increases Ml and Reduces M2 Macrophages in TME
[00590] This example investigated the effect of HC-5404 in combination with axitinib and aPD-1 on RENCA RCC model following procedures analogous to those described above. HC-5404 + axitinib + aPD-1 triple combination showed trend of increased Ml macrophages and decreased M2 macrophages compared to dual combinations.
[00591] Mice with established subcutaneous syngeneic RENCA RCC tumors were treated with vehicle (0.5% methylcellulose in water, BID, PO), HC-5404 (30 mg/kg, BID, PO), axitinib (30 mg/kg, BID, PO), anti-PD-1 Ab (10 mg/kg, BIW), or combinations of these treatments. Following 13 days of treatment, animals were euthanized, and tumors were evaluated by multicolor flow cytometry.
[00592] Treatment with HC-5404 + axitinib + aPD-1 triple combination resulted in the following: Ml -like macrophages are increased and M2 -like macrophages are decreased beyond either duo treatment (FIG. 65, FIG. 66).
Example 23: HC-5404, VEGFR-TKI, and Immune Checkpoint Inhibitor Combination Increases T-Cell Infiltration and Cytokine Expression in TME
[00593] This example investigated the effect of HC-5404, axitinib, aPD-1, and HC-5404 in combination with axitinib and aPD-1 on T-Cell infiltration following procedures analogous to those described above. HC-5404 + axitinib + aPD-1 triple combination increased CD8 T-cell tumor infiltration and expression of stimulatory cytokines.
[00594] Mice with established subcutaneous syngeneic RENCA RCC tumors were treated with vehicle (0.5% methylcellulose in water, BID, PO), HC-5404 (30 mg/kg, BID, PO), axitinib (30 mg/kg, BID, PO), anti-PD-1 Ab (10 mg/kg, BIW) or combinations of these treatments. Following 13 days of treatment, animals were euthanized, and tumors were evaluated by multicolor flow cytometry.
[00595] T-cell infiltration and activation was observed with HC-5404 + axitinib + ICI triple combination treatment. Increased frequency and number of CD8 T-cells were observed, as well as increased expression of inflammatory cytokines on CD8 T-cells, including IFNy and TNFa. These increases were greater than any monotherapy or duo combination (FIG. 67). Example 24: HC-5404 and Immune Checkpoint Inhibitor Combination Improves Efficacy and Immune Activation in Tumor Microenvironment
[00596] This example investigated the effect of HC-5404, aPD-1, and HC-5404 in combination with aPD-1 on myeloid cells in tumor microenvironment and T-cell activation following procedures analogous to those described above using MB49 syngeneic model. HC-5404 + anti-PD- 1 combination showed improved efficacy and immune stimulation in syngeneic MB49 model. Mice with established syngeneic MB49 tumors were randomized 6 days post tumor inoculation into groups that received either vehicle (0.5% methylcellulose in water, BID, PO), HC-5404 (10 or 30 mg/kg, BID, PO), anti-PD-1 Ab (10 mg/kg, BIW), or both HC-5404 and anti-PD-1.
[00597] Combination efficacy of HC-5404 + aPD-1 was observed in multiple studies. Myeloid activation: increased type 1 interferon receptor (IFNAR1) expression on macrophages and MDSCs. T-cell activation: increased cytotoxic T and NK infiltration into tumor tissue and increased DC frequency and T-cell activation in draining lymph node (FIG. 68, FIG. 69).
Example 25: Single Agent Efficacy of HC-5404
[00598] This example investigated the effect of HC-5404 on RT4 Bladder, HCT116 CRC, A549 NSCLC, and A549 NSCLC following procedures analogous to those described above (FIG. 70, FIG. 71, FIG. 72, FIG. 73)
Example 26: HC-5404 Sensitizes Antiangiogenics Across Multiple Indications
[00599] This example investigated the effect of HC-5404 in combination with anti angiogenic agents for endometrial cancer, gastric cancer, and pancreatic cancer following procedures analogous to those described above. HC-5404 combines with anti angiogenic agents in tumor models of endometrial cancer, gastric cancer and pancreatic neuroendocrine cancers.
