Combinations for cancer treatment

Abstract

Providing combinations for cancer treatment. [Solution] The present invention relates to a combination comprising a checkpoint inhibitor, a c-Met inhibitor, and compound 1. The present invention also relates to the crystalline form of the free base of compound 1 and the crystalline form of the salt of compound 1 in combination with a checkpoint inhibitor. The present invention also relates to a pharmaceutical composition comprising these combinations. The present invention further relates to a method for treating cancer by administering compound 1 as a monotherapy or in combination as described herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 148,921 filed on 12 February 2021, U.S. Provisional Application No. 63 / 113,556 filed on 13 November 2020, and U.S. Provisional Application No. 63 / 059,601 filed on 31 July 2020, all of which are incorporated herein by reference in their entirety.

[0002] This invention relates to a combination comprising an immune checkpoint inhibitor (ICI) and compound 1. The invention also relates to the crystalline form of the free base of compound 1 in combination with a checkpoint inhibitor, and the crystalline form of a salt of compound 1. The invention also relates to a pharmaceutical composition of compound 1 used in combination with a checkpoint inhibitor. Furthermore, the invention relates to a method for treating cancer by administering compound 1 as a monotherapy or in combination as described herein. [Background technology]

[0003] Cancer is a major cause of morbidity and mortality worldwide. While standard treatments for many different cancer types have improved significantly over the years, current standards still fail to meet the need for more effective therapies to improve cancer treatment. The clinical use of immuno-oncological agents targeting cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death receptor-1 (PD-1) and its ligand PD-L1 has resulted in improvements beyond standard treatment in the treatment of many cancer types. While these checkpoint inhibitors have produced improved clinical responses in certain cancers, sustained clinical responses occur in only about 10–45% of patients. Furthermore, a significant number of tumors become resistant or refractory.

[0004] In recent years, TAM tyrosine kinases, particularly AXL receptor tyrosine kinases, have emerged as promising targets for cancer therapy. AXL is a cell surface receptor tyrosine kinase that is part of the TAM family of kinases, which also includes TYRO3 and MERTK. Several drugs classified as "AXL inhibitors" are in clinical trials, but there are many other target multiple kinase receptors besides AXL.

[0005] Human Axl belongs to the Tyro3, ​​Axl, and Mer(TAM) subfamily of receptor tyrosine kinases, which includes Mer. TAM kinases are characterized by an extracellular ligand-binding domain consisting of two immunoglobulin-like domains and two fibronectin type III domains. Axl is overexpressed in numerous tumor cell types and was initially cloned from patients with chronic myeloid leukemia. When overexpressed, Axl exhibits transformative potential. Axl signaling is thought to induce tumor growth by activating proliferative and anti-apoptotic signaling pathways. Axl is associated with cancers such as lung cancer, myeloid leukemia, uterine cancer, ovarian cancer, glioma, melanoma, thyroid cancer, renal cell carcinoma, osteosarcoma, gastric cancer, prostate cancer, and breast cancer. Overexpression of Axl is associated with a poor prognosis in patients with the indicated cancers.

[0006] Activation of Mer, such as Axl, transmits downstream signaling pathways that trigger tumor growth and activation. Mer binds to ligands such as the soluble protein Gas-6. Gas-6 binding to Mer induces autophosphorylation of Mer on its intracellular domain, leading to downstream signal activation. Overexpression of Mer in cancer cells leads to increased metastasis, most likely due to the production of soluble Mer extracellular domain proteins acting as decoy receptors. Tumor cells secrete a soluble form of the extracellular Mer receptor that reduces the ability of soluble Gas-6 ligands to activate Mer on endothelial cells, thus causing cancer progression.

[0007] Therefore, there is a need for novel therapies in the art, including, for example, combination therapies for the treatment of cancer. The present invention provides solutions to these problems and other problems in the art. [Overview of the project] [Means for solving the problem]

[0008] In one aspect, the present invention is a method for treating cancer in a subject, (i) administering to a subject a dose of compound 1 or a pharmaceutically acceptable salt thereof in an amount of approximately 5 mg to approximately 100 mg, wherein compound 1 has the structure: [ka] Having, to administer, (ii) A method comprising administering a therapeutically effective dose of a checkpoint inhibitor to a subject.

[0009] In another embodiment, the present invention relates to a method for treating cancer in a subject, wherein the subject requiring such treatment is given a dose of compound 1: [ka] Alternatively, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, The method includes administering a therapeutically effective dose of a checkpoint inhibitor or a pharmaceutical composition containing a checkpoint inhibitor in combination with the inhibitor.

[0010] In one aspect, the present invention is a method for treating urothelial carcinoma in a subject, (i) administering to a subject a dose of compound 1 or a pharmaceutically acceptable salt thereof in an amount of approximately 5 mg to approximately 100 mg, wherein compound 1 has the structure: [ka] Having, to administer, (ii) A method comprising administering a therapeutically effective dose of a checkpoint inhibitor to a subject.

[0011] In another aspect, the present invention relates to a method for treating urothelial carcinoma in a subject, wherein the subject requiring such treatment is given a dose of compound 1: [ka] Alternatively, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, The method includes administering a therapeutically effective dose of a checkpoint inhibitor or a pharmaceutical composition containing a checkpoint inhibitor in combination with the inhibitor.

[0012] In these and other embodiments, the checkpoint inhibitor is selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors. In these and other embodiments and examples, the checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, atezolizumab (TECENTRIQ®), durvalumab, avelumab (BAVENCIO®), semiprimab, camrelizumab, cintilimab, tislerizumab, tripalimab, spartalizumab, dostallimab, KN035 (Jiangsu Alphamb Biopharmaceuticals Co.), kosivelimab (formerly CK-301), CA-170 (Curis, Inc.), BMS-986189 (Bristol Myers Squibb Co.), and ipilimumab (Yervoy, Bristol Myers Squibb Co.).

[0013] In a further embodiment, what is provided is a method for treating cancer in a subject, (i) administering to a subject a dose of compound 1 or a pharmaceutically acceptable salt thereof in an amount of approximately 5 mg to approximately 100 mg, wherein compound 1 has the structure: [ka] Having, to administer, (ii) A method comprising administering to a subject a therapeutically effective dose of nivolumab and at least one additional immunomodulator.

[0014] In one embodiment of this design, the immunomodulator is selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, and IL-2 targeting agents.

[0015] In these and other embodiments, the subjects are human subjects in need of treatment.

[0016] In these and other embodiments, the checkpoint inhibitor is selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors. In these and other embodiments and examples, the checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, atezolizumab (TECENTRIQ®), durvalumab, avelumab (BAVENCIO®), semiprimab, camrelizumab, cintilimab, tislerizumab, tripalimab, spartalizumab, dostallimab, KN035 (Jiangsu Alphamb Biopharmaceuticals Co.), kosivelimab (formerly CK-301), CA-170 (Curis, Inc.), BMS-986189 (Bristol Myers Squibb Co.), and ipilimumab (Yervoy, Bristol Myers Squibb Co.).

[0017] In these and other embodiments, the IL-2 targeting agent is a CD122-preferential IL-2 pathway agonist, a PEG-IL-2Rαβ-biased agonist, an IL-2Rβ-biased agonist, or an IL-2Rβγ-biased agonist. cBiased agonists, IL-2v / IL-2α fusion proteins, anti-EDB mAb (L19) / IL-2v fused to L19 / TNFv, anti-GD2 mAb / IL-2v, anti-FAP mAb / IL-2v, anti-CEA mAb / IL-2v, anti-PD-1 mAb / IL-2v, patient-derived tumor cell vaccine + HD-IL-2, adoptive cell therapy + IL-2 injection, adoptive cell therapy + IL-2 injection + anti-PD-1 mAb, orthogonal IL-2v / IL-2Rβ variant pairs, anti-IL-2Rα mAb / PBD conjugate, PEG-IL-2Rα biased agonist, IL-2v / human Fc fusion protein, PEG-IL-2Rα biased (N88D) / IgG1 fusion protein, anti-IL-2 The group consists of recombinant plasmids encoding mAb / IL-2v, IL-2, PPI, TGF-β1, and IL-10, as well as IL-2Rβ antagonists.

[0018] In one embodiment, the IL-2 targeting agent is a CD122-preferred IL-2 pathway agonist. In one embodiment, the CD122-preferred IL-2 pathway agonist is benpegaldesleukin (BEMPEG; NKTR-214; Bristol Myers Squibb Co.).

[0019] In one embodiment, the IL-2 targeting agent is a PEG-IL-2Rα biased agonist. In one embodiment, the PEG-IL-2Rα biased agonist is NKTR-358 (Bristol Myers Squibb Co.).

[0020] In a further embodiment, the present invention includes a method for treating cancer in a subject, comprising administering to the subject a dose of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, in an amount of about 5 mg to about 100 mg.

[0021] In these and other embodiments, Compound 1 is administered as a crystalline solid of the free base or as a crystalline, pharmaceutically acceptable salt. To avoid misunderstanding, unless otherwise specified, “Compound 1” means these crystalline free base forms and crystalline salt forms.

[0022] In these and other embodiments, compound 1 is a crystalline solid form characterized as form A, form B, form C, form D, form E, form F, form G, form H, form K, form O, or form Q.

[0023] In these and other embodiments, compound 1 is a crystalline HCl salt of compound 1.

[0024] In these and other embodiments, compound 1 is a crystalline fumarate of compound 1, or a hydrate or solvate thereof.

[0025] In these and other embodiments, compound 1 is a crystalline phosphate of compound 1, or a hydrate or solvate thereof. [Brief explanation of the drawing]

[0026] [Figure 1A] The presence of tumor microvessels is shown by CD31 staining after treatment with compound 1. The horizontal bars represent the mean value for n=3 tumors per condition. [Figure 1B] This shows the presence of tumor microvessels by CD31 staining after treatment with compound 1. Paraffin-embedded tumor tissue was stained with the vascular marker CD31, and the density of vessels was scored across different conditions. [Figure 2A] The presence of cytotoxic T cells is shown by CD8 staining after treatment with compound 1, PD-1, and a combination of compound 1 + PD-1. The horizontal bars represent the median number of tumors per condition (n=8-12). [Figure 2B] The presence of cytotoxic T cells is shown by CD8 staining after treatment with PD-1 and a combination of compound 1 + PD-1. Paraffin-embedded tumor tissue was stained with the vascular marker CD8, and vascular density was scored across conditions. [Figure 3] The tumor volume after treatment with the combination therapy of compound 1+PD-1, compound 1+PD-L1, and compound 1+CTLA-4 is shown. [Figure 4]The growth curves of subcutaneously transplanted CT26 colon cells in mice treated with compound 1 as a monotherapy or in combination with an anti-PD-1 inhibitor are shown (40-day treatment period). [Figure 5] The Kaplan-Meier survival curves for mice with CT26 colon tumors treated with compound 1, an anti-PD-1 inhibitor, and a combination of compound 1 and an anti-PD-1 inhibitor are shown (40-day treatment period). [Figure 6A] This study compares tumor growth after treatment with either the vehicle, compound 1 at 30 mg / kg, anti-PD-1 at 10 mg / kg, or both. The symbol represents the median tumor volume. [Figure 6B] A Kaplan-Meier plot showing conditional survival for CT26 tumor-bearing mice after treatment is shown. In the case of conditional survival, the treatment group was excluded from the study when 40% of the relevant animals reached the tumor size threshold. [Figure 7A] This shows that compound 1 dose-dependently inhibited efferocytosis using 25k apoptotic Jurkat. [Figure 7B] This shows that compound 1 dose-dependently inhibited efferocytosis using 50k apoptotic Jurkat. [Figure 8] The mean (SD) plasma concentration-time profiles of compound 1 after the initial administration of compound 1 as a monotherapy agent and after daily administration for 28 days in subjects with solid tumors are shown. [Modes for carrying out the invention]

[0027] [Table 1] [Table 2] [Table 3] [Table 4] As used herein, unless otherwise specified, the following definitions apply:

[0028] For the purposes of this invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 95th Ed. In addition, the general principles of organic chemistry are incorporated herein by reference in their entirety by reference from "Organic Chemistry", 2 nd This is described in Thomas Sorrell (ed.), University Science Books, Sausalito: 2006, and in "March's Advanced Organic Chemistry," 7th Ed., Ed.: Smith, MB and March, J., John Wiley & Sons, New York: 2013.

[0029] As used herein, the terms “low / limited / significant hygroscopicity” refer to materials exhibiting water uptake of <0.5 / <2.0 / ≧2.0 wt% across a specific RH range.

[0030] As used herein, the term “stoichiometric hydrate” refers to a crystalline material having a defined water content over a wide RH range. Typical stoichiometric hydrates include hemihydrates, monohydrates, sesquihydrates, and dihydrates.

[0031] As used herein, the term "variable hydrate" refers to a crystalline material that has a variable water content over a wide RH range without undergoing a phase change.

[0032] As used herein, the chemical term “form” refers to a single-phase chemical compound or a salt thereof.

[0033] As used herein, the terms “low / limited / intermediate / good / high solubility” refer to materials having a solubility of <1 / 1-20 / 20-100 / 100-200 / >200 mg / mL.

[0034] As used herein, the term "crystalline" refers to a material that produces an XRPD pattern with sharp peaks (similar to instrumental peak widths) and weaker diffuse scattering compared to those peaks.

[0035] As used herein, the term “disordered crystallinity” refers to a material that produces an XRPD pattern with broad peaks (compared to instrumental peak widths) and / or strong diffuse scattering compared to peaks. Disordered materials are: 1) Microcrystalline, 2) Crystalline with a high defect density, 3) A mixture of crystalline phase and X-ray amorphous phase, 4) The above combinations are possible.

[0036] As used herein, the term “insufficient signal” means that the spectroscopic analysis of a sample produces a spectrum or pattern (output) with an insufficient signal that exceeds the expected background noise.

[0037] As used herein, the term “single-crystal phase” refers to an XRPD pattern that is determined to contain evidence of a single crystalline form due to Bragg peaks indexed by a single unit cell. Indexing is the process of assigning Miller index labels to each peak in the diffraction pattern. The size and shape of the crystalline unit cell are also determined during the indexing process.

[0038] As used herein, the term “slurry” refers to a suspension prepared by adding a sufficient amount of solid to a given solvent under ambient conditions such that undissolved solids are present. A typical slurry involves stirring (typically by agitation or vibration), which is also referred to as “slurring” in a sealed vial at a given temperature over a long period of time. Typically, the solids are recovered after a given period using the methods described herein.

[0039] As used herein, the terms "X-ray amorphous" or "amorphous" refer to materials in which diffuse scattering is present but there is no evidence of a Bragg peak in the XRPD pattern.

[0040] As used herein, the term "crystalline" refers to a compound in a solid state having a periodic and repeating three-dimensional internal arrangement of atoms, ions, or molecules characteristic of a crystal, arranged in a fixed geometric pattern or lattice with a rigid long-range order, for example. The term crystalline does not necessarily mean that the compound exists as a crystal, but rather that it has this crystalline internal structure arrangement.

[0041] As used herein, the term “substantially crystalline” refers to a solid material that is primarily arranged in a fixed geometric pattern or lattice with a rigid long-range order. For example, a substantially crystalline material has a degree of crystallinity greater than about 85% (e.g., greater than about 90%, greater than about 95%, or greater than about 99%). Note that the term “substantially crystalline” also includes the descriptor “crystalline” as defined in the preceding paragraph.

[0042] For the purposes of this invention, “patient” includes humans and any other animals, particularly mammals, and other living organisms. Therefore, this method is applicable to both human therapeutic and veterinary uses. In preferred embodiments, the patient is a mammal, and in most preferred embodiments, the patient is a human. Examples of preferred mammals include mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, and primates.

[0043] A "kinase-dependent disease or condition" refers to a pathological condition that depends on the activity of one or more kinases. Kinases are directly or indirectly involved in signaling pathways for various cellular activities, including proliferation, adhesion, migration, differentiation, and invasion. Diseases related to kinase activity include tumor growth, support for solid tumor growth, and pathological angiogenesis associated with other diseases involving excessive local angiogenesis, such as eye diseases (e.g., diabetic retinopathy, age-related macular degeneration) and inflammation (e.g., psoriasis, rheumatoid arthritis).

[0044] The "therapeutic dose" is the amount of the crystalline form or crystalline salt form of the present invention that, when administered to a patient, improves the symptoms of the disease. The amount of the crystalline form or crystalline salt form of the present invention that constitutes the "therapeutic dose" varies depending on the compound, the disease state and its severity, the age of the patient being treated, and so on. The therapeutic dose can be routinely determined by those skilled in the art, taking into account their knowledge and this disclosure.

[0045] The term "pharmaceutically acceptable" is used herein to mean a compound, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues in proportion to a reasonable benefit-risk ratio, without excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications.

[0046] As used herein, the term “pharmaceutically acceptable excipient” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients generally include those that are safe, non-toxic, biologically and otherwise undesirable, and acceptable for veterinary and human pharmaceutical uses. In one embodiment, each component is “pharmaceutically acceptable” as defined herein. For example, Remington:The Science and Practice of Pharmacy,21st ed.;Lippincott Williams & Wilkins:Philadelphia,Pa.,2005;Handbook of 'Pharmaceutical Excipients,6th ed.;Rowe et al,Eds.;The Pharmaceutical Press and the American Pharmaceutical Association:2009;Handbook of Pharmaceutical Additives,3rd ed.;Ash and Ash Eds.;Gower Publishing Company: 2007; Pharmaceutical Pref or mulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, Fla., 2009.

[0047] As used herein, the term “concurrently” means at the same time. For example, if two treatment regimens are being administered concurrently to a single patient, they are being administered at the same time. It will be understood that the simultaneous administration of two treatment regimens does not necessarily mean that the actual delivery of the two drugs occurs at the same time, as each regimen may require a different dosing schedule and / or a different mode of delivery.

[0048] As used herein and as provided by the National Cancer Institute, “checkpoint inhibitor” refers to any drug that blocks, inhibits, or modulates checkpoint proteins. Checkpoint inhibitors are produced by several types of immune system cells, such as T cells, and by some cancer cells. Examples of checkpoint proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2. Examples of checkpoint inhibitors include pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, cemiprimab, camrelizumab, cintilimab, tislerizumab, tripalimab, spartalizumab, dostallimab, KN035 (Jiangsu Alphamb Biopharmaceuticals Co.), cosivelimab (formerly CK-301), CA-170 (Curis, Inc.), and BMS-986189 (Bristol Myers Squibb Co.). Examples of FDA-approved checkpoint inhibitors include the products pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, and cemiprimab. Dosage and other information about approved checkpoint inhibitors is available from the FDA, EMEA, or other national health regulatory agencies.

[0049] "Cancer" is any physiological condition in mammals characterized by uncontrolled cell growth, particularly but not limited to: Cardiac: sarcomas (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyomas, fibromas, lipomas and teratomas; Head and neck: squamous cell carcinoma of the head and neck, pharyngeal and hypopharyngeal carcinoma, nasal and paranasal sinus carcinoma, nasopharyngeal carcinoma, salivary gland carcinoma, oral and oropharyngeal carcinoma; Lung: bronchogenic carcinoma (squamous cell, anaplastic small cell, anaplastic large cell, adenocarcinoma, non-small cell lung carcinoma), alveolar (bronchiolar) carcinoma, alveolar sarcoma, alveolar soft part sarcoma, bronchial adenoma, sarcoma, li Lymphoma, chondrodic hamartoma, mesothelioma; Colon: colorectal cancer, adenocarcinoma, gastrointestinal stromal tumor, lymphoma, carcinoid, Turcot syndrome; Gastrointestinal tract: gastric cancer, gastroesophageal junction adenocarcinoma, esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (pancreatic ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, bipoma), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, chorioadenoma, hamartoma, Leiomyoma; Breast: Metastatic breast cancer, ductal carcinoma in situ, invasive ductal carcinoma, tubular carcinoma, medullary carcinoma, mucinous carcinoma, lobular carcinoma in situ, triple-negative breast cancer; Urogenital tract: Kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia, renal cell carcinoma, metastatic renal cell carcinoma), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, urothelial carcinoma), prostate (adenocarcinoma, sarcoma, castration-resistant prostate cancer, bone metastasis, bone metastasis associated with castration-resistant prostate cancer), testes (seminocarcinoma, teratoma, fetal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor) , lipoma), clear cell carcinoma, papillary carcinoma, penile carcinoma, penile squamous cell carcinoma; liver: liver cancer (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticular cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochondroma (osteochondrosis exostosis), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor; thyroid: medullary thyroid carcinoma, differentiated thyroid carcinoma, papillary thyroid carcinoma, follicular thyroid carcinoma, Haasle cell carcinoma, and anaplastic thyroid carcinoma;Nervous system: Skull (osteoma, hemangioma, granuloma, xanthomas, degenerative osteitis), meninges (meningioma, meningiosarcoma, glioma), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor [pineal glandoma], glioblastoma multiforme, oligodendroglioma, Schwann cell tumor, retinoblastoma, congenital tumor), spinal neurofibroma, meningioma, glioma, sarcoma), NF1, neurofibromatosis, plexiform neurofibroma; Gynecology: Uterus (endometrial cancer), cervix (cervical cancer, pretumescent cervical cancer) Dysplasia), ovaries (ovarian cancer [serous cystadenocarcinoma, mucocystadenocarcinoma, unclassified cancer], granulosa-theca cell tumor, Sertoli-Leydig cell tumor, undifferentiated germ cell tumor, malignant teratoma), vulva (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, staphyloid sarcoma (embryonic rhabdomyosarcoma)], fallopian duct carcinoma (carcinoma); hematology: blood (myeloid leukemia [acute and Diffuse leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, spinal dysplasia syndrome, myelofibrosis, polycythemia vera, essential thrombocythemia, Hodgkin's disease, non-Hodgkin lymphoma [malignant lymphoma]; skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, lentiginous dysplastic nevus, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and adrenal gland: including neuroblastoma, which refers to cell proliferative disorder conditions. Therefore, the term “cancer cell” as provided herein includes cells suffering from any one of the conditions identified above. In some embodiments, compounds or combinations as disclosed herein may be used to treat diseases including HIV, sickle cell disease, graft-versus-host disease, acute graft-versus-host disease, chronic graft-versus-host disease, and sickle cell anemia.