[00600] HC-5404 synergized with lenvatinib to induce an average 34% tumor regression in MFE280 MSS model of endometrial cancer (FIG. 74).
[00601] The combination of HC-5404 and DC-101, a surrogate VEGFR2 antibody for ramucirumab, resulted in tumor growth inhibition of 80% in GAO 114 PDX model of gastric cancer (FIG. 75).
[00602] The combination of HC-5404 and sunitinib resulted in tumor growth inhibition of -50% in a TM01125 PDX model of pancreatic cancer (FIG. 76). Example 27: HC-5404 and Antiangiogenic Agent Efficacy on Human Endometrial Cancer Model
[00603] This example investigated the effect of HC-5404 in combination with Lenvatinib in MFE280 human endometrial cancer model following procedures analogous to those described above.
[00604] HC-5404 at 30 mg/kg, PO, BID showed only a trend for tumor growth inhibition with a TGI% value of -37%, while lenvatinib significantly inhibited tumor growth with a TGI% value of -85%. The combination treatment resulted in average tumor regression of -34% (P>0.05 vs. Lenvatinib). Treatments were well tolerated: no treatment related BWL was observed. On day 7, 2 h post dose, tumors from 6 animals per group were collected: 1/3 FFPE, 1/3 Frozen and 1/3 RNALater (FIG. 77, FIG. 78).
Example 28: ASNS and PSAT1 Induced by HC-5404 and Antiangiogenic Agent Human Endometrial Cancer Model
[00605] This example investigated the effect of HC-5404 in combination with lenvatinib in MFE280 human endometrial cancer model following procedures analogous to those described above.
[00606] MFE280 tumors sampled after 7 days of treatment: HC-5404 30 mpk BID; Lenvatinib 10 mpk QD. HC-5404 as single-agent or in combination with Lenvatinib, induces ASNS in endometrial cancer tumor model MFE280 (FIG. 79) and PSAT1 in endometrial cancer tumor model MFE280 (FIG. 80). PD modulation consistent across RCC and endometrial (MSS) cancer model.
Example 29: HC-5404 Induces ATF4 Targets In Gastric PDX Model
[00607] This example investigated the effect of HC-5404 in combination with DC-101 in gastric PDX model following procedures analogous to those described above. HC-5404 induces proteins involved in amino acid metabolism in gastric PDX (GA0114-R19P6).
[00608] Gastric PDX model (GA0114-R19P6) PD arm sampled after 7 days, Ih post-dose.
Similar to observations in ccRCC and endometrial models, HC-5404 induces ATF4 target genes, including ASNS, PSAT1 and other enzymes involved in amino acid metabolism (FIG. 81, FIG. 82). Example 30: HC-5404 and ICIs
[00609] This example investigated the effect of HC-5404 in combination with immune checkpoint inhibitor in bladder cancer, PDAC, and prostate cancer following procedures analogous to those described above.
[00610] HC-5404 shows combination benefit in bladder cancer and melanoma with immune checkpoint inhibitor, anti-PDl, -50% increased TGI benefit over ICI alone. Combination with docetaxel in neuroendocrine (castration-resistant) prostate cancer leads to 45% TGI benefit. Addition of HC-5404 with gemcitabine in PDAC leads to a delayed recurrence compared to gemcitabine alone (FIG. 83, FIG. 84, FIG. 85).
Example 31: HC-5404 and VEGFR-TKI Combination Decreases Pro-angiogenic cytokines [00611] This example investigated the effect of HC-5404 in combination with VEGFR-TKIs in modulating pro-angiogenic cytokines in plasma and in tumor tissue following procedures analogous to those described above. HC-5404 and cabozantinib decrease pro-angiogenic cytokines in plasma and in tumor tissue.
[00612] Mouse plasma sampled following 7 days of treatment with HC-5404 (30 mpk, PO, BID) and cabozantinib (30 mpk, PO, QD). Multiple cytokines associated with angiogenesis were quantified by Illumina multiplex. Decreases in multiple pro-angiogenic cytokines were observed.
[00613] Plasma analysis from mice bearing 786-0 tumors, treated with HC-5404 at 3, 10, or 30 mg/kg (PO; BID) as a single agent or in combination with cabozantinib (30 mg/kg; PO; QD). Multiple cytokines associated with angiogenesis were quantified by Illumina multiplex (FIG. 86, FIG. 87, FIG. 88, FIG. 89, FIG. 90) Vascular endothelial growth factor-A (VEGF-A) was decreased in plasma by HC-5404 as a single agent in a dose-dependent fashion (FIG. 86). Hepatocyte growth factor (HGF) was decreased by the combination of HC-5404 and cabozantinib in mouse plasma (FIG. 87). Interleukin-8 (IL-8) was decreased by the combination of HC-5404 and cabozantinib in mouse plasma (FIG. 88). Lymphatic vessel endothelial hyaluronan receptor- 1 (LYVE-1) was decreased by HC-5404 in a dose-dependent manner as either monotherapy or in combination with cabozantinib (FIG. 89). Syndecan was decreased by HC-5404 as monotherapy or in combination with cabozantinib (FIG. 90).
Example 32: Tumors that Progress on Axitinib Retain Sensitivity To HC-5404 Combination Treatment
[00614] This example investigated the effect of HC-5404 in combination with VEGFR-TKIs in modulating tumor progression following procedures analogous to those described above. Tumors that progress on axitinib were sensitive to the addition of HC-5404 to the treatment regimen. [00615] Mice harboring 786-0 xenografts were treated for 14 days with axitinib (30 mg/kg; PO; BID), following which the mice with tumors that progressed (approximately doubled in size) were rerandomized into four groups as indicated. After enabling 786-0 xenografts to progress on axitinib for two weeks, HC-5404 was added to the regimen, resulting in 20% tumor regression and inhibition of several vascular markers associated with immature and mature blood vessels (FIG.
91, FIG. 92, FIG. 93)
[00616] HC-5404 and axitinib both decreased the proportion of smooth muscle actin (SMA) as monotherapy, which was further diminished by the combination treatment (FIG. 92). Proportion of cells expressing Meca32 was decreased in the combination group relative to vehicle. Meca32 is a marker of vascular endothelial cells (FIG. 92). Proportion of cells expressing the vascular marker CD31 is decreased by axitinib, an effect that is enhanced by addition of HC-5404 in the combination group (FIG. 92). Tumors randomized into the combination group have decreased proportion of cells expressing the pericyte markers NG2 and MCAM (FIG. 93).
Example 33: HC-5404 Combines with Antiangiogenic Therapies To Inhibit Tumor Growth [00617] This example investigated the effect of HC-5404 in combination with VEGFR-TKIs in inhibiting tumor growth following procedures analogous to those described above. Combination of HC-5404 with anti-VEGFR2 antibody DC 101 (mouse surrogate for ramucirumab) shows robust combinatorial benefit in RCC models.
[00618] HC-5404 combined with antiangiogenic monoclonal antibody (DC 101) selectively targeted mouse VEGFR-2. 786-0 and A498 RCC tumor models were treated with HC-5404 (30 mg/kg; PO; BID) and DC-101 (15 mg/kg; IP; BIW) for 28 days (FIG. 94, FIG. 95). No treatment- related BWL is observed in any of the groups.
Example 34: HC-5404 Shows Combination Benefit with VEGRD-TKIs in RENCA RCC Model
[00619] This example investigated the effect of HC-5404 in combination with VEGFR-TKIs in inhibiting tumor growth using RENCA RCC tumor model, following procedures analogous to those described above.