[0050] As defined by the National Cancer Institute, the term “solid tumor” typically refers to an abnormal mass of tissue that does not contain cysts or fluid areas. Solid tumors can be benign (not cancerous) or malignant (cancerous). Different types of solid tumors are named after the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas. Leukemia (cancer of the blood) does not generally form solid tumors.

[0051] As defined by the National Cancer Institute, "immunomodulators" are substances that stimulate or suppress the immune system.

[0052] The terms “to treat” or “treatment” mean any sign of success or improvement in the progression, severity, and / or duration of a disease, lesion, or condition, including any objective or subjective parameters such as reduction; remission; reduction of symptoms or causing the patient to develop a more tolerable injury, lesion, or condition; slowing the rate of degeneration or decay; reducing the terminal debilitation of degeneration; or improving the patient’s physical or mental health.

[0053] The term "enhance" refers to an increase or improvement in the function or activity of a protein or cell after such administration or contact with the combination described herein, compared to the protein or cell before administration or contact.

[0054] The term "administer" refers to the act of delivering any combination or composition described herein to a subject by route such as oral, mucosal, topical, suppository, intravenous, parenteral, intraperitoneal, intramuscular, intrafocal, subarachnoid, intranasal, or subcutaneous administration. Parenteral administration includes intravenous, intramuscular, arterioleal, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Administration is generally performed after the onset of a disease, disorder, or condition or its symptoms, but in certain cases it may be performed before the onset of a disease, disorder, or condition or its symptoms (for example, administration to a patient prone to such a disease, disorder, or condition).

[0055] The term "co-administration" refers to the administration of two or more drugs (e.g., the combination described herein and another active agent such as the anticancer agent described herein). The timing of co-administration depends on the combination and some of the compositions being administered and may include administration concurrently with, immediately before, or immediately after the administration of one or more additional therapies, such as chemotherapy, hormone therapy, radiation therapy, or immunotherapy. The compounds of the present invention can be administered to a patient alone or co-administered. Co-administration means the simultaneous or sequential administration of individual or combination (one or more compounds or drugs) of compounds. Accordingly, the preparations may also be combined with other active substances (e.g., to reduce metabolic degradation) if desired. The compounds described herein can be used in combination with other active agents known to be useful in treating cancer.

[0056] The term “anticancer agent” is used in its obvious and ordinary sense and refers to a composition having antineoplastic properties or the ability to inhibit cell growth or proliferation. In embodiments, the anticancer agent is a chemotherapeutic agent. In embodiments, the anticancer agent is a drug identified herein that has utility in a method of treating cancer. In embodiments, the anticancer agent is a drug approved by the FDA or similar regulatory agency in a country other than the United States for the treatment of cancer.

[0057] The terms “chemotherapeutic agent” or “chemotherapeutic drug” are used in their obvious and ordinary sense to refer to a chemical composition or compound having antineoplastic properties or the ability to inhibit cell growth or proliferation. “Chemotherapy” refers to a therapy or regimen comprising the administration of a chemotherapeutic agent or anticancer agent as described herein.

[0058] In general, the nomenclature used in this application is based on the nomenclature rules adopted by the International Union of Pure and Applied Chemistry (IUPAC). The chemical structures shown herein were prepared using CHEMDRAW®. Any open valencies appearing on carbon, oxygen, or nitrogen atoms in the structures herein indicate the presence of hydrogen atoms.

[0059] Appearances and Embodiments In one embodiment, the present invention relates to a method for treating cancer in a subject, wherein the subject requiring such treatment is given a therapeutically effective amount of compound 1: [ka] Alternatively, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising compound (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, The method includes administering a therapeutically effective dose of a checkpoint inhibitor or a pharmaceutical composition containing a checkpoint inhibitor in combination with the inhibitor.

[0060] In one embodiment, the present invention relates to a method for treating cancer in a subject, wherein the subject requiring such treatment is given a dose of compound 1: [ka] Alternatively, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising compound (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, The method includes administering a therapeutically effective dose of a checkpoint inhibitor or a pharmaceutical composition containing a checkpoint inhibitor in combination with the inhibitor.

[0061] In another aspect, the present invention relates to a method for treating cancer in a subject, (i) administering to a subject a therapeutically effective dose of compound 1 or a pharmaceutically acceptable salt thereof, wherein compound 1 has the structure: [ka] Having, to administer, (ii) A method comprising administering a therapeutically effective dose of a checkpoint inhibitor to a subject.

[0062] In another aspect, the present invention relates to a method for treating cancer in a subject, (i) administering to a subject a dose of compound 1 or a pharmaceutically acceptable salt thereof in an amount of approximately 5 mg to approximately 100 mg, wherein compound 1 has the structure: [ka] Having, to administer, (ii) A method comprising administering a therapeutically effective dose of a checkpoint inhibitor to a subject.

[0063] In another embodiment, the present invention includes a method for treating cancer in a subject, comprising administering to the subject in need of such treatment a therapeutically effective amount of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising compound (1) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0064] In another embodiment, the present invention includes a method for treating cancer in a subject, comprising administering to a subject in need of such treatment a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof in a dose of about 5 mg to about 100 mg, or compound (1) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0065] In one aspect, the present invention is a method for treating urothelial carcinoma in a subject, (i) administering to a subject a dose of compound 1 or a pharmaceutically acceptable salt thereof in an amount of approximately 5 mg to approximately 100 mg, wherein compound 1 has the structure: [ka] Having, to administer, (ii) A method comprising administering a therapeutically effective dose of a checkpoint inhibitor to a subject.

[0066] In another aspect, the present invention relates to a method for treating urothelial carcinoma in a subject, wherein the subject requiring such treatment is given a dose of compound 1: [ka] Alternatively, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising compound (1) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, The method includes administering a therapeutically effective dose of a checkpoint inhibitor or a pharmaceutical composition containing a checkpoint inhibitor in combination with the inhibitor.

[0067] In one embodiment, the checkpoint inhibitor is selected from PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors.

[0068] In one embodiment, the checkpoint inhibitor is selected from αPD-1 inhibitors, PD-L1 inhibitors, and αCTLA-4 inhibitors.

[0069] In another embodiment, the checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, semiprimab, camrelizumab, cintilimab, tislerizumab, tripalimab, spartalizumab, dostallimab, KN035 (Jiangsu Alphamb Biopharmaceuticals Co.), cosivelimab (formerly CK-301), CA-170 (Curis, Inc.), BMS-986189 (Bristol Myers Squibb Co.), and ipilimumab.

[0070] In one embodiment, the checkpoint inhibitor is selected from the group consisting of atezolizumab, avelumab, and nivolumab.

[0071] In one embodiment, the checkpoint inhibitor is pembrolizumab.

[0072] In one embodiment, the checkpoint inhibitor is nivolumab.

[0073] In one embodiment, the checkpoint inhibitor is atezolizumab.

[0074] In one embodiment, the checkpoint inhibitor is avelumab.

[0075] In one embodiment, the checkpoint inhibitor is semiprimab.

[0076] In one embodiment, the checkpoint inhibitor is camrelizumab.

[0077] In one embodiment, the checkpoint inhibitor is cintilimab.

[0078] In one embodiment, the checkpoint inhibitor is tislerizumab.

[0079] In one embodiment, the checkpoint inhibitor is tripalimab.

[0080] In one embodiment, the checkpoint inhibitor is spartalizumab.

[0081] In one embodiment, the checkpoint inhibitor is dostallimab.

[0082] In one embodiment, the checkpoint inhibitor is KN035.

[0083] In one embodiment, the checkpoint inhibitor is cosivelimab.

[0084] In one embodiment, the checkpoint inhibitor is CA-170.

[0085] In one embodiment, the checkpoint inhibitor is BMS-986189.

[0086] In one embodiment, the checkpoint inhibitor is ipilimumab.

[0087] In one embodiment of the above-described configuration, compound 1 or a pharmaceutically acceptable salt thereof is administered orally once daily (qd) or twice daily (bid). In one embodiment, compound 1 or a pharmaceutically acceptable salt thereof is administered orally once daily (qd). In one embodiment, compound 1 or a pharmaceutically acceptable salt thereof is administered orally twice daily (bid).

[0088] In this specification, the dosage of Compound 1 is expressed as free base equivalent (FBE) unless otherwise specified.

[0089] In some embodiments, the therapeutically effective amount of compound 1 is about 1 mg to about 500 mg, about 1 mg to about 300 mg, about 1 mg to about 200 mg, about 1 mg to about 150 mg, about 5 mg to about 150 mg, or about 5 mg to about 100 mg.

[0090] In one embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is about 5 mg to about 80 mg. In another embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is about 5 mg to about 50 mg.

[0091] In one embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is 8 mg to 12 mg. In one embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is 18 mg to 22 mg. In one embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is 38 mg to 40 mg. In one embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is about 10 mg. In one embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is about 20 mg. In one embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is about 40 mg. In one embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is about 60 mg. In one embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is about 80 mg. In one embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is about 100 mg. In one embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is about 120 mg. In one embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is about 140 mg. In one embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is selected from about 10 mg, 20 mg, and 40 mg. In one embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is selected from about 10 mg, 20 mg, 40 mg, 60 mg, and 80 mg. In one embodiment, the dose of compound 1 or a pharmaceutically acceptable salt thereof is selected from about 10 mg, 20 mg, 40 mg, 60 mg, 80 mg, 100 mg, 120 mg, and 140 mg. In one embodiment, the dosage of compound 1 or a pharmaceutically acceptable salt thereof is selected from approximately 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, and 300 mg.

[0092] In further embodiments, ●Administer compound 1 in an amount greater than 0 mg but less than or equal to 100 mg. ●Administer compound 1 in a dose between 0 mg and 95 mg. ●Administer compound 1 in a dose between 0 mg and 90 mg. ●Administer compound 1 in a dose between 0 mg and 85 mg. ●Administer compound 1 in a dose greater than 0 mg but less than or equal to 80 mg. ●Administer compound 1 in a dose between 0 mg and 75 mg. ●Administer compound 1 in a dose between 0 mg and 70 mg. ●Administer compound 1 in a dose between 0 mg and 65 mg. ●Administer compound 1 in a dose greater than 0 mg but less than or equal to 60 mg. ●Administer compound 1 in a dose greater than 0 mg but less than or equal to 55 mg. ●Administer compound 1 in a dose greater than 0 mg but less than or equal to 50 mg. ●Administer compound 1 in a dose between 0 mg and 45 mg. ●Administer compound 1 in a dose greater than 0 mg but less than or equal to 40 mg. ●Administer compound 1 in a dose greater than 0 mg but less than or equal to 35 mg. ●Administer compound 1 in a dose greater than 0 mg but less than or equal to 30 mg. ●Administer compound 1 in a dose greater than 0 mg but less than or equal to 25 mg. ●Administer compound 1 in an amount greater than 0 mg but less than or equal to 20 mg. ●Administer compound 1 in a dose greater than 0 mg but less than or equal to 15 mg. ●Administer compound 1 in an amount greater than 0 mg but less than or equal to 10 mg, or ●Administer compound 1 in a dose between 0 mg and 5 mg.

[0093] Solid form of compound 1 In the embodiments and examples described above, compound 1 may be administered in crystalline (free base) solid form or as a crystalline salt.

[0094] Crystalline (free base) solid of compound 1 In one embodiment, compound 1 is administered as a crystalline (free base) solid. In one embodiment, the crystalline solid form of compound 1 is characterized as morphology A, morphology B, morphology C, morphology D, morphology E, morphology F, morphology G, morphology H, morphology I, morphology J, morphology K, morphology L, morphology M, morphology N, morphology O, morphology P, or morphology Q. In another embodiment, the crystalline solid form of compound 1 is characterized as morphology A, morphology B, morphology C, morphology D, morphology E, morphology F, morphology G, morphology H, morphology K, morphology O, or morphology Q. In yet another embodiment, the crystalline solid form of compound 1 is characterized as morphology I, morphology J, morphology L, morphology M, morphology N, or morphology P. The crystalline solid forms of compound 1, characterized as form A, form B, form C, form D, form E, form F, form G, form H, form I, form J, form K, form L, form M, form N, form O, form P, or form Q, are disclosed in WO2020 / 123800, the contents of which are incorporated herein by reference in their entirety for any purpose.

[0095] In one embodiment, the crystalline solid is characterized as compound 1 form A.

[0096] In one embodiment, the crystalline solid is characterized as compound form 1B.

[0097] In one embodiment, the crystalline solid is characterized as compound 1 form C.

[0098] In one embodiment, the crystalline solid is characterized as compound form 1D.

[0099] In one embodiment, the crystalline solid is characterized as compound form 1E.

[0100] In one embodiment, the crystalline solid is characterized as compound form 1F.

[0101] In one embodiment, the crystalline solid is characterized as compound form 1G.

[0102] In one embodiment, the crystalline solid is characterized as compound form 1H.

[0103] In one embodiment, the crystalline solid is characterized as compound form I.

[0104] In one embodiment, the crystalline solid is characterized as compound form 1 J.

[0105] In one embodiment, the crystalline solid is characterized as compound form 1 K.

[0106] In one embodiment, the crystalline solid is characterized as compound form 1 L.

[0107] In one embodiment, the crystalline solid is characterized as compound form 1 M.

[0108] In one embodiment, the crystalline solid is characterized as compound 1 form N.

[0109] In one embodiment, the crystalline solid is characterized as compound 1 form O.

[0110] In one embodiment, the crystalline solid is characterized as compound form 1, P.

[0111] In one embodiment, the crystalline solid is characterized as compound form Q.

[0112] Crystalline salt of compound 1 In another embodiment, compound 1 is administered as a crystalline salt or its hydrate or solvate.

[0113] In one embodiment, the crystalline salt is characterized as HCl form A of compound 1, HCl form B of compound 1, HCl form C of compound 1, or HCl form D of compound 1. The crystalline salt forms characterized as HCl form A of compound 1, HCl form B of compound 1, HCl form C of compound 1, or HCl form D of compound 1 are disclosed in WO2020 / 123800, the contents of which are incorporated herein by reference in their entirety for any purpose.

[0114] In one embodiment, the crystalline salt is characterized as HCl form A of compound 1.

[0115] In one embodiment, the crystalline salt is characterized as HCl form B of compound 1.

[0116] In one embodiment, the crystalline salt is characterized as HCl form C of compound 1.

[0117] In one embodiment, the crystalline salt is characterized as HCl form D of compound 1.

[0118] In one embodiment, a pharmaceutical composition as disclosed herein comprises a crystalline fumarate of compound 1, or a hydrate or solvate thereof. In some embodiments, the crystalline fumarate of compound 1 is characterized as fumarate form A of compound 1 or hemifumarate form B of compound 1. The crystalline fumarate of compound 1 characterized as fumarate form A of compound 1 or hemifumarate form B of compound 1 is disclosed in WO2020 / 123800, the contents of which are incorporated herein by reference in whole for all purposes.

[0119] In one embodiment, the crystalline salt is characterized as fumarate form A of compound 1.

[0120] In one embodiment, the crystalline fumarate is characterized as hemifumarate form B of compound 1.

[0121] Pharmaceutical composition of compound 1 In the embodiments and examples described above, compound 1 may be administered as a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises compound 1 in crystalline (free base) solid form. In another embodiment, the pharmaceutical composition comprises compound 1 as a crystalline salt.

[0122] In further embodiments, the pharmaceutical composition is a tablet.

[0123] In further embodiments, the tablet pharmaceutical composition is a. About 20% to about 40% by weight of compound 1, either as a crystalline solid or as a crystalline salt selected from the group consisting of the HCl salt of compound 1, the fumarate of compound 1, and the phosphate of compound 1. b. Approximately 35% to 45% by weight of microcrystalline cellulose. c. Approximately 15% to 25% by weight of lactose, d. Approximately 2% to 8% by weight of hydroxypropyl cellulose, e. Approximately 4% to 8% by weight of croscarmellose sodium, f. Approximately 0.1% to 0.5% by weight of silicon dioxide, and g. Approximately 0.5% to 3.5% by weight of magnesium stearate, and optionally h. Includes film coating.

[0124] In one embodiment, the tablet pharmaceutical composition is a. About 20% to about 40% by weight of compound 1, either as a crystalline solid or as a crystalline salt selected from the group consisting of the HCl salt of compound 1, the fumarate of compound 1, and the phosphate of compound 1. b. Approximately 35% to 45% by weight of microcrystalline cellulose. c. Approximately 15% to 25% by weight of anhydrous lactose. d. Approximately 2% to 8% by weight of hydroxypropyl cellulose, e. Approximately 4% to 8% by weight of croscarmellose sodium, f. Approximately 0.1% to 0.5% by weight of colloidal silicon dioxide, g. Approximately 0.5% to 3.5% by weight of magnesium stearate, and optionally h. Includes film coating.

[0125] In one embodiment, the tablet pharmaceutical composition is a. About 25% to about 35% by weight of compound 1, either as a crystalline solid or as a crystalline salt selected from the group consisting of the HCl salt of compound 1, the fumarate of compound 1, and the phosphate of compound 1. b. Microcrystalline cellulose of approximately 37% to 43% by weight. c. Approximately 18% to 22% by weight of anhydrous lactose. d. Approximately 2% to 6% by weight of hydroxypropyl cellulose, e. Approximately 5% to 7% by weight of croscarmellose sodium, f. Colloidal silicon dioxide in an amount of approximately 0.2% to approximately 0.4% by weight, and g. Approximately 0.5% to 3.5% by weight of magnesium stearate, and optionally h. Includes film coating.

[0126] Therefore, in another embodiment, the tablet pharmaceutical composition, a. About 20% to about 40% by weight of compound 1, either as a crystalline solid or as a crystalline salt selected from the group consisting of the HCl salt of compound 1, the fumarate of compound 1, and the phosphate of compound 1. b. Approximately 35% to 45% by weight of microcrystalline cellulose. c. Approximately 15% to 25% by weight of lactose, d. Approximately 2% to 8% by weight of hydroxypropyl cellulose, e. Approximately 2% to 8% by weight of croscarmellose sodium. f. Approximately 0.1% to 0.5% by weight of silicon dioxide, and g. Approximately 1% to 5% by weight of stearic acid, and optionally h. Includes film coating.

[0127] Therefore, in another embodiment, the tablet pharmaceutical composition, a. About 20% to about 40% by weight of compound 1, either as a crystalline solid or as a crystalline salt selected from the group consisting of the HCl salt of compound 1, the fumarate of compound 1, and the phosphate of compound 1. b. Approximately 35% to 45% by weight of microcrystalline cellulose. c. Approximately 15% to 25% by weight of anhydrous lactose. d. Approximately 2% to 8% by weight of hydroxypropyl cellulose, e. Approximately 2% to 8% by weight of croscarmellose sodium. f. Approximately 0.1% to 0.5% by weight of colloidal silicon dioxide, g. Approximately 1% to 5% by weight of stearic acid, and optionally h. Includes film coating.

[0128] In one embodiment, the tablet pharmaceutical composition is a. About 25% to about 35% by weight of compound 1, either as a crystalline solid or as a crystalline salt selected from the group consisting of the HCl salt of compound 1, the fumarate of compound 1, and the phosphate of compound 1. b. Approximately 35% to 40% by weight of microcrystalline cellulose, c. Approximately 16% to 22% by weight of anhydrous lactose. d. Approximately 3% to 7% by weight of hydroxypropyl cellulose, e. Approximately 3% to 7% by weight of croscarmellose sodium f. Approximately 0.1% to 0.5% by weight of colloidal silicon dioxide, g. Approximately 0.5% to 3.5% by weight stearic acid, and optionally h. Includes film coating.

[0129] In one embodiment, the pharmaceutical composition of the present disclosure comprises compound 1 as a crystalline (free base) solid.

[0130] In one embodiment, the crystalline solid form of compound 1 is characterized as morphology A, morphology B, morphology C, morphology D, morphology E, morphology F, morphology G, morphology H, morphology I, morphology J, morphology K, morphology L, morphology M, morphology N, morphology O, morphology P, or morphology Q. In another embodiment, the crystalline solid form of compound 1 is characterized as morphology A, morphology B, morphology C, morphology D, morphology E, morphology F, morphology G, morphology H, morphology K, morphology O, or morphology Q. In yet another embodiment, the crystalline solid form of compound 1 is characterized as morphology I, morphology J, morphology L, morphology M, morphology N, or morphology P. The crystalline solid forms of compound 1, characterized as form A, form B, form C, form D, form E, form F, form G, form H, form I, form J, form K, form L, form M, form N, form O, form P, or form Q, are disclosed in WO2020 / 123800, the contents of which are incorporated herein by reference in their entirety for any purpose.

[0131] In one embodiment, the crystalline solid is characterized as compound 1 form A.

[0132] In one embodiment, the crystalline solid is characterized as compound form 1B.

[0133] In one embodiment, the crystalline solid is characterized as compound 1 form C.

[0134] In one embodiment, the crystalline solid is characterized as compound form 1D.

[0135] In one embodiment, the crystalline solid is characterized as compound form 1E.

[0136] In one embodiment, the crystalline solid is characterized as compound form 1F.

[0137] In one embodiment, the crystalline solid is characterized as compound form 1G.