[00620] Renca Syngeneic mouse RCC tumor model treated with either HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID, or a combination thereof. HC-5404 + Axitinib shows modest increase in efficacy compared to Axitinib alone PD and immunophenotyping samples were collected on day 13, after 4h treatment (FIG. 96). Example 35: Efficacy of HC-5404 with and without VEGFR-TKIs in RCC PDX Models - A Single Animal Trial
[00621] This example investigated the effect of HC-5404 in combination with VEGFR-TKIs in inhibiting tumor growth using 12 RCC PDX tumor models, following procedures analogous to those described above.
[00622] Single animal per group (n=l) trial of animals harboring diverse RCC PDX models were used in this study. Animals were treated with either HC-5404 (30 mg/kg; PO; BID), axitinib (30 mg/kg; PO; BID) or a combination thereof.
[00623] HC-5404 and axitinib showed combination benefits in RXF-393 and RXF-2282 models (FIG. 97, FIG. 98). No combination benefits were observed in RXF-2178 and RXF-2502 RCC PDX models (FIG. 99, FIG. 100). Treatments were well-tolerated as determined by BW measurement. No treatment related BW loss was observed.
[00624] HC-5404 and axitinib showed combination benefits in SMTCA75, RXF-616, RXF-2304 and RXF-488 models (FIG. 101, FIG. 102, FIG. 103, FIG. 104), resulting in tumor stasis or regressions. HC-5404 as a monotherapy exhibited antitumor efficacy in SMTCA75 and RXF-2304 models. Treatments were well-tolerated as determined by BW measurement. No treatment related BW loss was observed.
[00625] HC-5404 and axitinib showed combination benefits in RXF-2667 and RXF-2783 models (FIG. 105, FIG. 107), resulting in tumor regressions, while no combination benefit was observed in RXF-1220 and RXF-631 models (FIG. 106, FIG. 108). HC-5404 as a monotherapy exhibited antitumor efficacy in RXF-2667 and RXF-2540 models. Treatments were well-tolerated as determined by BW measurement. No treatment related BW loss was observed.
INCORPORATION BY REFERENCE
[00626] This application refers to various issued patents, published patent applications, journal articles, and/or other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art. EQUIVALENTS
[00627] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMS WHAT IS CLAIMED:
1. A method of activating the immune system of a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I)
Figure imgf000125_0001
or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD- 1 antibody.
2. A method of activating the immune system of a subject having cancer, comprising administering to the subject a therapeutically effective amount of a compound of formula (I)
Figure imgf000125_0002
or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD- 1 antibody.
3. The method of claim 1 or 2, wherein the method inhibits the activity of myeloid-derived suppressor cells (MDSCs).
4. The method of any one of claims 1-3, wherein the method increases the infiltration of T cells and NK cells into a tumor.
5. The method of claim 4, wherein the tumor is selected from the group consisting of bladder cancer, squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer
6. The method of any one of claims 1-5, wherein the method increases the frequency of dendritic cells in a draining lymph node.
7. A method of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I)
Figure imgf000126_0001
or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti-PD- 1 antibody.
8. The method of any one of claims 2-7, wherein the cancer is a solid tumor.
9. The method of any one of claims 2-4 and 6-8, wherein the cancer is selected from the group consisting of bladder cancer, squamous cell head and neck cancer, Merkel cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, triple negative breast cancer, gastric carcinoma, endometrial carcinoma, urothelial carcinoma, and neuroendocrine cancer.
10. The method of any one of claims 2-4, 6, and 7, wherein the cancer is a liquid tumor.
11. The method of any one of claims 2-4, 6, 7, and 10, wherein the cancer is selected from the group consisting of Classical Hodgkin’s lymphoma, primary thymic mediastinal lymphoma, multiple myeloma, and B cell malignancies selected from non-Hodgkin lymphomas and chronic lymphocytic leukemia.
12. The method of any one of claims 1-11, wherein the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, and cemiplimab.
13. The method of any one of claims 1-12, wherein the anti-PD-1 antibody is pembrolizumab.
14. The method of any one of claims 1-12, wherein the anti-PD-1 antibody is nivolumab.
15. The method of any one of claims 1-14, wherein the therapeutically effective amount of the anti-PD-1 antibody is administered once daily.
16. The method of any one of claims 1-14, wherein the therapeutically effective amount of the anti-PD-1 antibody is administered twice daily.
17. The method of any one of claims 1-16, wherein the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered simultaneously.
18. The method of any one of claims 1-16, wherein the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti-PD-1 antibody are administered sequentially.
19. The method of any one of claims 1-18, wherein the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount of about 22 mg to about 451 mg twice daily.
20. The method of any one of claims 1-18, wherein the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount of about 177 mg to about 900 mg twice daily.
21. The method of any one of claims 1-20, wherein the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally.
22. The method of any one of claims 1-21, wherein the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered in the form of a capsule.
23. The method of any one of claims 1-22, wherein the method increases the tumor cell expression of type 1 interferon receptor (INF ARI) in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
24. The method of any one of claims 1-23, wherein the method increases the peripheral blood monocyte surface expression of INF ARI in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
25. The method of any one of claims 1-24, wherein the method increases tumor cell expression of calreticulin in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
26. The method of any one of claims 1-25, wherein the method increases polymorphonuclear myeloid-derived suppressor cell (MDSC) and/or tumor associated macrophage (TAM) expression of INF ARI in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
27. The method of any one of claims 1-26, wherein the method increases TAM expression of PD-L1 in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
28. The method of any one of claims 1-27, wherein the method increases the infiltration of CD8 T cells and/or NK cells in a tumor in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
29. The method of any one of claims 1-28, wherein the method increases expression of CD69 on T cells in a tumor draining lymph node in the subject relative to a subject receiving placebo, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the therapeutically effective amount of the anti-PD-1 antibody.
30. The method of any one of claims 1-29, wherein the method reduces suppressive activity of MDSCs in the subject.
31. The method of any one of claims 1-30, wherein the method increases dendritic cell frequency in a tumor draining lymph node in the subject.
32. The method of any one of claims 1-31, wherein the method increases Ki67, GzmB, and memory phenotypic markers on CD4 T-cells in the subject.
33. The method of any one of claims 1-32, wherein the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered as a pharmaceutically acceptable salt of the compound of formula (I).
34. The method of claim 33, wherein the pharmaceutically acceptable salt is a hemifumarate salt.
35. A method of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I):
Figure imgf000129_0001
or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent.
36. A method of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I):
Figure imgf000129_0002
or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an anti angiogenic agent, wherein the subject has been treated with an anti-cancer therapy.
37. The method of claim 36, wherein the subject is resistant or has acquired resistance to the anti-cancer therapy.
38. The method of claim 36, wherein the anti-cancer therapy is selected from the group consisting of: administration of an immunotherapeutic agent, a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, an anti angiogenic agent, or a combination thereof; radiation therapy; surgery; and combinations thereof.
39. The method of any one of claims 35-38, wherein the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti angiogenic agent are administered simultaneously.
40. The method of any one of claims 35-38, wherein the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of the anti angiogenic agent are administered sequentially.
41. The method of any one of claims 35-40, wherein the therapeutically effective amount of the anti angiogenic agent is administered to the subject daily.
42. The method of any one of claims 35-41, wherein the therapeutically effective amount of the anti angiogenic agent is administered to the subject once daily.
43. The method of any one of claims 35-41, wherein the therapeutically effective amount of the anti angiogenic agent is administered to the subject twice daily.
44. The method of any one of claims 35-43, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of the anti angiogenic agent reduces p-PERK levels in the subject relative to administering a therapeutically effective amount of the anti angiogenic agent alone.