[0138] In one embodiment, the crystalline solid is characterized as compound form 1H.

[0139] In one embodiment, the crystalline solid is characterized as compound form I.

[0140] In one embodiment, the crystalline solid is characterized as compound form 1 J.

[0141] In one embodiment, the crystalline solid is characterized as compound form 1 K.

[0142] In one embodiment, the crystalline solid is characterized as compound form 1 L.

[0143] In one embodiment, the crystalline solid is characterized as compound form 1 M.

[0144] In one embodiment, the crystalline solid is characterized as compound 1 form N.

[0145] In one embodiment, the crystalline solid is characterized as compound 1 form O.

[0146] In one embodiment, the crystalline solid is characterized as compound form 1, P.

[0147] In one embodiment, the crystalline solid is characterized as compound form Q.

[0148] In another embodiment, the pharmaceutical composition of the present disclosure comprises compound 1 as a crystalline salt or its hydrate or solvate.

[0149] In one embodiment, the crystalline salt is characterized as HCl form A of compound 1, HCl form B of compound 1, HCl form C of compound 1, or HCl form D of compound 1. The crystalline salt forms characterized as HCl form A of compound 1, HCl form B of compound 1, HCl form C of compound 1, or HCl form D of compound 1 are disclosed in WO2020 / 123800, the contents of which are incorporated herein by reference in their entirety for any purpose.

[0150] In one embodiment, the crystalline salt is characterized as HCl form A of compound 1.

[0151] In one embodiment, the crystalline salt is characterized as HCl form B of compound 1.

[0152] In one embodiment, the crystalline salt is characterized as HCl form C of compound 1.

[0153] In one embodiment, the crystalline salt is characterized as HCl form D of compound 1.

[0154] In one embodiment, a pharmaceutical composition as disclosed herein comprises a crystalline fumarate of compound 1, or a hydrate or solvate thereof. In some embodiments, the crystalline fumarate of compound 1 is characterized as fumarate form A of compound 1 or hemifumarate form B of compound 1. The crystalline fumarate of compound 1 characterized as fumarate form A of compound 1 or hemifumarate form B of compound 1 is disclosed in WO2020 / 123800, the contents of which are incorporated herein by reference in whole for all purposes.

[0155] In one embodiment, the crystalline salt is characterized as fumarate form A of compound 1.

[0156] In one embodiment, the crystalline fumarate is characterized as hemifumarate form B of compound 1.

[0157] In one embodiment, the pharmaceutical composition comprises a crystalline phosphate of compound 1, or a hydrate or solvate thereof. In some embodiments, the crystalline phosphate of compound 1 is characterized as phosphate form A of compound 1. The crystalline phosphate of compound 1 characterized as phosphate form A of compound 1 is disclosed in WO2020 / 123800, the contents of which are incorporated herein by reference in whole for all purposes.

[0158] In one embodiment, the tablet pharmaceutical composition is a. Approximately 25% to 35% by weight of the hemifumarate of compound 1. b. Microcrystalline cellulose of approximately 37% to 43% by weight. c. Approximately 18% to 22% by weight of anhydrous lactose. d. Approximately 2% to 6% by weight of hydroxypropyl cellulose, e. Approximately 5% to 7% by weight of croscarmellose sodium, f. Colloidal silicon dioxide in an amount of approximately 0.2% to approximately 0.4% by weight, and g. Approximately 0.5% to 3.5% by weight of magnesium stearate, and optionally h. Includes film coating.

[0159] In one embodiment, the tablet pharmaceutical composition is a. Hemifumarate form B of compound 1 at a concentration of approximately 25% to 35% by weight. b. Microcrystalline cellulose of approximately 37% to 43% by weight. c. Approximately 18% to 22% by weight of anhydrous lactose. d. Approximately 2% to 6% by weight of hydroxypropyl cellulose, e. Approximately 5% to 7% by weight of croscarmellose sodium, f. Colloidal silicon dioxide in an amount of approximately 0.2% to approximately 0.4% by weight, and g. Approximately 0.5% to 3.5% by weight of magnesium stearate, and optionally h. Includes film coating.

[0160] In one embodiment, the tablet pharmaceutical composition is a. Approximately 27.75 weight percent of compound 1 hemifumarate, b. Approximately 41.47% by weight of microcrystalline cellulose, c. Approximately 20.73% by weight of anhydrous lactose, d. Approximately 3% by weight of hydroxypropyl cellulose, e. Approximately 6% by weight of croscarmellose sodium, f. Approximately 0.3 weight percent colloidal silicon dioxide, and g. Approximately 0.75 weight percent magnesium stearate, and optionally h. Includes film coating.

[0161] In one embodiment, the tablet pharmaceutical composition is a. Hemifumarate form B of compound 1 at approximately 27.75 weight percent. b. Approximately 41.47% by weight of microcrystalline cellulose, c. Approximately 20.73% by weight of anhydrous lactose, d. Approximately 3% by weight of hydroxypropyl cellulose, e. Approximately 6% by weight of croscarmellose sodium, f. Approximately 0.3 weight percent colloidal silicon dioxide, and g. Approximately 0.75 weight percent magnesium stearate, and optionally h. Includes film coating.

[0162] In one embodiment, the tablet pharmaceutical composition is a. 20-25 mg of compound 1 hemifumarate, b. 30-35 mg of microcrystalline cellulose, c. 15-18 mg of anhydrous lactose, d. 1.5-4.5 mg of hydroxypropylcellulose, e. 4-6 mg of croscarmellose sodium, f. 0.1~0.3 mg of colloidal silicon dioxide, and g. 0.5-0.7 mg of magnesium stearate, and optionally Contains 2-6 mg of film coating.

[0163] In one embodiment, the tablet pharmaceutical composition is a. Hemifumarate form B of compound 1, 20-25 mg b. 30-35 mg of microcrystalline cellulose, c. 15-18 mg of anhydrous lactose, d. 1.5-4.5 mg of hydroxypropylcellulose, e. 4-6 mg of croscarmellose sodium, f. 0.1~0.3 mg of colloidal silicon dioxide, and g. 0.5-0.7 mg of magnesium stearate, and optionally Contains 2-6 mg of film coating.

[0164] In one embodiment, the tablet pharmaceutical composition is a. Hemifumarate form B of Compound 1, 22.20 mg b. 30-35 mg of microcrystalline cellulose, c. 15-18 mg of anhydrous lactose, d. 1.5-4.5 mg of hydroxypropylcellulose, e. 4-6 mg of croscarmellose sodium, f. 0.1~0.3 mg of colloidal silicon dioxide, and g. 0.5-0.7 mg of magnesium stearate, and optionally h. It contains a film coating of 2 - 6 mg.

[0165] In one embodiment, the tablet pharmaceutical composition a. 22.20 mg of hemifumarate form B of Compound 1, b. 33.17 mg of microcrystalline cellulose, c. 16.59 mg of anhydrous lactose, d. 2.4 mg of hydroxypropyl cellulose, e. 4.8 mg of croscarmellose sodium, f. 0.24 mg of colloidal silicon dioxide, and g. 0.6 mg of magnesium stearate, and optionally h. It contains a film coating of 3.2 mg.

[0166] In one embodiment, the tablet pharmaceutical composition a. Approximately 25% to approximately 35% by weight of hemifumarate of Compound 1, b. Approximately 35% to approximately 40% by weight of microcrystalline cellulose, c. Approximately 16% to approximately 22% by weight of anhydrous lactose, d. Approximately 3% to approximately 7% by weight of hydroxypropyl cellulose, e. Approximately 3% to approximately 7% by weight of croscarmellose sodium f. Approximately 0.1% to approximately 0.5% by weight of colloidal silicon dioxide, and g. Approximately 0.5% to approximately 3.5% by weight of stearic acid, and optionally h. It contains a film coating.

[0167] In one embodiment, the tablet pharmaceutical composition a. Approximately 25% to approximately 35% by weight of hemifumarate form B of Compound 1, b. Approximately 35% to approximately 40% by weight of microcrystalline cellulose, c. Approximately 16% to 22% by weight of anhydrous lactose. d. Approximately 3% to 7% by weight of hydroxypropyl cellulose, e. Approximately 3% to 7% by weight of croscarmellose sodium f. Approximately 0.1% to 0.5% by weight of colloidal silicon dioxide, g. Approximately 0.5% to 3.5% by weight stearic acid, and optionally h. Includes film coating.

[0168] In one embodiment, the tablet pharmaceutical composition is a. Approximately 27.75 weight percent of compound 1 hemifumarate, b. Approximately 38.63 weight percent of microcrystalline cellulose, c. Approximately 19.32% by weight of anhydrous lactose, d. Approximately 5% by weight of hydroxypropyl cellulose, e. Approximately 6% by weight of croscarmellose sodium f. Approximately 0.3 weight percent colloidal silicon dioxide, and g. Approximately 3% by weight of stearic acid, and optionally h. Includes film coating.

[0169] In one embodiment, the tablet pharmaceutical composition is a. Hemifumarate form B of compound 1 at approximately 27.75 weight percent. b. Approximately 38.63 weight percent of microcrystalline cellulose, c. Approximately 19.32% by weight of anhydrous lactose, d. Approximately 5% by weight of hydroxypropyl cellulose, e. Approximately 6% by weight of croscarmellose sodium f. Approximately 0.3 weight percent colloidal silicon dioxide, and g. Approximately 3% by weight of stearic acid, and optionally h. It includes a film coating.

[0170] In one embodiment, the tablet pharmaceutical composition a. 20 - 25 mg of hemifumarate of Compound 1, b. 30 - 40 mg of microcrystalline cellulose, c. 15 - 20 mg of anhydrous lactose, d. 3 - 7 mg of hydroxypropyl cellulose, e. 3 - 7 mg of croscarmellose sodium, f. 0.1 - 0.3 mg of colloidal silicon dioxide, and g. 2 - 4 mg of stearic acid, and optionally h. 2 - 5 mg of film coating.

[0171] In one embodiment, the tablet pharmaceutical composition a. 20 - 25 mg of hemifumarate Form B of Compound 1, b. 30 - 40 mg of microcrystalline cellulose, c. 15 - 20 mg of anhydrous lactose, d. 3 - 7 mg of hydroxypropyl cellulose, e. 3 - 7 mg of croscarmellose sodium, f. 0.1 - 0.3 mg of colloidal silicon dioxide, and g. 2 - 4 mg of stearic acid, and optionally h. 2 - 5 mg of film coating.

[0172] In one embodiment, the tablet pharmaceutical composition a. 22.20 mg of hemifumarate Form B of Compound 1, b. 30 - 40 mg of microcrystalline cellulose, c. 15 - 20 mg of anhydrous lactose, d. 3 - 7 mg of hydroxypropyl cellulose, e. 3 - 7 mg of croscarmellose sodium, f. 0.1 - 0.3 mg of colloidal silicon dioxide, and g. 2-4 mg of stearic acid, and optionally Contains 2-5 mg of film coating.

[0173] In one embodiment, the tablet pharmaceutical composition is a. Hemifumarate form B of Compound 1, 22.20 mg b. 30.9 mg of microcrystalline cellulose, c. 15.46 mg of anhydrous lactose, d.4 mg of hydroxypropylcellulose, e. 4.8 mg of croscarmellose sodium, f. 0.24 mg of colloidal silicon dioxide, and g.2.4 mg of stearic acid, and optionally Contains 3.2 mg of h film coating.

[0174] In one embodiment, the tablet pharmaceutical composition is a. 83-93 mg of compound 1 hemi fumarate, b. 120-150 mg of microcrystalline cellulose, c. 60-80 mg of anhydrous lactose, d. 12-30 mg of hydroxypropylcellulose, e. 12-30 mg of croscarmellose sodium, f. 0.5 to 1.5 mg of colloidal silicon dioxide, and g. 8-16 mg of stearic acid, and optionally Contains 8-14 mg of film coating.

[0175] In one embodiment, the tablet pharmaceutical composition is a. Hemifumarate form B of compound 1, 83-93 mg. b. 120-150 mg of microcrystalline cellulose, c. 60-80 mg of anhydrous lactose, d. 12-30 mg of hydroxypropylcellulose, e. 12-30 mg of croscarmellose sodium, f. 0.5 to 1.5 mg of colloidal silicon dioxide, and g. 8-16 mg of stearic acid, and optionally Contains 8-14 mg of film coating.

[0176] In one embodiment, the tablet pharmaceutical composition is a. Hemifumarate form B of compound 1, 88.78 mg b. 120-150 mg of microcrystalline cellulose, c. 60-80 mg of anhydrous lactose, d. 12-30 mg of hydroxypropylcellulose, e. 12-30 mg of croscarmellose sodium, f. 0.5 to 1.5 mg of colloidal silicon dioxide, and g. 8-16 mg of stearic acid, and optionally Contains 8-14 mg of film coating.

[0177] In one embodiment, the tablet pharmaceutical composition is a. Hemifumarate form B of compound 1, 88.78 mg b. 123.62 mg of microcrystalline cellulose, c. 61.82 mg of anhydrous lactose, d. 16 mg of hydroxypropylcellulose, e. 19.2 mg of croscarmellose sodium, f. 0.96 mg of colloidal silicon dioxide, and g.9.6 mg of stearic acid, and optionally Contains a film coating of 12.8 mg of h.

[0178] In another embodiment, compound 1 is administered as a tablet pharmaceutical composition as provided in the following table. [Table 5]

[0179] In another embodiment, compound 1 is administered as a tablet pharmaceutical composition as provided in the following table. [Table 6]

[0180] Any of the formulations provided above can be adjusted according to the desired dose of compound 1. Thus, the amount of each formulation component can be proportionally adjusted to provide tablet formulations containing various amounts of compound 1, as provided in the previous paragraph.

[0181] Cancer treatment In the embodiments described above, compound 1 is administered together with a checkpoint inhibitor for treating cancer and optionally with additional immunomodulators.

[0182] In the embodiments and examples described above, compound 1 is administered as a monotherapy for treating cancer.

[0183] In one embodiment, the cancer is selected from cardiac cancer, head and neck cancer, lung cancer, colon cancer, gastrointestinal cancer, breast cancer, genitourinary tract cancer, liver cancer, bone cancer, thyroid cancer, nervous system cancer, gynecological cancer, hematological cancer, skin cancer, and adrenal gland cancer.

[0184] In further embodiments, cardiac cancer is selected from angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyosarcoma, fibroma, lipoma, and teratoma.

[0185] In another further embodiment, head and neck cancer is selected from squamous cell carcinoma of the head and neck, pharyngeal and hypopharyngeal cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, oral cavity and oropharyngeal cancer.

[0186] In another further embodiment, lung cancer is selected from bronchogenic carcinoma, alveolar (bronchiolar) carcinoma, bronchial adenoma, lymphoma, chondromatoid hamartoma, and mesothelioma, which are selected from squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma, and non-small cell lung cancer.

[0187] In another further embodiment, the colon cancer is selected from colorectal cancer, adenocarcinoma, gastrointestinal stromal tumor, lymphoma, carcinoid, and turcot syndrome.

[0188] In another further embodiment, gastrointestinal cancer is selected from gastric cancer, gastroesophageal junction adenocarcinoma, esophageal squamous cell carcinoma, esophageal adenocarcinoma, esophageal leiomyosarcoma, esophageal lymphoma, gastric carcinoma, gastric lymphoma, gastric leiomyosarcoma, pancreatic ductal adenocarcinoma, pancreatic insulinoma, pancreatic glucagonoma, pancreatic gastrinoma, pancreatic carcinoid tumor, vipoma, small intestinal adenocarcinoma, small intestinal lymphoma, small intestinal carcinoid tumor, small intestinal Kaposi's sarcoma, small intestinal leiomyoma, small intestinal hemangioma, small intestinal lipoma, small intestinal neurofibroma, small intestinal fibroma, colonic adenocarcinoma, colonic tubular adenoma, colonic villous adenoma, colonic hamartoma, and colonic leiomyoma.

[0189] In another further embodiment, breast cancer is selected from metastatic breast cancer, ductal carcinoma in situ, invasive ductal carcinoma, tubular carcinoma, medullary carcinoma, mucinous carcinoma, lobular carcinoma in situ, and triple-negative breast cancer.

[0190] In another further embodiment, urogenital tract carcinoma is selected from renal adenocarcinoma, nephroblastoma, renal lymphoma, renal cell carcinoma, squamous cell carcinoma of the bladder or urethra, transitional cell carcinoma of the bladder or urethra, adenocarcinoma of the bladder or urethra, urothelial carcinoma of the bladder or urethra, prostatic adenocarcinoma, prostatic sarcoma, castration-resistant prostate cancer, seminomas, testicular teratoma, embryonal carcinoma, testicular teratocarcinoma, testicular choriocarcinoma, testicular sarcoma, testicular stromal cell carcinoma, testicular fibroma, testicular fibroadenoma, testicular adenomatous tumor, testicular lipoma, clear cell carcinoma, and papillary carcinoma.

[0191] In another further embodiment, liver cancer is selected from hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma.

[0192] In another further embodiment, bone cancer is selected from osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma, reticular sarcoma, multiple myeloma, malignant giant cell tumor chordoma, osteochondroma, benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor.

[0193] In another further embodiment, the thyroid cancer is selected from medullary thyroid carcinoma, differentiated thyroid carcinoma, papillary thyroid carcinoma, follicular thyroid carcinoma, Haasle cell carcinoma, and undifferentiated thyroid carcinoma.

[0194] In another further embodiment, neurological cancers are selected from osteomas of the skull, hemangiomas of the skull, granulomas of the skull, xanthomas of the skull, degenerative osteitis of the skull, meningiomas, meningiosarcomas, gliomas of the meninges, astrocytomas, medulloblastomas, gliomas, ependymomas, germ cell tumors [pineal glands], glioblastoma multiforme, oligodendroglioma, Schwann cell tumors, retinoblastomas, congenital brain tumors, spinal neurofibromas, meningiomas, and cerebral sarcomas.

[0195] In another further embodiment, gynecological cancers are selected from endometrial cancer, cervical cancer, preneoplastic cervical malformations, serous cystadenocarcinoma, mucocystadenocarcinoma, and unclassified ovarian cancer, granulosa-theca cell tumors, Sertoli-Leydig cell tumors, undifferentiated germ cell tumors, and malignant teratomas; squamous cell carcinoma of the vulva, carcinoma in situ of the vulva, adenocarcinoma of the vulva, fibrosarcoma of the vulva, melanoma of the vulva, clear cell carcinoma of the vagina, squamous cell carcinoma of the vagina, embryonal rhabdomyosarcoma, and fallopian duct carcinoma.

[0196] In another further embodiment, the hematological cancer is selected from myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, spinal dysplasia syndromes), Hodgkin's disease, and non-Hodgkin lymphoma [malignant lymphoma].

[0197] In another further embodiment, the skin cancer is selected from malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, lentiginous dysplastic nevus, lipoma, hemangioma, dermatofibroma, keloid, and psoriasis.

[0198] In one embodiment, the cancer is selected from cardia cancer, head and neck cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, non-clear cell renal cell carcinoma, advanced clear cell renal cell carcinoma, castration-resistant prostate cancer, hormone receptor-positive breast cancer, prostate cancer, colon cancer, gastrointestinal cancer, breast cancer, urogenital tract cancer, liver cancer, bone cancer, thyroid cancer, nervous system cancer, gynecological cancer, hematological cancer, skin cancer, and adrenal gland cancer.

[0199] In further embodiments, the cancer is a solid tumor.

[0200] In further embodiments, the cancer is a solid tumor that is inoperable, locally progressive, metastatic, or recurrent.

[0201] In further embodiments, the solid tumor is unresectable or metastatic, and there is no life-sustaining therapy, or currently available therapies are unacceptable or no longer effective.

[0202] In another further embodiment, cancer or solid tumors are ICI-refractory.

[0203] In yet another further embodiment, cancer or solid tumors are platinum-refractory.

[0204] In further embodiments, the solid tumor is a sarcoma, carcinoma, or lymphoma.

[0205] In further embodiments, the cancer is advanced clear cell renal cell carcinoma, hormone receptor-positive breast cancer, or castration-resistant prostate cancer.

[0206] In one embodiment, the cancer is advanced clear cell renal carcinoma.

[0207] In one embodiment, the cancer is unresectable, advanced or metastatic clear cell renal cell carcinoma.

[0208] In one embodiment, the cancer is a non-clear cell renal cell carcinoma.

[0209] In one embodiment, the cancer is advanced non-clear cell renal cell carcinoma.

[0210] In one embodiment, the cancer is an unresectable, advanced or metastatic non-clear cell renal cell carcinoma.

[0211] In one embodiment, unresectable advanced or metastatic non-clear cell renal cell carcinoma includes papillary renal cell carcinoma, unclassifiable renal cell carcinoma, and sarcomatoid renal cell carcinoma.

[0212] In one embodiment, the cancer is hormone receptor-positive breast cancer.

[0213] In one embodiment, the cancer is castration-resistant prostate cancer.

[0214] In a further embodiment, castration-resistant prostate cancer is metastatic.

[0215] In yet another further embodiment, the adrenal carcinoma is a neuroblastoma.

[0216] In yet another further embodiment, the cancer is urothelial carcinoma.

[0217] In another further embodiment, urothelial carcinoma is a locally advanced or metastatic transitional cell carcinoma of the urothelium.

[0218] In another further embodiment, the cancer is advanced urothelial carcinoma.

[0219] In another further embodiment, the cancer is metastatic urothelial carcinoma.

[0220] In another further embodiment, the cancer is ICI-refractory urothelial carcinoma.

[0221] In another further embodiment, the cancer is platinum-refractory urothelial carcinoma.

[0222] In another further embodiment, the cancer is urothelial carcinoma of the renal pelvis, ureter, bladder, or urethra.

[0223] In yet another further embodiment, the cancer is urothelial carcinoma of the renal pelvis.

[0224] In yet another further embodiment, the cancer is urothelial carcinoma of the ureter.

[0225] In another further embodiment, the cancer is urothelial carcinoma of the urethra.

[0226] In yet another further embodiment, the cancer is urothelial carcinoma of the bladder.

[0227] In another embodiment, the cancer is selected from endometrial cancer, sarcoma, neuroendocrine tumor, ovarian cancer, colorectal cancer, HCC, NSCLC, gastric cancer, and melanoma.

[0228] In another embodiment, the cancer is endometrial cancer.

[0229] In another embodiment, the cancer is a sarcoma.

[0230] In another embodiment, the cancer is a neuroendocrine tumor.

[0231] In another embodiment, the cancer is ovarian cancer.

[0232] In another embodiment, the cancer is colorectal cancer.

[0233] In another embodiment, colorectal cancer is either right-sided colorectal cancer (RCRC) or left-sided colorectal cancer (LCRC).

[0234] In another embodiment, the cancer is hepatocellular carcinoma.

[0235] In another embodiment, the cancer is non-small cell lung cancer.

[0236] In another embodiment, the cancer is gastric cancer.

[0237] In another embodiment, the cancer is melanoma.

[0238] In another embodiment, the cancer is a solid tumor. In a further embodiment, the cancer is an unresectable, advanced or metastatic solid tumor. In a further embodiment, the solid tumor is a genitourinary cancer. In a further embodiment, the genitourinary cancer is selected from the group consisting of clear cell renal cell carcinoma (ccRCC), non-clear cell renal cell carcinoma (nccRCC), urothelial carcinoma (UC, ICI-never experienced, and experienced), and metastatic castration-resistant prostate cancer (mCRPC).

[0239] In one embodiment, the subject is a human being.

[0240] In one embodiment, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, and a checkpoint inhibitor or a pharmaceutical composition containing a checkpoint inhibitor are administered simultaneously, sequentially, or separately.

[0241] In one embodiment, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, and a pharmaceutical composition containing a checkpoint inhibitor or immunomodulator are administered simultaneously, sequentially, or separately. In one embodiment, the immunomodulator is selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, and IL-2 targeting agents.

[0242] In one embodiment, the method further includes evaluating treatment with the combination therapy by determining one or more of the following: inhibition of disease progression, inhibition of tumor growth, reduction of primary tumor, alleviation of tumor-related symptoms, inhibition of tumor secretory factors, delay of the appearance of primary or secondary tumors, slowing of the onset of primary or secondary tumors, reduction of the occurrence of primary or secondary tumors, slowing or reduction of the severity of secondary effects of the disease, cessation of tumor growth and regression of tumors, increase in time to progression (TTP), increase in progression-free survival (PFS), increase in overall response rate, increase in overall survival (OS), or increase in duration of response (DOR), or changes in tumor markers from baseline.

[0243] In one embodiment, the method includes the treatment of cancer that has not been previously treated with any other anticancer treatment. In another embodiment, the method includes the treatment of cancer that has not been previously treated with a PD-1 inhibitor. In yet another embodiment, the method includes the treatment of cancer that has been previously treated with a PD-1 inhibitor. In yet another embodiment, the method includes the treatment of cancer that has been previously treated with pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, semiprimab, camrelizumab, cintilimab, tislerizumab, tripalimab, spartalizumab, dostallimab, KN035, cosivelimab, CA-170 (Curis, Inc.), or BMS-986189. In another embodiment, the method includes the treatment of cancer that has not been previously treated with pembrolizumab, nivolumab, atezolizumab, durvalumab, avelumab, semiprimab, camrelizumab, cintilimab, tislerizumab, tripalimab, spartalizumab, dostallimab, KN035, cosivelimab, CA-170 (Curis, Inc.), or BMS-986189.

[0244] In one embodiment, the method includes the treatment of cancer previously treated with a PD-1 inhibitor, which initially yielded a partial response but later developed resistance to PD-1 as the disease progressed.

[0245] In one embodiment, the method includes the treatment of cancer previously treated with a PD-1 inhibitor, which initially resulted in a stable disease but later led to the development of resistance to PD-1 as the disease progressed.

[0246] In one embodiment, the method includes the treatment of cancer previously treated with a PD-1 inhibitor, which initially resulted in a complete response but later led to the development of resistance to PD-1 as the disease progressed.

[0247] In one embodiment, the method includes the treatment of cancer that has been previously treated with a PD-1 inhibitor, and this treatment was unsuccessful.

[0248] In one embodiment, the method includes the treatment of cancer that has not been previously treated with a PD-1 inhibitor, which initially yielded a partial response but later developed resistance to PD-1 as the disease progressed.

[0249] In one embodiment, the method includes the treatment of cancer that has not been previously treated with a PD-1 inhibitor, which initially resulted in a stable disease but later led to the development of resistance to PD-1 as the disease progressed.

[0250] In one embodiment, the method includes the treatment of cancer that has not been previously treated with a PD-1 inhibitor, which initially resulted in a complete response, but later developed resistance to PD-1 as the disease progressed.

[0251] In one embodiment, the method includes the treatment of cancer that has not been previously treated with a PD-1 inhibitor, and this treatment was unsuccessful.

[0252] In some embodiments of this disclosure, a checkpoint inhibitor or PD-1 inhibitor in combination with a non-polymorphic, crystalline, or crystalline salt form of compound 1 is used to reduce or inhibit the metastasis of a primary tumor or cancer to other sites, or to reduce or inhibit the formation or establishment of metastatic tumors or cancers at other sites distal to the primary tumor or cancer, thereby inhibiting or reducing the recurrence or progression of the tumor or cancer.

[0253] In further embodiments of the present disclosure, provided herein are combination therapies for treating cancer, comprising a non-polymorph, crystalline, or crystalline salt form of compound 1 and a checkpoint inhibitor or PD-1 inhibitor, which have the potential to induce a potent and sustained immune response with enhanced therapeutic benefits and more manageable toxicity.

[0254] In further embodiments of the present disclosure, provided herein are combination therapies for treating cancer, comprising a non-polymorph, crystalline, or crystalline salt form of compound 1 and a checkpoint inhibitor or PD-1 inhibitor. In certain embodiments of the present disclosure, provided herein are methods for treating cancer and / or preventing the establishment of metastasis by using a non-polymorph, crystalline, or crystalline salt form of compound 1 of the present invention that acts synergistically with a checkpoint inhibitor.

[0255] In further embodiments, the Disclosure provides methods for one or more of the following: 1) reducing or inhibiting the growth, proliferation, migration or invasion of tumor or cancer cells that are potentially metastatic or will develop metastases; 2) reducing or inhibiting the formation or establishment of metastases arising from a primary tumor or cancer to one or more other sites, locations or regions different from the primary tumor or cancer; 3) reducing or inhibiting the growth or proliferation of metastases at one or more other sites, locations or regions different from the primary tumor or cancer after metastases have been formed or established; 4) reducing or inhibiting the formation or establishment of additional metastases after metastases have been formed or established; 5) extending overall survival; 6) extending progression-free survival; or 7) stabilizing the disease. The Method comprises administering a non-polymorph, crystalline, or crystalline salt form of Compound 1 of the Invention to a subject in need, in combination with a checkpoint inhibitor as described herein.

[0256] In some embodiments of this disclosure, administration of compound 1 in a non-polymorphic, crystalline, or crystalline salt form in combination with a checkpoint inhibitor or PD-1 inhibitor provides a detectable or measurable improvement in a given subject's condition, such as the alleviation or recovery of one or more adverse (physical) symptoms or consequences associated with the presence of a cytoproliferative or hyperproliferative disorder, neoplasm, tumor or cancer, or metastasis, i.e., a therapeutic benefit or beneficial effect.

[0257] The benefit or beneficial effect of the therapy is any objective or subjective, transient, temporary or long-term improvement in a condition or pathology, or a reduction in the onset, severity, duration or frequency of adverse symptoms associated with or caused by cell proliferation or hyperproliferative disorders, e.g., neoplasm, tumor or cancer, or metastasis. This may lead to an improvement in survival. A satisfactory clinical endpoint of the therapeutic method according to this disclosure is achieved, for example, by an increasing or partial reduction in the severity, duration or frequency of one or more associated pathologies, adverse symptoms or complications, or by the inhibition or restoration of one or more physiological, biochemical or cellular signs or characteristics of cell proliferation or hyperproliferative disorders, e.g., neoplasm, tumor or cancer, or metastasis. Thus, the benefit or improvement of the therapy may be, but are not limited to, the destruction of target proliferating cells (e.g., neoplasm, tumor or cancer, or metastasis), or the ablation of one or more, almost or all, pathologies, adverse symptoms or complications associated with or caused by cell proliferation or hyperproliferative disorders, e.g., neoplasm, tumor or cancer, or metastasis. However, the benefits or improvements of therapy do not necessarily have to be the cure or complete destruction of all target proliferating cells (e.g., neoplasms, tumors or cancers, or metastases), or the ablation of all pathological conditions, adverse symptoms, or complications associated with or caused by cell proliferation or hyperproliferative disorders, such as neoplasms, tumors or cancers, or metastases. For example, partial destruction of a tumor or cancer cell mass, or stabilization of a tumor or cancer mass, size, or cell number by inhibiting the progression or worsening of the tumor or cancer, can reduce mortality and extend life, even if only by days, weeks, or months, even if some or most of the tumor or cancer mass, size, or cells remain.

[0258] Specific, non-limiting examples of the benefits of the therapy include reducing the volume (size or cell mass) or number of neoplasms, tumors or cancers, or metastases; inhibiting or preventing (e.g., stabilizing) the increase in volume of neoplasms, tumors or cancers; slowing or inhibiting the progression, worsening or metastasis of neoplasms, tumors or cancers; or inhibiting the proliferation, growth or metastasis of neoplasms, tumors or cancers.

[0259] In some embodiments of the present disclosure, administration of a checkpoint inhibitor or PD-1 inhibitor in combination therapy with a non-polymorphic, crystalline, or crystalline salt form of compound 1 provides a detectable or measurable improvement or overall response according to an irRC (derived from time-point response assessments and based on tumor burden), comprising one or more of the following: (i) irCR -- complete disappearance of all lesions, whether measurable or not, and no new lesions (confirmed by repeated sequential assessments at least four weeks from the first documented date); (ii) irPR -- a reduction of ≥50% of tumor burden compared to baseline (confirmed by sequential assessments at least four weeks after the first documented date).

[0260] Optionally, any method described herein does not have to be immediately effective. For example, an increase in the number or mass of neoplasm, tumor, or cancer cells may continue after treatment, but over time, a final stabilization or reduction in the mass, size, or number of tumor cells may subsequently occur in a given subject.

[0261] Additional adverse symptoms and complications associated with neoplasms, tumors, cancers, and metastases that can be inhibited, reduced, decreased, delayed, or prevented include, for example, nausea, loss of appetite, lethargy, pain, and discomfort. Therefore, partial or complete reduction or decrease in the severity, duration, or frequency of adverse symptoms or complications associated with or caused by cytohyperproliferative disorders, and improvements in the subject's quality of life and / or health, such as increased energy, appetite, and psychological health, are all specific, non-limiting examples of the benefits of the therapy.

[0262] Therefore, the benefits or improvements of the therapy may also include subjective improvements in the quality of life of the treated subject. In additional embodiments, the method extends or prolongs the subject's lifespan (survival period). In further embodiments, the method improves the subject's quality of life.

[0263] In one embodiment, administration of a checkpoint inhibitor or PD-1 inhibitor in combination therapy with a non-polymorphic, crystalline, or crystalline salt form of compound 1 results in clinically relevant improvements in: (i) overall survival, (ii) progression-free survival, (iii) overall response rate, (iv) reduction of metastatic disease, (v) circulating levels of tumor antigens such as carbohydrate antigen 19.9 (CA19.9) and carcinoembryonic antigen (CEA) or other tumor-dependent factors, (vii) nutritional status (body weight, appetite, serum albumin), (viii) pain management or use of analgesics, and (ix) one or more markers of disease status and progression selected from one or more of the CRP / albumin ratio.

[0264] Treatment with compound 1 in non-polymorphic, crystalline, or crystalline salt forms in combination with checkpoint inhibitors or PD-1 inhibitors induces a more complex immune response, including not only the development of innate and type 1 immunity, but also immunomodulation that more efficiently restores appropriate immune function.

[0265] Combination of compound 1 and checkpoint inhibitor Combination of compound 1 and atezolizumab In the embodiments described above, compound 1 is administered together with a checkpoint inhibitor for treating cancer, and optionally with additional immunomodulators.

[0266] In one embodiment, the checkpoint inhibitor is atezolizumab.

[0267] In one embodiment, the present invention is a method for treating cancer in a subject, (i) administering to the subject a therapeutically effective amount of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, (ii) A method comprising administering a therapeutically effective dose of atezolizumab to a subject.

[0268] In one embodiment, the present invention is a method for treating cancer in a subject, (i) administering to subjects a dose of compound 1 or a pharmaceutically acceptable salt, or a pharmaceutical composition containing compound 1, in an amount of approximately 5 mg to approximately 100 mg, (ii) A method comprising administering a therapeutically effective dose of atezolizumab to a subject.

[0269] In another embodiment, the present invention relates to a method for treating cancer in a subject, wherein the subject in need of such treatment is given a therapeutically effective amount of compound 1 or a pharmaceutically acceptable salt thereof, or compound (1) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, The method includes administering a therapeutically effective dose of atezolizumab or a pharmaceutical composition containing atezolizumab in combination with the atezolizumab.

[0270] In another embodiment, the present invention relates to a method for treating cancer in a subject, wherein the subject requiring such treatment is given a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof, or compound (1) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, in a dose of about 5 mg to about 100 mg. The method includes administering a therapeutically effective dose of atezolizumab or a pharmaceutical composition containing atezolizumab in combination with the atezolizumab.

[0271] In one embodiment, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, and atezolizumab or a pharmaceutical composition containing atezolizumab are administered simultaneously, sequentially, or separately.

[0272] In one embodiment, the cancer is selected from cardia cancer, head and neck cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, non-clear cell renal cell carcinoma, advanced clear cell renal cell carcinoma, castration-resistant prostate cancer, hormone receptor-positive breast cancer, prostate cancer, colon cancer, gastrointestinal cancer, breast cancer, urogenital tract cancer, liver cancer, bone cancer, thyroid cancer, nervous system cancer, gynecological cancer, hematological cancer, skin cancer, urothelial carcinoma, and adrenal gland cancer.

[0273] In one embodiment, the amount of compound 1 or a pharmaceutically acceptable salt of compound 1 administered is greater than 0.0 mg but less than or equal to 100 mg of compound 1, greater than 0.0 mg but less than or equal to 95 mg of compound 1, greater than 0.0 mg but less than or equal to 90 mg of compound 1, greater than 0.0 mg but less than or equal to 85 mg of compound 1, greater than 0.0 mg but less than or equal to 80 mg of compound 1, greater than 0.0 mg but less than or equal to 75 mg of compound 1, greater than 0.0 mg but less than or equal to 70 mg of compound 1, greater than 0.0 mg but less than or equal to 65 mg of compound 1, greater than 0.0 mg but less than or equal to 60 mg of compound 1. The amounts are greater than 0.0 mg to 55 mg of compound 1, greater than 0.0 mg to 50 mg of compound 1, greater than 0.0 mg to 45 mg of compound 1, greater than 0.0 mg to 40 mg of compound 1, greater than 0.0 mg to 35 mg of compound 1, greater than 0.0 mg to 30 mg of compound 1, greater than 0.0 mg to 25 mg of compound 1, greater than 0.0 mg to 20 mg of compound 1, greater than 0.0 mg to 15 mg of compound 1, greater than 0.0 mg to 10 mg of compound 1, or 5 mg or less of compound 1. In one embodiment, compound 1 is administered once daily. In another embodiment, compound 1 is administered twice daily.

[0274] In one embodiment, atezolizumab is administered intravenously (IV) to the subject. In another embodiment, atezolizumab is administered to the subject by parenteral infusion.

[0275] In one embodiment, atezolizumab is administered once every two weeks, once every three weeks, or once every four weeks during the treatment period. In a further embodiment, atezolizumab is administered once every two weeks during the treatment period. In another further embodiment, atezolizumab is administered once every three weeks during the treatment period. In yet another further embodiment, atezolizumab is administered once every four weeks during the treatment period.

[0276] In one embodiment, the dose of atezolizumab is approximately 800 mg to approximately 1700 mg.

[0277] In one embodiment, the dose of atezolizumab is approximately 840 mg administered once every two weeks, approximately 1200 mg administered once every three weeks, or 1680 mg administered once every four weeks.

[0278] In one embodiment, tezolizumab is administered to subjects in IV unit dosage forms, the dose forms comprising 840 mg, 1200 mg, or 1680 mg of atezolizumab, water, glacial acetic acid, L-histidine, polysorbate 20, and sucrose.

[0279] In one embodiment, atezolizumab is administered to subjects in IV units, and the dosage form is marketed as Tecentriq®.

[0280] Combination of compound 1 and avelumab In one embodiment, the checkpoint inhibitor is avelumab.

[0281] In one embodiment, the present invention is a method for treating cancer in a subject, (i) administering to the subject a therapeutically effective amount of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, (ii) A method comprising administering a therapeutically effective dose of avelumab to a subject.

[0282] In one embodiment, the present invention is a method for treating cancer in a subject, (i) administering to subjects a dose of compound 1 or a pharmaceutically acceptable salt, or a pharmaceutical composition containing compound 1, in an amount of approximately 5 mg to approximately 100 mg, (ii) A method comprising administering a therapeutically effective dose of avelumab to a subject.

[0283] In another embodiment, the present invention relates to a method for treating cancer in a subject, wherein the subject in need of such treatment is given a therapeutically effective amount of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. The method includes administering a therapeutically effective dose of avelumab or a pharmaceutical composition containing avelumab in combination with the avelumab.

[0284] In another embodiment, the present invention relates to a method for treating cancer in a subject, wherein the subject requiring such treatment is given a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof in a dose of about 5 mg to about 100 mg, or compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. The method includes administering a therapeutically effective dose of avelumab or a pharmaceutical composition containing avelumab in combination with the avelumab.

[0285] In one embodiment, the present invention is a method for treating urothelial carcinoma in a subject, (i) administering to subjects a dose of compound 1 or a pharmaceutically acceptable salt, or a pharmaceutical composition containing compound 1, in an amount of approximately 5 mg to approximately 100 mg, (ii) A method comprising administering a therapeutically effective dose of avelumab to a subject.

[0286] In another embodiment, the present invention relates to a method for treating urothelial carcinoma in a subject, wherein the subject in need of such treatment is given a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof in a dose of about 5 mg to about 100 mg, or compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. The method includes administering a therapeutically effective dose of avelumab or a pharmaceutical composition containing avelumab in combination with the avelumab.

[0287] In one embodiment, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, and avelumab or a pharmaceutical composition containing avelumab are administered simultaneously, sequentially, or separately.

[0288] In one embodiment, the cancer is selected from cardia cancer, head and neck cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, non-clear cell renal cell carcinoma, advanced clear cell renal cell carcinoma, castration-resistant prostate cancer, hormone receptor-positive breast cancer, prostate cancer, colon cancer, gastrointestinal cancer, breast cancer, urogenital tract cancer, liver cancer, bone cancer, thyroid cancer, nervous system cancer, gynecological cancer, hematological cancer, skin cancer, urothelial carcinoma, and adrenal gland cancer.

[0289] In one embodiment, urothelial carcinoma is locally progressive or metastatic transitional cell carcinoma of the urothelium. In another embodiment, urothelial carcinoma is progressive urothelial carcinoma. In yet another embodiment, urothelial carcinoma is urothelial carcinoma of the renal pelvis, ureter, bladder, or urethra.

[0290] In one embodiment, the amount of compound 1 or a pharmaceutically acceptable salt of compound 1 administered is greater than 0.0 mg but less than or equal to 100 mg of compound 1, greater than 0.0 mg but less than or equal to 95 mg of compound 1, greater than 0.0 mg but less than or equal to 90 mg of compound 1, greater than 0.0 mg but less than or equal to 85 mg of compound 1, greater than 0.0 mg but less than or equal to 80 mg of compound 1, greater than 0.0 mg but less than or equal to 75 mg of compound 1, greater than 0.0 mg but less than or equal to 70 mg of compound 1, greater than 0.0 mg but less than or equal to 65 mg of compound 1, greater than 0.0 mg but less than or equal to 60 mg of compound 1. The amounts are greater than 0.0 mg to 55 mg of compound 1, greater than 0.0 mg to 50 mg of compound 1, greater than 0.0 mg to 45 mg of compound 1, greater than 0.0 mg to 40 mg of compound 1, greater than 0.0 mg to 35 mg of compound 1, greater than 0.0 mg to 30 mg of compound 1, greater than 0.0 mg to 25 mg of compound 1, greater than 0.0 mg to 20 mg of compound 1, greater than 0.0 mg to 15 mg of compound 1, greater than 0.0 mg to 10 mg of compound 1, or 5 mg or less of compound 1. In one embodiment, compound 1 is administered once daily. In another embodiment, compound 1 is administered twice daily.

[0291] In one embodiment, avelumab is administered to the subject intravenously (IV). In another embodiment, avelumab is administered to the subject by intravenous infusion.

[0292] In one embodiment, avelumab is administered once every two weeks, once every three weeks, or once every four weeks during the treatment period. In a further embodiment, avelumab is administered once every two weeks during the treatment period. In another further embodiment, avelumab is administered once every three weeks during the treatment period. In yet another further embodiment, avelumab is administered once every four weeks during the treatment period.