45. The method of any one of claims 35-43, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of the anti angiogenic agent increases ATF4 levels in the subject relative to administering a therapeutically effective amount of the anti angiogenic agent alone.
46. The method of any one of claims 35-43, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of the anti angiogenic agent modulates the activation of unfolded protein response (UPR) relative to administering a therapeutically effective amount of the anti angiogenic agent alone.
47. A method of treating a cancer in a subject receiving a therapeutically effective amount of an anti angiogenic agent therapy, comprising administering to the subject a therapeutically effective amount of a compound of formula (I):
Figure imgf000131_0001
or a pharmaceutically acceptable salt thereof.
48. The method of claim 47, wherein the subject is resistant or has acquired resistance to the anti angiogenic agent therapy.
49. The method of any one of claims 35-48, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, reduces p- PERK levels in the subject relative to administering a therapeutically effective amount of the anti angiogenic agent alone.
50. The method of any one of claims 35-48 wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, increases ATF4 levels in the subject relative to administering a therapeutically effective amount of the anti angiogenic agent alone.
51. The method of any one of claims 35-48, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, modulates the activation of unfolded protein response (UPR) relative to administering a therapeutically effective amount of the anti angiogenic agent alone.
52. The method of any one of claims 35-48, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, decreases tumor vasculature in the subject relative to administering a therapeutically effective amount of the anti angiogenic agent alone.
53. The method of any one of claims 35-52, wherein the anti angiogenic agent is a vascular endothelial growth factor receptor (VEGFR) modulator.
54. The method of any one of claims 35-52, wherein the anti angiogenic agent is a vascular endothelial growth factor (VEGF) targeting antibody.
55. The method of any one of claims 35-52, wherein the anti angiogenic agent is a tyrosine kinase inhibitor (TKI).
56. The method of any one of claims 35-52, wherein the anti angiogenic agent is a VEGF receptor tyrosine kinase inhibitor (VEGFR- TKI).
57. The method of any one of claims 35-56, wherein the anti angiogenic agent is selected from the group consisting of: apatinib, axitinib, brivanib, cabozantinib, canertinib, cediranib, dasatinib, erlotinib, gefitinib, leflunomide, lenvatinib, motesanib, nilotinib, nintedanib, pazopanib, regorafenib, semaxinib, sorafenib, sunitinib, tivozanib, vandetanib, vatalanib, XL0192, bevacizumab, ramucirumab, and pharmaceutically acceptable salts or biosimilars thereof.
58. The method of any one of claims 35-56, wherein the anti angiogenic agent is selected from the group consisting of: bevacizumab, ramucirumab, and biosimilars thereof.
59. The method of any one of claims 35-56, wherein the anti angiogenic agent is selected from the group consisting of: apatinib, axitinib, brivanib, cabozantinib, canertinib, cediranib, dasatinib, erlotinib, gefitinib, leflunomide, lenvatinib, motesanib, nilotinib, nintedanib, pazopanib, regorafenib, semaxinib, sorafenib, sunitinib, tivozanib, vandetanib, vatalanib, XL0192, and pharmaceutically acceptable salts thereof.
60. The method of any one of claims 35-59, wherein the cancer is a metastatic cancer or a locally advanced cancer.
61. The method of any one of claims 35-60 wherein the cancer is a solid tumor.
62. The method of any one of claims 35-61, wherein the cancer is selected from the group consisting of: a bladder cancer, a breast cancer, a carcinoma, a cervical cancer, a colorectal cancer, a gastric cancer, a hepatocellular cancer, a kidney cancer, a lung cancer, a neuroendocrine cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a skin cancer, and a thyroid cancer.
63. The method of claim 62, wherein the breast cancer is triple negative breast cancer or metastatic breast cancer; the carcinoma is carcinoma of unknown primary (CUP), endometrial carcinoma, head and neck squamous cell carcinoma, Merkel cell carcinoma, squamous cell carcinoma, or urothelial carcinoma; the gastric cancer is adenocarcinoma or gastrointestinal stromal tumor; the kidney cancer is a renal cell carcinoma (RCC); or the lung cancer is small cell lung cancer (SCLC) or non-small cell lung cancer.