[0293] In one embodiment, the dose of avelumab is approximately 500 mg to approximately 1700 mg.

[0294] In one embodiment, the dose of avelumab is approximately 800 mg administered once every two weeks, approximately 1200 mg administered once every three weeks, or 1600 mg administered once every four weeks.

[0295] In one embodiment, the dose of avelumab is approximately 800 mg, administered once every two weeks.

[0296] In one embodiment, avelumab is administered to subjects in IV units, and the dosage form is marketed as Bavencio®.

[0297] In one embodiment, the subject is a human being.

[0298] In one embodiment, a subject with advanced urothelial carcinoma is classified as having stage IV disease according to the AJCC TNM staging criteria (8th edition, January 1, 2018).

[0299] In one embodiment, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, and avelumab or a pharmaceutical composition containing avelumab are administered simultaneously, sequentially, or separately.

[0300] In one embodiment, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, and a combination of avelumab or a pharmaceutical composition containing avelumab are administered as maintenance therapy to a subject with advanced urothelial carcinoma.

[0301] In one embodiment, a subject with advanced urothelial carcinoma received first-line platinum-based doublet chemotherapy prior to maintenance therapy.

[0302] In one embodiment, the first-line platinum-based doublet chemotherapy comprises gemcitabine plus cisplatin and / or gemcitabine plus carboplatin.

[0303] In one embodiment, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, and a combination of avelumab or a pharmaceutical composition containing avelumab are administered to subjects with advanced urothelial carcinoma as second-line or third-line therapy.

[0304] In one embodiment, a subject with advanced urothelial carcinoma received first-line platinum-based doublet chemotherapy before receiving second-line or third-line therapy.

[0305] In one embodiment, the first-line platinum-based doublet chemotherapy comprises gemcitabine plus cisplatin and / or gemcitabine plus carboplatin.

[0306] In one embodiment, subjects with advanced urothelial carcinoma received first-line platinum-based doublet chemotherapy for at least four cycles, but no more than six cycles.

[0307] In one embodiment, the subject's disease has progressed after first-line platinum-based doublet chemotherapy.

[0308] In one embodiment, the method includes the treatment of cancer that has not been previously treated with prior immunotherapy using IL-2, IFN-α, or any anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CD137, or CTLA-4 antibody (including ipilimumab), or any other antibody or drug that specifically targets T cell costimulation or immune checkpoints. In another embodiment, the method includes the treatment of cancer that has not been previously treated with a PD-1 or PD-L1 inhibitor.

[0309] Combination of compound 1 and nivolumab In one embodiment, the present invention is a method for treating cancer in a subject, (i) administering to the subject a therapeutically effective amount of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, (ii) A method comprising administering a therapeutically effective dose of nivolumab to a subject.

[0310] In one embodiment, the present invention is a method for treating cancer in a subject, (i) administering to subjects a dose of compound 1 or a pharmaceutically acceptable salt, or a pharmaceutical composition containing compound 1, in an amount of approximately 5 mg to approximately 100 mg, (ii) A method comprising administering a therapeutically effective dose of nivolumab to a subject.

[0311] In another embodiment, the present invention includes a method for treating cancer in a subject, comprising administering to the subject in need of such treatment a therapeutically effective amount of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient, in combination with a therapeutically effective amount of nivolumab or a pharmaceutical composition comprising nivolumab.

[0312] In another embodiment, the present invention includes a method for treating cancer in a subject, comprising administering to a subject in need of such treatment a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof in a dose of about 5 mg to about 100 mg, or compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient, in combination with a therapeutically effective dose of nivolumab or a pharmaceutical composition comprising nivolumab.

[0313] In one embodiment, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, and nivolumab or a pharmaceutical composition containing nivolumab are administered simultaneously, sequentially, or separately.

[0314] In these and other embodiments, niborum is administered at approximately 360 mg every three weeks.

[0315] In one embodiment, the cancer is selected from melanoma, cardia cancer, head and neck cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, non-clear cell renal cell carcinoma, advanced clear cell renal cell carcinoma, castration-resistant prostate cancer, hormone receptor-positive breast cancer, prostate cancer, colon cancer, gastrointestinal cancer, breast cancer, urogenital tract cancer, liver cancer, bone cancer, thyroid cancer, nervous system cancer, gynecological cancer, hematological cancer, skin cancer, and adrenal gland cancer. In another embodiment, the cancer is selected from melanoma, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, classical Hodgkin lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, microsatellite-unstable high colorectal cancer, and hepatocellular carcinoma.

[0316] In one embodiment, the cancer is a solid tumor. In another embodiment, the solid tumor is selected from the group consisting of sarcomas, carcinomas, and lymphomas. In a further embodiment, the solid tumor is a genitourinary cancer. In a further embodiment, the genitourinary cancer is selected from the group consisting of clear cell renal cell carcinoma (ccRCC), non-clear cell renal cell carcinoma (nccRCC), urothelial carcinoma (UC, ICI-never experienced, and experienced), and metastatic castration-resistant prostate cancer (mCRPC).

[0317] The amount of compound 1 or its pharmaceutically acceptable salt administered is greater than 0.0 mg but less than or equal to 100 mg of compound 1, greater than 0.0 mg but less than or equal to 95 mg of compound 1, greater than 0.0 mg but less than or equal to 90 mg of compound 1, greater than 0.0 mg but less than or equal to 85 mg of compound 1, greater than 0.0 mg but less than or equal to 80 mg of compound 1, greater than 0.0 mg but less than or equal to 75 mg of compound 1, greater than 0.0 mg but less than or equal to 70 mg of compound 1, greater than 0.0 mg but less than or equal to 65 mg of compound 1, greater than 0.0 mg but less than or equal to 60 mg of compound 1, compound The amounts are greater than 0.0 mg and up to 55 mg of compound 1, greater than 0.0 mg and up to 50 mg of compound 1, greater than 0.0 mg and up to 45 mg of compound 1, greater than 0.0 mg and up to 40 mg of compound 1, greater than 0.0 mg and up to 35 mg of compound 1, greater than 0.0 mg and up to 30 mg of compound 1, greater than 0.0 mg and up to 25 mg of compound 1, greater than 0.0 mg and up to 20 mg of compound 1, greater than 0.0 mg and up to 15 mg of compound 1, greater than 0.0 mg and up to 10 mg of compound 1, or 5 mg or less of compound 1. In one embodiment, compound 1 is administered once daily. In another embodiment, compound 1 is administered twice daily.

[0318] In one embodiment, nivolumab is administered to the subject intravenously (IV). In another embodiment, nivolumab is administered to the subject by intravenous infusion.

[0319] In one embodiment, nivolumab is administered once every two weeks, once every three weeks, or once every four weeks during the treatment period. In a further embodiment, nivolumab is administered once every two weeks during the treatment period. In another further embodiment, nivolumab is administered once every three weeks during the treatment period. In yet another further embodiment, nivolumab is administered once every four weeks during the treatment period.

[0320] In one embodiment, the dose of nivolumab is approximately 50 mg to approximately 500 mg.

[0321] In one embodiment, nivolumab is administered to subjects in IV unit dosage form, and the dosage form is marketed as OPDIVO®.

[0322] Compound 1 in combination with other checkpoint inhibitors In one embodiment, the checkpoint inhibitor is pembrolizumab.

[0323] In one embodiment, the present invention is a method for treating cancer in a subject, (i) administering to subjects a dose of compound 1 or a pharmaceutically acceptable salt, or a pharmaceutical composition containing compound 1, in an amount of approximately 5 mg to approximately 100 mg, (ii) A method comprising administering a therapeutically effective dose of pembrolizumab to a subject.

[0324] In another embodiment, the present invention relates to a method for treating cancer in a subject, wherein the subject requiring such treatment is given a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof in a dose of about 5 mg to about 100 mg, or compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. The method includes administering a therapeutically effective dose of pembrolizumab or a pharmaceutical composition containing pembrolizumab in combination with the drug.

[0325] In one embodiment, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, and pembrolizumab or a pharmaceutical composition containing pembrolizumab are administered simultaneously, sequentially, or separately.

[0326] In one embodiment, the cancer is selected from melanoma, cardia cancer, head and neck cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, non-clear cell renal cell carcinoma, advanced clear cell renal cell carcinoma, castration-resistant prostate cancer, hormone receptor-positive breast cancer, prostate cancer, colon cancer, gastrointestinal cancer, breast cancer, urogenital tract cancer, liver cancer, bone cancer, thyroid cancer, nervous system cancer, gynecological cancer, hematological cancer, skin cancer, and adrenal cancer. In another embodiment, the cancer is selected from melanoma, non-small cell lung cancer, small cell lung cancer, head and neck squamous cell carcinoma, classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, microsatellite instability hypercarcinoma, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma, urothelial carcinoma, or endometrial cancer.

[0327] The amount of compound 1 or its pharmaceutically acceptable salt administered is greater than 0.0 mg but less than or equal to 100 mg of compound 1, greater than 0.0 mg but less than or equal to 95 mg of compound 1, greater than 0.0 mg but less than or equal to 90 mg of compound 1, greater than 0.0 mg but less than or equal to 85 mg of compound 1, greater than 0.0 mg but less than or equal to 80 mg of compound 1, greater than 0.0 mg but less than or equal to 75 mg of compound 1, greater than 0.0 mg but less than or equal to 70 mg of compound 1, greater than 0.0 mg but less than or equal to 65 mg of compound 1, greater than 0.0 mg but less than or equal to 60 mg of compound 1, compound The amounts are greater than 0.0 mg and up to 55 mg of compound 1, greater than 0.0 mg and up to 50 mg of compound 1, greater than 0.0 mg and up to 45 mg of compound 1, greater than 0.0 mg and up to 40 mg of compound 1, greater than 0.0 mg and up to 35 mg of compound 1, greater than 0.0 mg and up to 30 mg of compound 1, greater than 0.0 mg and up to 25 mg of compound 1, greater than 0.0 mg and up to 20 mg of compound 1, greater than 0.0 mg and up to 15 mg of compound 1, greater than 0.0 mg and up to 10 mg of compound 1, or 5 mg or less of compound 1. In one embodiment, compound 1 is administered once daily. In another embodiment, compound 1 is administered twice daily.

[0328] In one embodiment, pembrolizumab is administered to the subject intravenously (IV). In another embodiment, pembrolizumab is administered to the subject by intravenous infusion.

[0329] In one embodiment, pembrolizumab is administered once every two weeks, once every three weeks, or once every four weeks during the treatment period. In a further embodiment, pembrolizumab is administered once every two weeks during the treatment period. In another further embodiment, pembrolizumab is administered once every three weeks during the treatment period. In yet another further embodiment, pembrolizumab is administered once every four weeks during the treatment period.

[0330] In one embodiment, the dose of pembrolizumab is approximately 50 mg to approximately 250 mg.

[0331] In one embodiment, pembrolizumab is administered to subjects in IV units, and the dosage form is marketed as KETRUDA®.

[0332] In one embodiment, the checkpoint inhibitor is durvalumab.

[0333] In one embodiment, the present invention is a method for treating cancer in a subject, (i) administering to subjects a dose of compound 1 or a pharmaceutically acceptable salt, or a pharmaceutical composition containing compound 1, in an amount of approximately 5 mg to approximately 100 mg, (ii) A method comprising administering a therapeutically effective dose of durvalumab to a subject.

[0334] In another embodiment, the present invention relates to a method for treating cancer in a subject, wherein the subject requiring such treatment is given a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof in a dose of about 5 mg to about 100 mg, or compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. The method includes administering a therapeutically effective dose of durvalumab or in combination with a pharmaceutical composition containing durvalumab.

[0335] In one embodiment, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, and durvalumab or a pharmaceutical composition containing durvalumab are administered simultaneously, sequentially, or separately.

[0336] In one embodiment, the cancer is selected from melanoma, cardia cancer, head and neck cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, non-clear cell renal cell carcinoma, advanced clear cell renal cell carcinoma, castration-resistant prostate cancer, hormone receptor-positive breast cancer, prostate cancer, colon cancer, gastrointestinal cancer, breast cancer, urogenital tract cancer, liver cancer, bone cancer, thyroid cancer, nervous system cancer, gynecological cancer, hematological cancer, skin cancer, urothelial carcinoma, and adrenal gland cancer. In another embodiment, the cancer is selected from the group consisting of urothelial carcinoma and non-small cell lung cancer.

[0337] The amount of compound 1 or its pharmaceutically acceptable salt administered is greater than 0.0 mg but less than or equal to 100 mg of compound 1, greater than 0.0 mg but less than or equal to 95 mg of compound 1, greater than 0.0 mg but less than or equal to 90 mg of compound 1, greater than 0.0 mg but less than or equal to 85 mg of compound 1, greater than 0.0 mg but less than or equal to 80 mg of compound 1, greater than 0.0 mg but less than or equal to 75 mg of compound 1, greater than 0.0 mg but less than or equal to 70 mg of compound 1, greater than 0.0 mg but less than or equal to 65 mg of compound 1, greater than 0.0 mg but less than or equal to 60 mg of compound 1, compound The amounts are greater than 0.0 mg and up to 55 mg of compound 1, greater than 0.0 mg and up to 50 mg of compound 1, greater than 0.0 mg and up to 45 mg of compound 1, greater than 0.0 mg and up to 40 mg of compound 1, greater than 0.0 mg and up to 35 mg of compound 1, greater than 0.0 mg and up to 30 mg of compound 1, greater than 0.0 mg and up to 25 mg of compound 1, greater than 0.0 mg and up to 20 mg of compound 1, greater than 0.0 mg and up to 15 mg of compound 1, greater than 0.0 mg and up to 10 mg of compound 1, or 5 mg or less of compound 1. In one embodiment, compound 1 is administered once daily. In another embodiment, compound 1 is administered twice daily.

[0338] In one embodiment, durvalumab is administered to the subject intravenously (IV). In another embodiment, durvalumab is administered to the subject by parenteral infusion.

[0339] In one embodiment, durvalumab is administered once every two weeks, once every three weeks, or once every four weeks during the treatment period. In a further embodiment, durvalumab is administered once every two weeks during the treatment period. In another further embodiment, durvalumab is administered once every three weeks during the treatment period. In yet another further embodiment, durvalumab is administered once every four weeks during the treatment period.

[0340] In one embodiment, the dose of durvalumab is approximately 10 mg / kg every two weeks.

[0341] In one embodiment, durvalumab is administered to subjects in IV units, and the dosage form is marketed as IMFINZI®.

[0342] In one embodiment, the checkpoint inhibitor is semiprimab.

[0343] In one embodiment, the present invention is a method for treating cancer in a subject, (i) administering to subjects a dose of compound 1 or a pharmaceutically acceptable salt, or a pharmaceutical composition containing compound 1, in an amount of approximately 5 mg to approximately 100 mg, (ii) A method comprising administering to a subject a therapeutically effective dose of cemiprimab or a pharmaceutically acceptable salt or prodrug thereof.

[0344] In another embodiment, the present invention relates to a method for treating cancer in a subject, wherein the subject requiring such treatment is given a pharmaceutical composition comprising compound 1 or a pharmaceutically acceptable salt thereof in a dose of about 5 mg to about 100 mg, or compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. The method includes administering a therapeutically effective dose of cemiprimab or in combination with a pharmaceutical composition containing cemiprimab.

[0345] In one embodiment, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, and semiprimab or a pharmaceutical composition containing semiprimab are administered simultaneously, sequentially, or separately.

[0346] In one embodiment, the cancer is selected from melanoma, cardia cancer, head and neck cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, non-clear cell renal cell carcinoma, advanced clear cell renal cell carcinoma, castration-resistant prostate cancer, hormone receptor-positive breast cancer, prostate cancer, colon cancer, gastrointestinal cancer, breast cancer, urogenital tract cancer, liver cancer, bone cancer, thyroid cancer, nervous system cancer, gynecological cancer, hematological cancer, skin cancer, urothelial carcinoma, and adrenal gland cancer. In another embodiment, the cancer is cutaneous squamous cell carcinoma.

[0347] The amount of compound 1 or its pharmaceutically acceptable salt administered is greater than 0.0 mg but less than or equal to 100 mg of compound 1, greater than 0.0 mg but less than or equal to 95 mg of compound 1, greater than 0.0 mg but less than or equal to 90 mg of compound 1, greater than 0.0 mg but less than or equal to 85 mg of compound 1, greater than 0.0 mg but less than or equal to 80 mg of compound 1, greater than 0.0 mg but less than or equal to 75 mg of compound 1, greater than 0.0 mg but less than or equal to 70 mg of compound 1, greater than 0.0 mg but less than or equal to 65 mg of compound 1, greater than 0.0 mg but less than or equal to 60 mg of compound 1, compound The amounts are greater than 0.0 mg and up to 55 mg of compound 1, greater than 0.0 mg and up to 50 mg of compound 1, greater than 0.0 mg and up to 45 mg of compound 1, greater than 0.0 mg and up to 40 mg of compound 1, greater than 0.0 mg and up to 35 mg of compound 1, greater than 0.0 mg and up to 30 mg of compound 1, greater than 0.0 mg and up to 25 mg of compound 1, greater than 0.0 mg and up to 20 mg of compound 1, greater than 0.0 mg and up to 15 mg of compound 1, greater than 0.0 mg and up to 10 mg of compound 1, or 5 mg or less of compound 1. In one embodiment, compound 1 is administered once daily. In another embodiment, compound 1 is administered twice daily.

[0348] In one embodiment, semiprimab is administered intravenously (IV) to the subject. In another embodiment, semiprimab is administered by parenteral infusion to the subject.

[0349] In one embodiment, cemiplimab is administered once every two weeks, once every three weeks, or once every four weeks during the treatment period. In a further embodiment, cemiplimab is administered once every two weeks during the treatment period. In another further embodiment, cemiplimab is administered once every three weeks during the treatment period. In yet another further embodiment, cemiplimab is administered once every four weeks during the treatment period.

[0350] In one embodiment, the dose of semiprimab is approximately 350 mg / 7 mL every three weeks.

[0351] In one embodiment, semiprimab is administered to subjects in IV units, and the dosage form is marketed as LIBTAYO®.

[0352] Combination of compound 1 with a checkpoint inhibitor and additional immunomodulators In the embodiments described above, compound 1 is administered together with a checkpoint inhibitor for treating cancer, and optionally with additional immunomodulators. In one embodiment, the checkpoint inhibitor is selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors. In these and other embodiments, the checkpoint inhibitors include pembrolizumab, nivolumab, atezolizumab (TECENTRIQ®), durvalumab, avelumab (BAVENCIO®), semiprimab, camrelizumab, cintilimab, tislerizumab, tripalimab, spartalizumab, dostallimab, KN035 (Jiangsu Alphamb Biopharmaceuticals Co.), kosiberimab (formerly CK-301), CA-170 (Curis, Inc.), BMS-986189 (Bristol Myers Squibb Co.), and ipilimumab (Yervoy, Bristol Myers Squibb Co.). Selected from the group consisting of Co.)

[0353] In one embodiment, the checkpoint inhibitor is nivolumab.

[0354] In one embodiment, the additional immunomodulator is ipilimumab.

[0355] In another embodiment, the additional immunomodulator is an IL-2 targeting agent. In a further embodiment, the IL-2 targeting agent is a CD122-preferential IL-2 pathway agonist, a PEG-IL-2Rαβ biased agonist, an IL-2Rβ biased agonist, or an IL-2Rβγ c Biased agonists, IL-2v / IL-2α fusion proteins, anti-EDB mAb (L19) / IL-2v fused to L19 / TNFv, anti-GD2 mAb / IL-2v, anti-FAP mAb / IL-2v, anti-CEA mAb / IL-2v, anti-PD-1 mAb / IL-2v, patient-derived tumor cell vaccine + HD-IL-2, adoptive cell therapy + IL-2 injection, adoptive cell therapy + IL-2 injection + anti-PD-1 mAb, orthogonal IL-2v / IL-2Rβ variant pairs, anti-IL-2Rα mAb / PBD conjugate, PEG-IL-2Rα biased agonist, IL-2v / human Fc fusion protein, PEG-IL-2Rα biased (N88D) / IgG1 fusion protein, anti-IL-2 The group consists of recombinant plasmids encoding mAb / IL-2v, IL-2, PPI, TGF-β1, and IL-10, as well as IL-2Rβ antagonists.

[0356] In one embodiment, the IL-2 targeting agent is a CD122-preferential IL-2 pathway agonist.

[0357] In one embodiment, the IL-2 targeting agent is benpegaldesleukine (BEMPEG; NKTR-214; Bristol Myers Squibb Co.).

[0358] In one embodiment, the IL-2 targeting agent is a PEG-IL-2Rαβ biased agonist.

[0359] In one embodiment, the IL-2 targeting agent is THOR-707 (Sanofi).

[0360] In one embodiment, the IL-2 targeting agent is TransCon IL-2β / γ (Ascendis Pharma).

[0361] In one embodiment, the IL-2 targeting agent is an IL-2Rβ bias agonist.

[0362] In one embodiment, the IL-2 targeting agent is MDNA-19 (Medicenna).

[0363] In one embodiment, the IL-2 targeting agent is IL-2Rβγ c He is a biased agonist.

[0364] In one embodiment, the IL-2 targeting agent is Neo-2 / 15(Neoleukin).

[0365] In one embodiment, the IL-2 targeting agent is an IL-2v / IL-2Rα fusion protein.

[0366] In one embodiment, the IL-2 targeting agent is an anti-EDB mAb(L19) / IL-2v fused to L19 / TNFv.

[0367] In one embodiment, the IL-2 targeting agent is daromum (Philogen).

[0368] In one embodiment, the IL-2 targeting agent is an anti-EDB mAb(L19) / IL-2v.

[0369] In one embodiment, the IL-2 targeting agent is darleukin (Philogen).

[0370] In one embodiment, the IL-2 targeting agent is an anti-GD2 mAb / IL-2v.

[0371] In one embodiment, the IL-2 targeting agent is APN-301 (APerion).

[0372] In one embodiment, the IL-2 targeting agent is RG-7461 (Roche).

[0373] In one embodiment, the IL-2 targeting agent is an anti-CEA mAb / IL-2v.

[0374] In one embodiment, the IL-2 targeting agent is cergutuzumab amanaleukin (Roche).

[0375] In one embodiment, the IL-2 targeting agent is an anti-PD-1 mAb / IL-2v. In another embodiment, the IL-2 targeting agent is PD1-IL2v(Roche).

[0376] In one embodiment, the IL-2 targeting agent is a vaccine of patient-derived tumor cells + HD-IL-2.

[0377] In one embodiment, the IL-2 targeting agent is the Oncoquest-L vaccine (Xemebiopharma.com).

[0378] In one embodiment, the IL-2 targeting agent is adoptive cell therapy + IL-2 injection.

[0379] In one embodiment, the IL-2 targeting agent is lifileucel (Iovance).

[0380] In one embodiment, the IL-2 targeting agent is adoptive cell therapy + IL-2 injection + anti-PD-1 mAb.

[0381] In one embodiment, the IL-2 targeting agent is rifeureucel + pembrolizumab.

[0382] In one embodiment, the IL-2 targeting agent is an orthogonal IL-2v / IL-2Rβ mutant pair.

[0383] In one embodiment, the IL-2 targeting agent is an anti-IL-2Rα mAb.PBD conjugate.

[0384] In one embodiment, the IL-2 targeting agent is camidanlumab tesirine (ADC Therapeutics).

[0385] In one embodiment, the IL-2 targeting agent is a PEG-IL2-Rα biased agonist.

[0386] In one embodiment, the IL-2 targeting agent is NKTR-358 (Bristol Myers Squibb).

[0387] In one embodiment, the IL-2 targeting agent is THOR-809 (Sanofi).

[0388] In one embodiment, the IL-2 targeting agent is an IL-2v / human fusion protein.

[0389] In one embodiment, the IL-2 targeting agent is efavaleukin alfa (AMG592) (Amgen).

[0390] In one embodiment, the IL-2 targeting agent is an IL-2Rα bias (N88D) / IgG1 fusion protein.

[0391] In one embodiment, the IL-2 targeting agent is RG-7835 (RO7049665) (Roche).

[0392] In one embodiment, the IL-2 targeting agent is an IL-2 mutein / Fc fusion protein.

[0393] In one embodiment, the IL-2 targeting agent is CC-92252 (Bristol Myers Squibb).

[0394] In one embodiment, the IL-2 targeting agent is an anti-IL-2 mAb / IL-2v.

[0395] In one embodiment, the IL-2 targeting agent is F5111.2 (Creative Biolabs).

[0396] In one embodiment, the IL-2 targeter is a recombinant plasmid encoding IL-2, PPI, TGF-β1, and IL-10.

[0397] In one embodiment, the IL-2 targeting agent is NNC0361-0041(NIDDK).

[0398] In one embodiment, the IL-2 targeting agent is an IL-2Rβ antagonist.

[0399] In one embodiment, the IL-2 targeting agent is MDNA-209 (Medicenna).

[0400] In one embodiment, the present invention is a method for treating cancer in a subject, (i) administering to the subject a therapeutically effective amount of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, (ii) administering to the subject a therapeutically effective dose of nivolumab or a pharmaceutical composition containing nivolumab, (iii) A method comprising administering to a subject a therapeutically effective amount of an immunomodulator or a pharmaceutical composition comprising a therapeutically effective amount of an immunomodulator.

[0401] In one embodiment, the present invention is a method for treating cancer in a subject, (i) administering to subjects a dose of compound 1 or a pharmaceutically acceptable salt, or a pharmaceutical composition containing compound 1, in an amount of approximately 5 mg to approximately 100 mg, (ii) administering to the subject a therapeutically effective dose of nivolumab or a pharmaceutical composition containing nivolumab, (iii) A method comprising administering to a subject a therapeutically effective amount of an immunomodulator or a pharmaceutical composition comprising a therapeutically effective amount of an immunomodulator.

[0402] In one embodiment, the present invention is a method for treating cancer in a subject, (i) administering to the subject a therapeutically effective amount of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, (ii) administering to the subject a therapeutically effective dose of nivolumab or a pharmaceutical composition containing nivolumab, (iii) a method comprising administering to a subject a therapeutically effective dose of ipilimumab or a pharmaceutical composition comprising a therapeutically effective dose of ipilimumab.

[0403] In one embodiment, the present invention is a method for treating cancer in a subject, (i) administering to subjects a dose of compound 1 or a pharmaceutically acceptable salt, or a pharmaceutical composition containing compound 1, in an amount of approximately 5 mg to approximately 100 mg, (ii) administering to the subject a therapeutically effective dose of nivolumab or a pharmaceutical composition containing nivolumab, (iii) a method comprising administering to a subject a therapeutically effective dose of ipilimumab or a pharmaceutical composition comprising a therapeutically effective dose of ipilimumab.

[0404] In one embodiment, the present invention is a method for treating cancer in a subject, (i) administering to the subject a therapeutically effective amount of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, (ii) administering to the subject a therapeutically effective dose of nivolumab or a pharmaceutical composition containing nivolumab, (iii) A method comprising administering to a subject a therapeutically effective amount of BEMPEG or a pharmaceutical composition containing a therapeutically effective amount of BEMPEG.

[0405] In one embodiment, the present invention is a method for treating cancer in a subject, (i) administering to subjects a dose of compound 1 or a pharmaceutically acceptable salt, or a pharmaceutical composition containing compound 1, in an amount of approximately 5 mg to approximately 100 mg, (ii) administering to the subject a therapeutically effective dose of nivolumab or a pharmaceutical composition containing nivolumab, (iii) A method comprising administering to a subject a therapeutically effective amount of BEMPEG or a pharmaceutical composition containing a therapeutically effective amount of BEMPEG.

[0406] In one embodiment, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, nivolumab or a pharmaceutical composition containing nivolumab, and ipilimumab or a pharmaceutical composition containing ipilimumab are administered simultaneously, sequentially, or separately.

[0407] In one embodiment, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing compound 1, nivolumab or a pharmaceutical composition containing nivolumab, and BEMPEG or a pharmaceutical composition containing BEMPEG are administered simultaneously, sequentially, or separately.

[0408] In these and other embodiments, nivolumab is administered intravenously at approximately 360 mg every three weeks, or at approximately 240 mg every two weeks.

[0409] In these and other embodiments, ipilimumab is administered as four IV doses at approximately 1 mg / kg every three weeks.

[0410] In these and other embodiments, BEMPEG is administered intravenously at approximately 0.003 mg / kg every two weeks, approximately 0.006 mg / kg every three weeks, or approximately 0.009 mg / kg every three weeks.

[0411] In one embodiment, the cancer is selected from melanoma, cardia cancer, head and neck cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, non-clear cell renal cell carcinoma, advanced clear cell renal cell carcinoma, castration-resistant prostate cancer, hormone receptor-positive breast cancer, prostate cancer, colon cancer, gastrointestinal cancer, breast cancer, urogenital tract cancer, liver cancer, bone cancer, thyroid cancer, nervous system cancer, gynecological cancer, hematological cancer, skin cancer, and adrenal gland cancer. In another embodiment, the cancer is selected from melanoma, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, classical Hodgkin lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, microsatellite-unstable high colorectal cancer, and hepatocellular carcinoma.

[0412] In one embodiment, the cancer is selected from cardia cancer, head and neck cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, non-clear cell renal cell carcinoma, advanced or metastatic clear cell renal cell carcinoma, castration-resistant prostate cancer, hormone receptor-positive breast cancer, prostate cancer, colon cancer, gastrointestinal cancer, breast cancer, urogenital tract cancer, liver cancer, bone cancer, thyroid cancer, nervous system cancer, gynecological cancer, hematological cancer, skin cancer, urothelial carcinoma, adrenal gland cancer, endometrial cancer, sarcoma, neuroendocrine tumor, ovarian cancer, hepatocellular carcinoma, gastric cancer, colorectal cancer, and melanoma.

[0413] In one embodiment, the cancer is selected from endometrial cancer, sarcoma, neuroendocrine tumor, ovarian cancer, colorectal cancer, HCC, NSCLC, gastric cancer, and melanoma.

[0414] In one embodiment, the cancer is a solid tumor. In another embodiment, the solid tumor is selected from the group consisting of sarcomas, carcinomas, and lymphomas. In a further embodiment, the solid tumor is a genitourinary cancer. In a further embodiment, the genitourinary cancer is selected from the group consisting of clear cell renal cell carcinoma (ccRCC), non-clear cell renal cell carcinoma (nccRCC), urothelial carcinoma (UC, ICI-never experienced, and experienced), and metastatic castration-resistant prostate cancer (mCRPC).

[0415] The amount of compound 1 or its pharmaceutically acceptable salt administered is greater than 0.0 mg but less than or equal to 100 mg of compound 1, greater than 0.0 mg but less than or equal to 95 mg of compound 1, greater than 0.0 mg but less than or equal to 90 mg of compound 1, greater than 0.0 mg but less than or equal to 85 mg of compound 1, greater than 0.0 mg but less than or equal to 80 mg of compound 1, greater than 0.0 mg but less than or equal to 75 mg of compound 1, greater than 0.0 mg but less than or equal to 70 mg of compound 1, greater than 0.0 mg but less than or equal to 65 mg of compound 1, greater than 0.0 mg but less than or equal to 60 mg of compound 1, compound The amounts are greater than 0.0 mg and up to 55 mg of compound 1, greater than 0.0 mg and up to 50 mg of compound 1, greater than 0.0 mg and up to 45 mg of compound 1, greater than 0.0 mg and up to 40 mg of compound 1, greater than 0.0 mg and up to 35 mg of compound 1, greater than 0.0 mg and up to 30 mg of compound 1, greater than 0.0 mg and up to 25 mg of compound 1, greater than 0.0 mg and up to 20 mg of compound 1, greater than 0.0 mg and up to 15 mg of compound 1, greater than 0.0 mg and up to 10 mg of compound 1, or 5 mg or less of compound 1. In one embodiment, compound 1 is administered once daily. In another embodiment, compound 1 is administered twice daily.

[0416] In one embodiment, nivolumab is administered to the subject intravenously (IV). In another embodiment, nivolumab is administered to the subject by intravenous infusion.

[0417] In one embodiment, nivolumab is administered once every two weeks, once every three weeks, or once every four weeks during the treatment period. In a further embodiment, nivolumab is administered once every two weeks during the treatment period. In another further embodiment, nivolumab is administered once every three weeks during the treatment period. In yet another further embodiment, nivolumab is administered once every four weeks during the treatment period.

[0418] In one embodiment, the dose of nivolumab is approximately 50 mg to approximately 500 mg.

[0419] In one embodiment, nivolumab is administered to subjects in IV unit dosage form, and the dosage form is marketed as OPDIVO®.

[0420] In these and other embodiments, nivolumab is administered intravenously at approximately 3 mg / kg every two weeks.

[0421] In these and other embodiments, ipilimumab is administered as four IV doses of approximately 3 mg / kg every three weeks.

[0422] In some embodiments, nivolumab is administered intravenously at approximately 1 mg / kg every three weeks, and ipilimumab is administered intravenously at approximately 3 mg / kg on the same day for up to four doses.

[0423] In some embodiments, nivolumab is administered intravenously at approximately 3 mg / kg every three weeks, and ipilimumab is administered intravenously four times on the same day at approximately 1 mg / kg.

[0424] In some embodiments, nivolumab is administered in four doses of approximately 3 mg / kg intravenously every three weeks, followed by 480 mg every four weeks, while ipilimumab is administered in four doses of approximately 1 mg / kg intravenously every three weeks.

[0425] In some embodiments, nivolumab is administered intravenously at approximately 3 mg / kg every two weeks, and ipilimumab is administered intravenously at approximately 1 mg / kg every six weeks.

[0426] In some embodiments, nivolumab is administered at approximately 360 mg every three weeks, and ipilimumab is administered IV at approximately 1 mg / kg four times on the same day.

[0427] In some embodiments, nivolumab is administered at approximately 240 mg every two weeks, and BEMPEG is administered at approximately 0.006 mg / kg every three weeks.

[0428] In some embodiments, nivolumab is administered at approximately 240 mg every two weeks, and BEMPEG is administered at approximately 0.003 mg / kg every two weeks.

[0429] In some embodiments, nivolumab is administered at approximately 240 mg every two weeks, and BEMPEG is administered at approximately 0.006 mg / kg every two weeks.

[0430] In some embodiments, nivolumab is administered at approximately 360 mg every three weeks, and BEMPEG is administered at approximately 0.006 mg / kg every three weeks.

[0431] In some embodiments, nivolumab is administered at approximately 360 mg every three weeks, and BEMPEG is administered at approximately 0.009 mg / kg every three weeks.

[0432] Combination of Compound 1 and other immunomodulators Non-polymorphic, crystalline, or crystalline salt forms of Compound 1 as disclosed herein are administered concurrently with checkpoint inhibitors or PD-1 inhibitors. Hereinafter referred to as the “combination.” The combination may be administered with one or more additional therapies for the treatment of a disease or disorder, such as cancer or other disease or disorder associated with overgrowth. One or more additional therapies include (i) surgery, (ii) radiotherapy (e.g., gamma radiation, neutron radiation therapy, electron radiation therapy, proton therapy, close-range radiotherapy, and whole-body radioisotopes), (iii) endocrine therapy, (iv) adjuvant therapy, immunotherapy, CAR T-cell therapy, and (v) other chemotherapeutic agents.

[0433] The term “co-administered” (or “co-administered”) refers to any combination as disclosed herein, as well as any form of simultaneous administration or any separate, sequential administration of further active pharmaceutical components, including cytotoxic agents and radiotherapy. When administration is not simultaneous, the compounds are administered in close proximity to each other for a short period of time. Furthermore, it is not relevant whether the compounds are administered in the same dosage form; for example, one compound may be administered topically while another is administered orally.

[0434] Typically, any drug that is active against the disease or condition being treated may be administered concurrently. Examples of such drugs for cancer treatment can be found, for example, at https: / / www.cancer.gov / about-cancer / treatment / drugs (last accessed January 22, 2019) and in Cancer Principles and Practice of Oncology by VTDevita and S. Hellman (editors), 11 th This can be found in publicly available sources such as the 2018 edition, Lippincott Williams & Wilkins Publishers. Those skilled in the art will be able to identify which drug combinations are useful based on the specific characteristics of the drugs and diseases involved.

[0435] In one embodiment, the treatment method includes combination therapy and co-administration of one or more additional therapies, including immunotherapy. Immunotherapy (also called biological response modifier therapy, biologic therapy, biotherapy, immunotherapy, or biological therapy) is a treatment that uses a part of the immune system to fight disease. Immunotherapy can help the immune system recognize cancer cells or enhance its response to cancer cells. Immunotherapy includes active and passive immunotherapy. Active immunotherapy stimulates the body's own immune system, while passive immunotherapy uses immune system components that are generally produced outside the body.

[0436] Examples of active immunotherapy include, but are not limited to, cancer vaccines, tumor cell vaccines (autologous or allogeneic), dendritic cell vaccines, antigen vaccines, anti-idiotype vaccines, DNA vaccines, viral vaccines, or vaccines including tumor-infiltrating lymphocyte (TIL) vaccines using interleukin-2 (IL-2) or lymphokine-activated killer (LAK) cell therapy.

[0437] Examples of passive immunotherapy include, but are not limited to, targeted therapies involving monoclonal antibodies and toxins. Monoclonal antibodies include naked antibodies and conjugated monoclonal antibodies (also called tagged, labeled, or packed antibodies). Naked monoclonal antibodies do not have a drug or radioactive material to which they attach, while conjugated monoclonal antibodies are bound to, for example, chemotherapeutic drugs (chemically labeled), radioactive particles (radioactively labeled), or toxins (antitoxins). Examples of these naked monoclonal antibody drugs include, but are not limited to, rituximab (Rituxan), an antibody against the CD20 antigen used to treat B-cell non-Hodgkin lymphoma; trastuzumab (Herceptin), an antibody against the HER2 protein used to treat advanced breast cancer; alemtuzumab (Campath), an antibody against the CD52 antigen used to treat B-cell chronic lymphocytic leukemia (B-CLL); cetuximab (Erbitux), an antibody against the EGFR protein used, for example, in combination with irinotecan, to treat advanced colorectal and head and neck cancers; and bevacizumab (Avastin), an anti-angiogenic therapy that acts against the VEGF protein and is used, for example, in combination with chemotherapy, to treat metastatic colorectal cancer. Examples of conjugate monoclonal antibodies include, but are not limited to, ibritumomab tiuxetan (Zevalin), a radiolabeled antibody that delivers radioactivity directly to cancerous B lymphocytes and is used, for example, to treat B-cell non-Hodgkin lymphoma; tocitumomab (Bexxar), a radiolabeled antibody used, for example, to treat certain types of non-Hodgkin lymphoma; and gemtuzumab ozogamicin (Mylotarg), an immunotoxin containing calicheamicin and used, for example, to treat acute myeloid leukemia (AML).BL22 includes, for example, conjugated monoclonal antibodies for treating hair cell leukemia, immunotoxins for treating leukemia, lymphoma, and brain tumors, and radiolabeled antibodies such as OncoScint for colorectal cancer and ovarian cancer, and ProstaScint for prostate cancer.

[0438] Further examples of therapeutic antibodies that may be used include, but are not limited to, HERCEPTIN® (trastuzumab) (Genentech, Calif.), a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; REOPRO.RTM. (absiximab) (Centocor), an anti-glycoprotein IIb / IIIa receptor in platelets for the prevention of blood clot formation; ZENAPAX® (daclizumab) (Roche Pharmaceuticals, Switzerland), a humanized anti-CD25 monoclonal antibody for immunosuppression to prevent acute renal allograft rejection; PANOREX® (Glaxo Wellcome / Centocor), a mouse anti-17-IA cell surface antigen IgG2a antibody; BEC2 (ImClone System), a mouse anti-idiotype (GD3 epitope) IgG antibody; and IMC-C225 (ImClone System), a chimeric anti-EGFR IgG antibody. This includes VITAXIN® (Applied Molecular Evolution / Medlmmune), a humanized anti-alpha-V-beta-3 integrin antibody; Campath 1H / LDP-03 (Leukosite), a humanized anti-CD52IgG1 antibody; Smart M195 (Protein Design Lab / Kanebo), a humanized anti-CD33IgG antibody; RITUXAN® (IDEC Pharm / Genentech, Roche / Zettyaku), a chimeric anti-CD20IgG1 antibody; LYMPHOCIDE® (Immunomedics), LYMPHOCIDE® Y-90 (Immunomedics), Lymphoscan (Tc-99m labeled, radioimaging, Immunomedics), and Nuvion (against CD3, Protein Design Labs), all humanized anti-CD22IgG antibodies. CM3 is a humanized anti-ICAM3 antibody (ICOS Pharm). IDEC-114 is a primate-like anti-CD80 antibody (IDEC Pharm / Mitsubishi). ZEVALIN® is a radiolabeled mouse anti-CD20 antibody (IDEC / Schering AG). IDEC-131 is a humanized anti-CD40L antibody (IDEC / Eisai).IDEC-151 is a primate-modified anti-CD4 antibody (IDEC). IDEC-152 is a primate-modified anti-CD23 antibody (IDEC / Seikagaku). SMART anti-CD3 is a humanized anti-CD3IgG (Protein Design Lab). 5G1.1 is a humanized anti-complement factor 5 (C5) antibody (Alexion Pharm). D2E7 is a humanized anti-TNF-alpha antibody (CAT / BASF). CDP870 is a humanized anti-TNF-alpha Fab fragment (Celltech). IDEC-151 is a primate-modified anti-CD4IgG1 antibody (IDEC Pharm / SmithKline Beecham). MDX-CD4 is a human anti-CD4IgG antibody (Medarex / Eisai / Genmab). CD20-streptavidin (sreptdavidin) (+biotin-yttrium 90;NeoRx). CDP571 is a humanized anti-TNF-alpha-IgG4 antibody (Celltech). LDP-02 is a humanized anti-alpha-4-beta-7 antibody (LeukoSite / Genentech). OrthoClone OKT4A is a humanized anti-CD4-IgG antibody (Ortho Biotech). ANTOVA® is a humanized anti-CD40-L-IgG antibody (Biogen). ANTEGREN® is a humanized anti-VLA-4-IgG antibody (Elan). CAT-152 is a humanized anti-TGF-beta-2 antibody (Cambridge Ab Tech). Others are provided in a later paragraph.

[0439] One or more additional therapies also include adjuvant immunotherapy. Examples include cytokines, e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1-alpha, interleukins (including IL-1, IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27), tumor necrosis factor (including TNF-alpha), and interferons (including IFN-alpha, IFN-beta, and IFN-gamma); aluminum hydroxide (alum); Bacille This includes Calmette-Guerin (BCG); Keyhole Limpet Hemocyanin (KLH); Incomplete Freund's Adjuvant (IFA); QS-21; DETOX; Levamisole; and Dinitrophenyl (DNP), as well as combinations thereof, such as combinations of other cytokines, such as interleukins with IFN-alpha, such as IL-2.