64. The method of claim 62, wherein the renal cell carcinoma is clear cell renal cell carcinoma (ccRCC).
65. The method of claim 62, wherein the cancer is selected from the group consisting of: a breast cancer, a gastric cancer, a kidney cancer, and a lung cancer.
66. The method of any one of claims 35-65, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
67. The method of any one of claims 35-66, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
68. The method of any one of claims 35-66, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, daily.
69. The method of any one of claims 35-66, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, once daily.
70. The method of any one of claims 35-66, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 22 mg to about 798 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, twice daily.
71. The method of any one of claims 35-66, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
72. The method of any one of claims 35-66, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof.
73. The method of any one of claims 35-66, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, daily.
74. The method of any one of claims 35-66, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, once daily.
75. The method of any one of claims 35-66, wherein administering a therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises administering orally to the subject about 44 mg to about 177 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, twice daily.
76. The method of any one of claims 35-75, wherein the subject is in a fasting state.
77. The method of any one of claims 35-75, wherein the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered with food.
78. The method of any one of claims 35-75, wherein the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered about 30 minutes to about 1 hour after food.
79. The method of any one of claims 35-78, wherein the compound of formula (I) is administered as a pharmaceutically acceptable salt.
80. The method of claim 79, wherein the pharmaceutically acceptable salt is a hemifumarate salt.
81. The method of any one of claims 35-80, wherein the method further comprises administering to the subject a therapeutically effective amount of an immunotherapy.
82. A method of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I):
Figure imgf000135_0001
or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent, and a therapeutically effective amount of an immunotherapy.
83. A method of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (I):
Figure imgf000135_0002
or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of an anti angiogenic agent, and a therapeutically effective amount of an immunotherapy, wherein the subject has been treated with an anti-cancer therapy.
84. The method of claim 83, wherein the subject is resistant or has acquired resistance to the anti-cancer therapy.
85. The method of claim 83, wherein the anti-cancer therapy is selected from the group consisting of: surgery; radiation therapy; administration of an immunotherapeutic agent, a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, an anti angiogenic agent, or a combination thereof; and combinations thereof.
86. A method of treating a cancer in a subject receiving a therapeutically effective amount of an anti angiogenic agent therapy, comprising administering to the subject a therapeutically effective amount of a compound of formula (I):
Figure imgf000136_0001
or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immunotherapy.
87. The method of any one of claims 81-86, wherein the immunotherapy is an immune checkpoint inhibitor.
88. The method of any one of claims 81-86, wherein the immunotherapy is an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody.
89. The method of claim 88, wherein the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, and cemiplimab.
90. The method of claim 88 or 89, wherein the anti-PD-1 antibody is pembrolizumab.
91. The method of claim 88 or 89, wherein the anti-PD-1 antibody is nivolumab.
92. The method of any one of claims 81-91, wherein the therapeutically effective amount of an immunotherapy is administered daily.
93. The method of any one of claims 81-91, wherein the therapeutically effective amount of an immunotherapy is administered once daily.
94. The method of any one of claims 81-91, wherein the therapeutically effective amount of an immunotherapy is administered twice daily.
95. The method of any one of claims 81-94, wherein the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of an immunotherapy are administered simultaneously.
96. The method of any one of claims 81-94, wherein the therapeutically effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the therapeutically effective amount of an immunotherapy are administered sequentially.
97. The method of any one of claims 81-94, wherein the therapeutically effective amount of the anti angiogenic agent and the therapeutically effective amount of an immunotherapy are administered simultaneously.
98. The method of any one of claims 81-94, wherein the therapeutically effective amount of the anti angiogenic agent and the effective amount of an immunotherapy are administered sequentially.
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