[0440] In various embodiments, one or more additional therapies may include one or more of the following: adoptive cell transplantation, angiogenesis inhibitors, Bacillus Calmette-Guerin therapy, biochemotherapy, cancer vaccines, chimeric antigen receptor (CAR) T-cell therapy, cytokine therapy, gene therapy, immune checkpoint modulators, immune conjugates, radioconjugates, oncolytic virus therapy, or targeted drug therapy. Immunological therapies or immunotherapeutic agents are collectively referred to herein as “immunotherapy agents.”

[0441] This disclosure provides a method for preventing, treating, reducing, inhibiting or controlling a neoplasm, tumor or cancer in a subject who requires such treatment, the method comprising administering a therapeutically effective combination and one or more additional therapies. In various embodiments, treatment with the combination and one or more additional therapies provides a synergistic, additive, or synergistic effect in reducing the number of cancer cells when treated by combination, compared to each therapy alone. In some embodiments, treatment with the combination and one or more additional therapies results in a more potent synergistic antitumor activity and / or antitumor activity than the added effect of administering compound 1 or the immunotherapy agent alone in a non-polymorphic, crystalline, or crystalline salt form.

[0442] Human cancers possess numerous genetic and epigenetic alterations that generate neoantigens potentially recognizable by the immune system (Sjoblom et al. (2006) Science 314:268-74). The adaptive immune system, composed of T and B lymphocytes, has potent anti-cancer potential due to its broad ability to respond to diverse tumor antigens and its excellent specificity. Furthermore, the immune system exhibits considerable plasticity and memory components. The successful utilization of all these attributes of the adaptive immune system makes immunotherapy unique among all cancer treatment modalities.

[0443] In various embodiments, one or more additional therapies include adoptive cell transplantation, angiogenesis inhibitors, Bacillus Calmette-Guerin therapy, biochemotherapy, cancer vaccines, chimeric antigen receptor (CAR) T-cell therapy, cytokine therapy, gene therapy, immune checkpoint modulators, such as immune checkpoint inhibitors, immune conjugates, radioconjugates, oncolytic virus therapy, or targeted drug therapy.

[0444] In certain embodiments of each of the embodiments described above, as well as in other embodiments and embodiments described elsewhere in this specification, one or more additional therapies enhance the activity of the combination.

[0445] In certain embodiments of each of the embodiments described above, as well as in other embodiments and embodiments described elsewhere in this specification, one or more additional therapies are immune cell (e.g., T cells, dendritic cells, natural killer cells, etc.) modifiers selected from agonists or activators of costimulatory molecules, the modifiers being monoclonal antibodies known in the art, bispecific antibodies, tripspecific antibodies containing one or more immune checkpoint antigen-binding sites, or immune cell-engagement polyvalent antibodies / fusion proteins / constructs. In some embodiments, the immunotherapy agent may be an antibody that modulates a costimulatory molecule that binds to an antigen on the surface of an immune cell or cancer cell. In each of these different embodiments, the antibody modifier may be a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a tripspecific or multispecific format antibody, a fusion protein, or a fragment thereof, such as a diabody, a single-chain (sc)-diabody (scFv)2, a mini-antibody, a mini-body, a barnase barster, scFv-Fc, sc(Fab)2, a trimer antibody construct, a triabody antibody construct, a trimer body antibody construct, a triabody antibody construct, a collaborative antibody construct, (scFv-TNFa)3, or an F(ab)3 / DNL antibody construct.

[0446] In certain embodiments of each of the embodiments described above, as well as in other embodiments and embodiments described elsewhere in this specification, one or more additional therapies are immunotherapeutic agents that modulate the immune response, such as checkpoint inhibitors or checkpoint agonists. In some embodiments, one or more additional therapies are immunotherapeutic agents that enhance the antitumor immune response. In some embodiments, one or more additional therapies are immunotherapeutic agents that increase cell-mediated immunity. In some embodiments, one or more additional therapies are immunotherapeutic agents that increase T cell activity. In some embodiments, one or more additional therapies are immunotherapeutic agents that increase cytolytic T cell (CTL) activity.

[0447] In some embodiments, one or more additional therapies, administered concurrently with atezolizumab, may include molecules, e.g., binders, e.g., antibodies or functional fragments thereof that modulate (activate or inhibit) checkpoint proteins. Checkpoint inhibitors may be any molecules, drugs, treatments, and / or methods that inhibit immune checkpoints and / or promote immune checkpoint inhibitors, for example, by promoting endogenous immune checkpoint inhibitors, inhibiting transcription factors involved in immune checkpoint expression, and / or acting in coordination with several additional exogenous factors. For example, checkpoint inhibitors may include therapies that inhibit transcription factors involved in the expression of immune checkpoint genes or promote the expression of transcription factors for tumor-suppressor genes, e.g., BACH2 (Luan et al., (2016). Transcription Factors and Checkpoint Inhibitor Expression with Age: Markers of Immunosenescence. Blood, 128(22), 5983). Furthermore, checkpoint inhibitors may inhibit the transcription of immune checkpoint genes; modification and / or processing of immune checkpoint mRNA; translation of immune checkpoint proteins; and / or activation of molecules involved in the immune or immune checkpoint pathway, such as PD-1 transcription factors, e.g., HIF-1, STAT3, NF-κB, and AP-1, or common oncogenic pathways, e.g., JAK / STAT, RAS / ERK, or PI3K / AKT / mTOR (Zerdes et al., Genetic, transcriptional and post-translational regulation of the programmed death protein ligand 1 in cancer: biology and clinical correlations, Oncogene volume 37, pages 4639-4661 (2018), the disclosure thereof is incorporated herein by reference in its entirety).

[0448] Checkpoint inhibitors may include therapies, molecules, drugs, and / or methods that modulate immune checkpoints at the transcriptional level using, for example, co-repression of RNA interference pathways and / or post-transcriptional gene silencing (PTGS) (e.g., microRNA, miRNA; silencing RNA, small interfering RNA, or short interfering RNA (siRNA)). Transcriptional regulation of checkpoint molecules has been shown to involve mir-16, which has been shown to target the 3'UTR of checkpoint mRNA CD80, CD274 (PD-L1), and CD40 (Leibowitz et al., Post-transcriptional regulation of immune checkpoint genes by mir-16 in melanoma, Annals). of Oncology (2017) 28; v428-v448). Mir-33a has also been shown to be involved in regulating PD-1 expression in lung adenocarcinoma (Boldini et al., Role of microRNA-33a in Regulating the expression of PD-1 in lung adenocarcinoma, Cancer Cell Int. 2017;17:105 (the disclosure is incorporated herein by reference in its entirety).

[0449] T cell-specific aptamer-siRNA chimeras have been suggested as a highly specific method for inhibiting molecules in the immune checkpoint pathway (Hossain et al., The aptamer-siRNA conjugates: reprogramming T cells for cancer therapy, Ther. Deliv. 2015 Jan;6(1):1-4, the disclosure is incorporated herein by reference in its entirety).

[0450] Alternatively, members of the immune checkpoint pathway can be inhibited using therapies that affect related pathways, such as metabolism. For example, an oversupply of the glycolytic intermediate pyruvate in mitochondria from CAD macrophages promoted PD-L1 expression via the induction of the bone morphogenesis protein 4 / phosphorylated SMAD1 / 5 / IFN regulator 1 (BMP4 / p-SMAD1 / 5 / IRF1) signaling pathway. Therefore, implementing therapies that modulate metabolic pathways may lead to subsequent modulation of the immunoinhibitory PD-1 / PD-L1 checkpoint pathway (Watanabe et al., Pyruvate controls the checkpoint inhibitor PD-L1 and suppresses T cell immunity, J Clin Invest. 2017 Jun 30;127(7):2725-2738).

[0451] Checkpoint immunity can be regulated via oncolytic viruses that selectively replicate within tumor cells to induce an acute immune response in the tumor microenvironment, that is, by acting as gene vectors to deliver specific drugs (e.g., antibodies, miRNAs, siRNAs, etc.) to cancer cells and by causing the tumor breakdown and secretion of cytokines and chemokines, thereby providing a synergistic effect with immune checkpoint inhibition (Shi et al., Cancer Immunotherapy: A Focus on the Regulation of Immune Checkpoints, Int J Mol Sci. 2018 May;19(5):1389). Currently, clinical trials are underway using the following viruses as checkpoint inhibitors: poliovirus, measles virus, adenovirus, poxvirus, herpes simplex virus (HSV), coxsackievirus, reovirus, Newcastle disease virus (NDV), T-VEC (herpesvirus encoded by GM-CSF (granulocyte-macrophage colony-stimulating factor)), and H101 (Shi et al., above).

[0452] Checkpoint inhibitors can operate at the translational level of checkpoint immunity. Since the translation of mRNA into protein represents a critical event in the regulation of gene expression, inhibiting immune checkpoint translation is a way to inhibit the immune checkpoint pathway.

[0453] Inhibition of the immune checkpoint pathway can occur at any stage of the immune checkpoint translation process. For example, drugs, molecules, medications, therapies, and / or methods can inhibit the initiation process (which recruits the 40S ribosomal subunit to the 5' end of mRNA and scans the 5'UTR of mRNA toward its 3' end). Inhibition can occur by targeting the anticodon of the initiator methionyl-transfer RNA (tRNA) (Met-tRNAi), its base pairing with the start codon, or the recruitment of the 60S subunit, thereby initiating amino acid elongation and sequential addition in the translation of immune checkpoint-specific genes. Alternatively, checkpoint inhibitors can inhibit checkpoints at the translational level by interfering with the formation of the ternary complex (TC), i.e., eukaryotic initiation factor (eIF) 2 (or one or more of its α, β, and γ subunits), GTP, and Met-tRNAi.

[0454] Checkpoint inhibition can occur via destabilization of eIF2α by inhibiting the eIF4F complex and / or its cap-binding protein eIF4E, scaffolding protein eIF4G, or eIF4A helicase, by eliminating its phosphorylation via protein kinase R (PKR), PERK, GCN2, or HRI, or by preventing TC from binding to the 40S ribosome and / or other initiation factors, thus preventing the formation of the pre-initiation complex (PIC). Methods for considering translational control in cancer are discussed in Truitt et al., New frontiers in translational control of the cancer genome, Nat Rev Cancer. 2016 Apr 26;16(5):288-304, the contents of which are incorporated herein by reference in their entirety.

[0455] Checkpoint inhibitors may also include therapies, molecules, drugs, and / or methods that modulate immune checkpoints at the cellular and / or protein levels, for example, by inhibiting immune checkpoint receptors. Checkpoint inhibition can be achieved through the use of antibodies, antibody fragments, antigen-binding fragments, small molecules, and / or other drugs, drugs, therapies, and / or methods.

[0456] Immune checkpoints refer to suppressive pathways in the immune system that are involved in maintaining self-tolerance and regulating the degree of the immune system's response to minimize damage to peripheral tissues. However, tumor cells can also activate immune system checkpoints to reduce the effectiveness of the immune response against tumor tissue ("blocking" the immune response). In contrast to most anticancer drugs, checkpoint inhibitors do not directly target tumor cells to enhance the endogenous antitumor activity of the immune system, but rather target lymphocyte receptors or their ligands. (Pardoll, 2012, Nature Reviews Cancer 12:252-264).

[0457] In some embodiments, one or more additional therapies are modifiers of PD-1 activity, PD-L1 activity, PD-L2 activity, CTLA-4 activity, CD28 activity, CD80 activity, CD86 activity, 4-1BB activity, OX40 activity, KIR activity, Tim-3 activity, LAG3 activity, CD27 activity, CD40 activity, GITR activity, TIGIT activity, CD20 activity, CD96 activity, IDO1 activity, cytokines, chemokines, interferons, interleukins, lymphokines, members of the tumor necrosis factor (TNF) family, or immunostimulatory oligonucleotides. In some embodiments, the immune checkpoint modifier is, i.e., an inhibitor or antagonist, or an activator or agonist, such as a CD28 modifier, a 4-1BB modifier, an OX40 modifier, a CD27 modifier, a CD80 modifier, a CD86 modifier, a CD40 modifier, or a GITR modifier, a Lag-3 modifier, a 41BB modifier, a LIGHT modifier, a CD40 modifier, a GITR modifier, a TGF-beta modifier, a TIM-3 modifier, a SIRP-alpha modifier, a TIGIT modifier, a VSIG8 modifier, a BTLA modifier, a SIGLEC7 modifier, a SIGLEC9 modifier, an ICOS modifier, a B7H3 modifier, a B7H4 modifier, a FAS modifier, and / or a BTNL2 modifier. In some embodiments, the immunotherapy agent is an immune checkpoint modulator (for example, an immune checkpoint modulator antibody, which may be in the form of a monoclonal antibody, a bispecific antibody containing one or more immune checkpoint antigen binding sites, a triplicate antibody, or an immune cell engagement polyvalent antibody / fusion protein / construct, as known in the art).

[0458] In some embodiments, one or more additional therapies are agents that inhibit the activity of PD-1. In some embodiments, one or more additional therapies are agents that inhibit the activity of PD-L1 and / or PD-L2. In some embodiments, one or more additional therapies are agents that inhibit the activity of CTLA-4. In some embodiments, one or more additional therapies are agents that inhibit the activity of CD80 and / or CD86. In some embodiments, one or more additional therapies are agents that inhibit the activity of TIGIT. In some embodiments, one or more additional therapies are agents that inhibit the activity of KIR. In some embodiments, one or more additional therapies are agents that enhance or stimulate the activity that activates immune checkpoint receptors.

[0459] PD-1 (also known as programmed death 1, CD279, or PDCD1) is a cell surface receptor that plays a crucial role in regulating the balance between stimulative and inhibitory signals in the immune system and maintaining peripheral tolerance (Ishida, Y et al. 1992 EMBO J.11 3887; Kier, Mary E et al. 2008 Annu Rev Immunol 26 677-704; Okazaki, Taku et al. 2007 International Immunology 19 813-824). PD-1 is an inhibitory member of the immunoglobulin superfamily homologous to CD28. PD-1 is a monomeric type 1 transmembrane protein consisting of a single immunoglobulin-variable extracellular domain and a cytoplasmic domain containing an immunoreceptor tyrosine system inhibitory motif (ITIM) and an immunoreceptor tyrosine system switch motif (ITSM). PD-1 expression can be induced in T cells, B cells, natural killer (NK) cells, and monocytes during lymphocyte activation, for example, via T cell receptor (TCR) or B cell receptor (BCR) signaling (Kier, Mary E et al. 2008 Annu Rev Immunol 26 677-704; Agata, Y et al 1996 Int Immunol 8 765-72). PD-1 is a receptor for ligands CD80, CD86, PD-L1 (B7-H1, CD274), and PD-L2 (B7-DC, CD273), which are cell surface expression members of the B7 family (Freeman, Gordon et al. 2000 J Exp Med 192 1027; Latchman, Y et al. 2001 Nat Immunol 2:261). During ligand engagement, PD-1 recruits phosphatases, such as SHP-1 and SHP-2, to its intracellular tyrosine motif, and subsequently dephosphorylates effector molecules activated by TCR or BCR signaling (Chemnitz, J et al. 2004 J Immunol). 173:945-954; Riley, James L 2009 Immunological Reviews 229:114-125). Thus, PD-1 transduces inhibitory signals into T and B cells only when it is simultaneously engaged with the TCR or BCR.

[0460] PD-1 has been demonstrated to downmodulate effector T cell responses through both intracellular and extracellular functional mechanisms. Inhibitory signaling via PD-1 induces a state of unresponsiveness in T cells, resulting in cells unable to clonally expand or produce optimal levels of effector cytokines. PD-1 can also induce apoptosis in T cells through its ability to inhibit survival signals from co-stimulation, leading to a reduction in the expression of key anti-apoptotic molecules, such as Bcl-XL (Kier, Mary E et al. 2008 Annu Rev Immunol 26:677-704). In addition to these direct effects, recent publications suggest that PD-1 is involved in effector cell repression by promoting the induction and maintenance of regulatory T cells (TREGs). For example, PD-L1 expressed in dendritic cells has been shown to synergistically act with TGF-β to promote the induction of CD4+FoxP3+TREG through enhanced suppressor function (Francisco, Loise M et al. 2009 J Exp Med 206:3015-3029).

[0461] TIM-3 (also known as T-cell immunoglobulin and mucin domain-containing-3, TIM-3, hepatitis A virus cell receptor 2, HAVCR2, HAVcr-2, KIM-3, TIMD-3, TIMD3, Tim-3, and CD366) is a 33.4 kDa single-pass type I membrane protein involved in the immune response (Sanchez-Fueyo). et al., Tim-3 inhibits T helper type 1-mediated auto- and alloimmune responses and promotes immunological tolerance, Nat. Immunol. 4:1093-1101 (2003)).

[0462] TIM-3 is selectively expressed in Th1 cells, as well as phagocytic cells (e.g., macrophages and dendritic cells). The use of siRNA or blocking antibodies to reduce human TIM-3 expression resulted in increased interferon-gamma (IFN-γ) secretion from CD4-positive T cells, suggesting an inhibitory role of TIM-3 in human T cells. Analysis of clinical samples from patients with autoimmune diseases did not show TIM-3 expression in CD4-positive cells. In particular, T cell clones derived from cerebrospinal fluid of patients with multiple sclerosis showed lower TIM-3 expression levels and higher IFN-γ secretion than clones derived from normal healthy individuals (Koguchi K et al., J Exp Med. 203:1413-8. (2006)).

[0463] TIM-3 is a receptor for the ligand galectin-9, a member of the galectin family and a molecule universally expressed in various cell types, including β-galactosides; phosphatidylserine (PtdSer) (DeKryff et al., T cell / transmembrane, Ig, and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells, J Immunol. 2010 Feb 15;184(4):1918-30), and high mobility group protein 1 (also known as HMGB1, HMG1, HMG3, SBP-1, HMG-1, and high mobility group box 1) (Chiba et al., Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1 (Nat Immunol. 2012 Sep;13(9):832-42), and carcinoembryonic antigen-associated cell adhesion molecule 1 (also known as CEACAM1, BGP, BGP1, BGPI, or carcinoembryonic antigen-associated cell adhesion molecule 1) (Huang et al., CEACAM1 regulates TIM-3-mediated tolerance). It binds to (and exhaustion, Nature. 2015 Jan 15;517(7534):386-90).

[0464] BTLA (also known as B and T lymphocyte attenuator, BTLA1, CD272, and B and T lymphocyte-associated) is a 27.3 kDa single-pass type I membrane protein involved in lymphocyte inhibition during immune responses. BTLA is constitutively expressed in both B and T cells. BTLA interacts with HVEM (herpesvirus entry mediator) and members of the tumor-necrosis factor receptor (TNFR) family (Gonzalez et al., Proc. Natl. Acad. Sci. USA, 2005, 102:1116-21). The interaction between BTLA, which belongs to the CD28 family of the immunoglobulin superfamily, and HVEM and the costimulatory tumor-necrosis factor (TNF) receptor (TNFR) is unique in that it defines crosstalk between these two receptor families. BTLA contains a membrane-proximal immunoreceptor tyrosine system inhibitory motif (ITIM) and a membrane-distal immunoreceptor tyrosine system switch motif (ITSM). Division of either ITIM or ITSM destroys BTLA's ability to recruit either SHP1 or SHP2, suggesting that BTLA recruits SHP1 and SHP2 in a manner different from PD-1, and that both tyrosine motifs are required to block T cell activation. The BTLA cytoplasmic tail also contains a third conserved tyrosine-containing motif within its cytoplasmic domain, which is sequence-similar to the Grb-2 recruitment site (YXN). Furthermore, phosphorylated peptides containing the BTLA N-terminal tyrosine motif can interact in vitro with the p85 subunit of GRB2 and PI3K, but the functional effects of this interaction remain uninvestigated in vivo (Gavrieli et al., Bioochem. Biophysi Res Commun, 2003, 312, 1236-43). BTLA is a receptor for ligands PTPN6 / SHP-1, PTPN11 / SHP-2, TNFRSF14 / HVEM, and B7H4.

[0465] VISTA (also known as the V-domain Ig suppressor of T cell activation, VSIR, B7-H5, B7H5, GI24, PP2135, SISP1, DD1 alpha, VISTA, C10orf54, chromosome 10 open reading frame 54, PD-1H, and V-set immunomodulatory receptor) is an approximately 33.9 kDa single-pass type I membrane protein involved in T cell inhibition responses, embryonic stem cell differentiation via BMP4 signaling inhibition, and MMP2 activation mediated by MMP14 (Yoon et al., Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53, Science. 2015 Jul 31;349(6247):1261669). VISTA interacts with the ligand VSIG-3 (Wang et al., VSIG-3 as a ligand of VISTA inhibits human T-cell function, Immunology. 2019 Jan;156(1):74-85).

[0466] LAG-3 (also known as lymphocyte activation gene 3, LAG3, CD223, and lymphocyte activation 3) is a 57.4 kDa single-pass type I membrane protein involved in lymphocyte activation that also binds to HLA class II antigens. LAG-3 is a member of the immunoglobulin supergene family and is expressed in activated T cells (Huard et al., 1994, Immunogenetics 39:213), NK cells (Triebel et al., 1990, J.Exp.Med.171:1393-1405), regulatory T cells (Huang et al., 2004, Immunity 21:503-513; Camisaschi et al., 2010, J Immunol.184:6545-6551; Gagliani et al., 2013, Nat Med 19:739-746), and plasmacytoid dendritic cells (DCs) (Workman et al., 2009, J Immunol 182:1885-1891). LAG-3 is a membrane protein encoded by a gene located on chromosome 12 and is structurally and genetically related to CD4. Like CD4, LAG-3 can interact with MHC class II molecules on the cell surface (Baixeras et al., 1992, J.Exp.Med.176:327-337; Huard et al., 1996, Eur.J.Immunol.26:1180-1186). Direct binding of LAG-3 to MHC class II has been suggested to play a role in the downregulatory antigen-dependent stimulation of CD4+ T lymphocytes (Huard et al., 1994, Eur. J. Immunol. 24:3216-3221), and LAG-3 blockade has also been shown to reactivate CD8+ lymphocytes in both tumor or autoantigen (Gross et al., 2007, J Clin Invest. 117:3383-3392) and viral models (Blackburn et al., 2009, Nat. Immunol. 10:29-37).Furthermore, the cytoplasmic domain of LAG-3 can interact with LAP (LAG-3-related protein), a signaling molecule involved in the downregulation of the CD3 / TCR activation pathway (Iouzalen et al., 2001, Eur. J. Immunol. 31:2885-2891). In addition, CD4+CD25+ regulated T cells (Tregs) have been shown to express LAG-3 upon activation, contributing to the suppressor activity of Treg cells (Huang, C. et al., 2004, Immunity). (21:503-513). LAG-3 can also negatively regulate T cell homeostasis by Treg cells through both T cell-dependent and T cell-independent mechanisms (Workman, C.J. and Vignali, DA, 2005, J.Immunol. 174:688-695).

[0467] LAG-3 has been shown to interact with MHC class II molecules (Huard et al., CD4 / major histocompatibility complex class II interaction analyzed with CD4- and lymphocyte activation gene-3(LAG-3)-Ig fusion proteins, Eur J Immunol.1995 Sep;25(9):2718-21).

[0468] In addition, several kinases, such as CHEK-1, CHEK-2, and A2aR, are known to be checkpoint inhibitors.

[0469] CHEK-1 (also known as CHK1 kinase, CHK1, and checkpoint kinase 1) is a serine / threonine protein kinase of approximately 54.4 kDa that is involved in checkpoint-mediated cell cycle arrest and the activation of DNA repair in response to DNA damage and / or non-replicating DNA.

[0470] CHEK-2 (also known as CHK2 kinase, CDS1, CHK2, HuCds1, LFS2, PP1425, RAD53, hCds1, and checkpoint kinase 2) is a serine / threonine protein kinase of approximately 60.9 kDa that is involved in checkpoint-mediated cell cycle arrest, DNA repair activation, and double-strand break-mediated apoptosis.

[0471] A2aR (also known as adenosine A2A receptor, ADORA2A, adenosine A2a receptor, A2aR, ADORA2, and RDC8) is a multipath membrane receptor of approximately 44.7 kDa for adenosine and other ligands.

[0472] In some embodiments, exemplary immunotherapeutic agents may include one or more antibody modifiers targeting PD-1, PD-L1, PD-L2, CEACAM (e.g., CEACAM-1, -3, and / or -5), CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGF-beta, OX40, 41BB, LIGHT, CD40, GITR, TGF-beta, TIM-3, SIRP-alpha, VSIG8, BTLA, SIGLEC7, SIGLEC9, ICOS, B7H3, B7H4, FAS, and / or BTNL2, among others known in the art. In some embodiments, the immunotherapeutic agent is a drug that increases natural killer (NK) cell activity. In some embodiments, one or more additional therapies are drugs that inhibit the suppression of the immune response. In some embodiments, one or more additional therapies are drugs that inhibit suppressor cells or suppressor cell activity. In some embodiments, one or more additional therapies are agents or therapies that inhibit Treg activity. In some embodiments, one or more additional therapies are agents that inhibit the activity of an inhibitory immune checkpoint receptor.

[0473] In some embodiments, one or more additional therapies include T-cell modulators selected from agonists or activators of costimulatory molecules, administered concurrently with checkpoint inhibitors. In one embodiment, the costimulatory molecule agonist is selected from agonists of GITR, OX40, SLAM (e.g., SLAMF7), HVEM, LIGHT, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), CD30, CD40, BAFFR, CD7, NKG2C, NKp80, CD160, B7-H3, or CD83 ligands (e.g., agonist antibodies or their antigen-binding fragments, or soluble fusions). In other embodiments, the effector cell combination includes a bispecific T cell engager (e.g., a bispecific antibody molecule that binds to CD3 and tumor antigens (e.g., EGFR, PSCA, PSMA, EpCAM, HER2, in particular)).

[0474] In some embodiments, one or more additional therapies modulate PD-1 activity, PD-L1 activity, PD-L2 activity, CTLA-4 activity, CD28 activity, CD80 activity, CD86 activity, 4-1BB activity, OX40 activity, KIR activity, Tim-3 activity, LAG3 activity, CD27 activity, CD40 activity, GITR activity, TIGIT activity, CD20 activity, and CD96 activity. These include nodal substances, modifiers of IDO1 activity, modifiers of SIRP-alpha activity, modifiers of TIGIT activity, modifiers of VSIG8 activity, modifiers of BTLA activity, modifiers of SIGLEC7 activity, modifiers of SIGLEC9 activity, modifiers of ICOS activity, modifiers of B7H3 activity, modifiers of B7H4 activity, modifiers of FAS activity, modifiers of BTNL2 activity, cytokines, chemokines, interferons, interleukins, lymphokines, members of the tumor necrosis factor (TNF) family, or immunostimulatory oligonucleotides.

[0475] In some embodiments, one or more additional therapies are immune checkpoint modulators (e.g., immune checkpoint inhibitors, e.g., inhibitors of PD-1 activity, modulators of PD-L1 activity, modulators of PD-L2 activity, modulators of CTLA-4, or CD40 agonists (e.g., anti-CD40 antibody molecules), (xi)OX40 agonists (e.g., anti-OX40 antibody molecules), or (xii)CD27 agonists (e.g., anti-CD27 antibody molecules). In one embodiment, the immunotherapy agent is PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, CEA Inhibitors of CAM (e.g., CEACAM-1, -3 and / or -5), VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and / or TGF-beta, galectin-9, CD69, galectin-1, CD113, GPR56, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4. In one embodiment, an inhibitor of an immune checkpoint molecule inhibits PD-1, PD-L1, LAG-3, TIM-3, CEACAM (e.g., CEACAM-1, -3 and / or -5), CTLA-4, or any combination thereof.

[0476] In one embodiment, the immunotherapy agent is a protein agonist that stimulates T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD28H.

[0477] In some embodiments, one or more additional therapies are activators or agonists of the co-stimulatory molecule. In one embodiment, the agonist of the co-stimulatory molecule is selected from agonists of CD2, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligands (e.g., agonist antibodies or their antigen-binding fragments, or soluble fusions).

[0478] Inhibition of inhibitory molecules can be carried out at the DNA, RNA, or protein level. In embodiments, inhibitory nucleic acids (e.g., dsRNA, siRNA, or shRNA) may be used to inhibit the expression of inhibitory molecules. In other embodiments, the inhibitor of the inhibitory signal is a polypeptide, for example, a soluble ligand (e.g., PD-1-Ig or CTLA-4Ig), or an antibody or its antigen-binding fragment, for example, a monoclonal antibody, a bispecific antibody, a tripspecific antibody, or an immune cell-engagement polyvalent antibody / fusion protein / construct known in the art that binds to an inhibitory molecule; for example, an antibody or fragment thereof that binds to PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, CEACAM (e.g., CEACAM-1, -3 and / or -5), VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and / or TGF-beta, galectin-9, CD69, galectin-1, CD113, GPR56, CD48, GARP, PD1H, LAIR1, TIM-1, TIM-4, or a combination thereof (also referred to herein as “antibody molecule”).

[0479] In some embodiments, one or more additional therapies are monoclonal or bispecific antibodies administered concurrently with checkpoint inhibitors. For example, monoclonal or bispecific antibodies may specifically bind to members of the c-Met pathway and / or immune checkpoint modulators (e.g., bispecific antibodies that bind to both hepatocyte growth factor receptor (HGFR) and immune checkpoint modulators described herein, e.g., antibodies that bind to PD-1, PD-L1, PD-L2, or CTLA-4, LAG-3, OX40, 41BB, LIGHT, CD40, GITR, TGF-beta, TIM-3, SIRP-alpha, TIGIT, VSIG8, BTLA, SIGLEC7, SIGLEC9, ICOS, B7H3, B7H4, FAS, BTNL2, or CD27). In certain embodiments, the bispecific antibody specifically binds to the human HGFR protein, as well as to one of PD-1, PD-L1, and CTLA-4.

[0480] In some embodiments of the methods described herein, one or more additional therapies are PD-1 antagonists, PD-L1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, CD80 antagonists, CD86 antagonists, KIR antagonists, Tim-3 antagonists, LAG3 antagonists, TIGIT antagonists, CD20 antagonists, CD96 antagonists, or IDO1 antagonists.

[0481] In some embodiments, the PD-1 antagonist is an antibody that specifically binds to PD-1. In some embodiments, the antibody that binds to PD-1 is pembrolizumab (KEYTRUDA®, MK-3475; Merck), pidilizumab (CT-011; Curetech Ltd.), nivolumab (OPDIVO®, BMS-936558, MDX-1106; Bristol Myer Squibb), MEDI0680 (AMP-514; AstraZenenca / MedImmune), REGN2810 (Regeneron Pharmaceuticals), BGB-A317 (BeiGene Ltd.), PDR-001 (Novartis), or STI-A1110 (Sorrento Therapeutics). In some embodiments, the antibody that binds to PD-1 is an antibody identified as, for example, APE2058, APE1922, APE1923, APE1924, APE1950, or APE1963 (Anaptysbio), as described in PCT Publication 2014 / 179664, or an antibody containing the CDR region of any of these antibodies. In other embodiments, the PD-1 antagonist is a fusion protein containing the extracellular domain of PD-L1 or PD-L2, for example, AMP-224 (AstraZeneca / MedImmune). In other embodiments, the PD-1 antagonist is a peptide inhibitor, for example, AUNP-12 (Aurigene).

[0482] In some embodiments, the PD-L1 antagonist is an antibody that specifically binds to PD-L1. In some embodiments, the antibody that binds to PD-L1 is MEDI4736 (AstraZeneca / MedImmune), BMS-936559 (MDX-1105; Bristol Myers Squibb), avelumab (MSB0010718C; Merck KGaA), KD033 (Kadmon), the antibody portion of KD033, or STI-A1014 (Sorrento Therapeutics). In some embodiments, the antibodies that bind to PD-L1 are described in PCT Publication No. 2014 / 055897 and are, for example, Ab-14, Ab-16, Ab-30, Ab-31, Ab-42, Ab-50, Ab-52, or Ab-55, or antibodies containing the CDR region of any of these antibodies, the disclosure of which is incorporated herein by reference in its entirety.

[0483] In some embodiments, the CTLA-4 antagonist is an antibody that specifically binds to CTLA-4. In some embodiments, the antibody that binds to CTLA-4 is ipilimumab (YERVOY®; Bristol Myer Squibb) or tremelimumab (CP-675, 206; Pfizer). In some embodiments, the CTLA-4 antagonist is a CTLA-4 fusion protein or a soluble CTLA-4 receptor, such as KARR-102 (Kahr Medical Ltd.).

[0484] In some embodiments, the LAG3 antagonist is an antibody that specifically binds to LAG3. In some embodiments, antibodies that bind to LAG3 include IMP701 (Prima BioMed), IMP731 (Prima BioMed / GlaxoSmithKline), BMS-986016 (Bristol Myer Squibb), LAG525 (Novartis), and GSK2831781 (GlaxoSmithKline). In some embodiments, the LAG3 antagonist includes a soluble LAG3 receptor, such as IMP321 (Prima BioMed).

[0485] In some embodiments, the KIR antagonist is an antibody that specifically binds to KIR. In some embodiments, the antibody that binds to KIR is lirilumab (Bristol Myer Squibb (Innate Pharma)

[0486] In some embodiments, the immunotherapy agent is a cytokine, such as a chemokine, interferon, interleukin, lymphokine, or member of the tumor necrosis factor family. In some embodiments, the cytokine is IL-2, IL-15, or interferon-gamma.

[0487] In some embodiments of the above-described aspects or any other part of this specification, cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC)), kidney cancer (e.g., renal urothelial carcinoma), bladder cancer (e.g., bladder urothelial (transitional cell) carcinoma), breast cancer, colorectal cancer (e.g., colon adenocarcinoma), ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, melanoma (e.g., cutaneous melanoma), head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), thyroid cancer, sarcoma (e.g., soft tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma) (Tumors, osteosarcoma, chondrosarcoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangiosarcoma, endolymphatic sarcoma, leiomyosarcoma, or rhabdomyosarcoma), prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic eosinophilic leukemia, or chronic lymphocytic leukemia (CLL)), lymphoma (e.g., Hodgkin lymphoma or non-Hodgkin lymphoma (NHL)), myeloma (e.g., multiple myeloma (MM)), mycosis fungoides, Merkel cell carcinoma, hematological malignancies, blood tissue Cancers of the body, B-cell carcinoma, bronchial cancer, gastric cancer, brain or central nervous system cancer, surrounding nervous system cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, adrenal gland cancer, adrenocortical carcinoma, adenocarcinoma, inflammatory myofibroblastoma, gastrointestinal stromal tumor (GIST), colon cancer, spinal cord dysplasia syndrome (MDS), myeloproliferative disorder (MPD), polycythemia vera, chordoma, synoviomas, Ewing's tumor, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer The following are selected from the group consisting of cholangiocarcinoma, choriocarcinoma, seminomas, embryonal carcinoma, Wilms' tumor, bladder cancer, epithelial carcinoma, glioma, undifferentiated astrocytoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, hepatocellular carcinoma, thyroid cancer, small cell carcinoma, essential thrombocythemia, myeloid metaplasia of unknown cause, eosinophilic syndrome, systemic mastocytosis, familial hypereosinophilia, neuroendocrine carcinoma, or carcinoid tumors.

[0488] In some embodiments of the above-described aspects or any other part of this specification, the subject's cancer or tumor does not respond to immune checkpoint inhibition (e.g., to any immune checkpoint inhibitor described herein, e.g., a PD-1 antagonist or a PD-L1 antagonist), or the subject's cancer or tumor progresses after an initial response to immune checkpoint inhibition (e.g., to any immune checkpoint inhibitor described herein, e.g., a PD-1 antagonist or a PD-L1 antagonist).

[0489] In various embodiments, one or more additional therapies may include an antibody or its antigen-binding fragment. Within this definition, immune checkpoint inhibitors include bispecific antibodies and immune cell-engagement polyvalent antibodies / fusion proteins / constructs known in the art. In some embodiments, one or more additional therapies including a bispecific antibody may be bivalent and include a bispecific antibody bound to either the same epitope of an immune checkpoint molecule, two different epitopes of the same immune checkpoint molecule, or different epitopes of two different immune checkpoints.

[0490] Those skilled in the art can implement several bispecific antibody formats known in the art to target one or more of the following: CTLA4, PD1, PD-L1, TIM-3, LAG-3, various B-7 ligands, B7H3, B7H4, CHK1 and CHK2 kinases, BTLA, A2aR, OX40, 41BB, LIGHT, CD40, GITR, TGF-beta, SIRP-alpha, TIGIT, VSIG8, SIGLEC7, SIGLEC9, ICOS, FAS, BTNL2, and others for use in combination as described herein.

[0491] In various embodiments, one or more additional therapies may include immune cell engagement multivalent antibodies / fusion proteins / constructs.

[0492] In some embodiments of this disclosure, one or more additional therapies are populations of immune cells that may be administered in combination with a non-pleomorphic, crystalline, or crystalline salt form of compound 1 to treat a subject with cancer in co-administration with a checkpoint inhibitor. In some embodiments, the immunotherapy agent is a population of immune cells, such as leukocytes (nucleated white blood cells) that contain (e.g., express) receptors that bind to the antigen of interest. The leukocytes in this disclosure may be, for example, neutrophils, eosinophils, basophils, lymphocytes, or monocytes. In some embodiments, the leukocytes are lymphocytes. Examples of lymphocytes include T cells, B cells, natural killer (NK) cells, or NKT cells. In some embodiments, the T cells are CD4+ Th (T helper) cells, CD8+ cytotoxic T cells, γδ T cells, or regulatory (suppressor) T cells. In some embodiments, the immune cells are dendritic cells.

[0493] In some embodiments, the immune cells of the Disclosure are genetically engineered to express antigen-binding receptors. Cells are considered "engineered" if they contain engineered (exogenous) nucleic acids. Engineered nucleic acids of the Disclosure can be introduced into cells by any known (e.g., conventional) method. For example, manipulated nucleic acids can be processed by electroporation (see, e.g., Heiser W. C-Transcription Factor Protocols: Methods in Molecular Biology. TM. 2000; 130: 117-134), chemical (e.g., calcium phosphate or lipid), transfection (see, e.g., Lewis WH, et al., Somatic Cell Genet. 1980 May; 6(3): 333-47, Chen C., et al., Mol Cell Biol. 1987 August; 7(8): 2745-2752), fusion with bacterial protoplasts containing recombinant plasmids (see, e.g., Schaffner W. Proc Natl Acad Sci USA. 1980 April; 77(4): 2163-7), or by microinjecting purified DNA directly into the cell nucleus (see, e.g., Capecchi MRCell. 1980 November; 22(2 Pt). (See 2):479-88), or it can be introduced into cells by retroviral transduction.

[0494] Some aspects of this disclosure, including one or more additional therapies, provide a “adoptive cell” approach, which involves isolating immune cells (e.g., T cells) from a subject with cancer, genetically engineering the immune cells (e.g., expressing antigen-binding receptors such as chimeric antigen receptors), expanding the cells ex vivo, and then reintroducing the immune cells into the subject. This method results in a larger number of engineered immune cells in the subject compared to what can be achieved by conventional gene delivery and vaccination methods. In some embodiments, immune cells are isolated from the subject, expanded ex vivo without genetic modification, and then reintroduced into the subject.

[0495] The immune cells of this disclosure constitute receptors that bind to antigens, such as antigens encoded by exogenously delivered nucleic acids, as provided herein. In some embodiments, leukocytes are modified (e.g., genetically modified) to express receptors that bind to antigens. In some embodiments, the receptors may be naturally occurring antigen receptors (usually expressed on immune cells), recombinant antigen receptors (not usually expressed on immune cells), or chimeric antigen receptors (CARs). Naturally occurring and recombinant antigen receptors encompassed by this disclosure include T cell receptors, B cell receptors, NK cell receptors, NKT cell receptors, and dendritic cell receptors. "Chimeric antigen receptor" refers to an artificial immune cell receptor that is engineered to recognize and bind to an antigen expressed by tumor cells. Generally, CARs are designed for T cells and are chimeric of the signaling domain and antigen recognition domain (e.g., single-chain fragment (scFv) of an antibody) of the T cell receptor (TcR) complex (Enblad et al., Human Gene Therapy. 2015;26(8):498-505), the disclosure thereof is incorporated herein by reference in its entirety.

[0496] In some embodiments, the antigen-binding receptor is a chimeric antigen receptor (CAR). T cells expressing a CAR are referred to as "CAR T cells." In some embodiments, the CAR T cell receptor comprises a signaling domain and an antigen-recognition domain of a T cell receptor (TcR) complex (e.g., a single-chain fragment (scFv) of an antibody (Enblad et al., Human Gene Therapy. 2015;26(8):498-505), the disclosure thereof is incorporated herein by reference in its entirety.

[0497] There are four generations of CARs, each containing different components. The first generation of CARs binds antibody-derived scFv to the CD3 zeta (zeta or z) intracellular signaling domain of the T cell receptor through a hinge and transmembrane domain. The second generation of CARs incorporate additional domains, such as CD28, 4-1BB (41BB), or ICOS, to supply a co-stimulatory signal. The third generation of CARs contains two co-stimulatory domains fused to the TcR CD3-zeta chain. The third generation of co-stimulatory domains may include, for example, a combination of CD3z, CD27, CD28, 4-1BB, ICOS, or OX40. In some embodiments, the CAR contains an ectodomain (e.g., CD3), hinge, transmembrane domain, and endodomain commonly derived from a single-chain variable fragment (scFv), along with 1 (first generation), 2 (second generation), or 3 (third generation) signaling domains derived from CD3Z and / or a co-stimulatory molecule (Maude et al., Blood. 2015; 125(26): 4017-4023; Kakarla and Gottschalk, Cancer J. 2014; 20(2): 151-155), the disclosure thereof, is incorporated herein by reference in its entirety.

[0498] In some embodiments, the chimeric antigen receptor (CAR) is a T cell redirected for universal cytokine death (TRUCK), also known as a fourth-generation CAR. TRUCK is a CAR-redirected T cell used as a vehicle to produce and release transgenic cytokines that accumulate in target tissue, such as target tumor tissue. The transgenic cytokines are released upon CAR engagement with the target. TRUCK cells can deposit various therapeutic cytokines at the target, resulting in therapeutic concentrations at the target site and potentially avoiding systemic toxicity.

[0499] CARs typically differ in their functional properties. The CD3 zeta signaling domain of the T cell receptor, when engaged, activates and induces T cell proliferation, but can lead to anergy (directly inducing peripheral lymphocyte tolerance as a result of a loss of response by the body's defense mechanisms). Lymphocytes are considered anerious when they are unable to respond to a specific antigen. The addition of a costimulatory domain in second-generation CARs improved the replication capacity and persistence of modified T cells. Similar antitumor effects are observed in vitro with CD28 or 4-1BB CARs, but preclinical in vivo trials suggest that 4-1BB CARs may produce superior proliferation and / or persistence. Clinical trials suggest that both of these second-generation CARs are capable of inducing substantial T cell proliferation in vivo, but CARs containing the 4-1BB costimulatory domain appear to be longer-lasting. Third-generation CARs combine multiple signaling domains (co-stimuli) to enhance their potency. Fourth-generation CARs are further modified by constitutive or inducible expression cassettes for transgenic cytokines released by CAR T cells to modulate T cell responses. See, for example, Enblad et al., Human Gene Therapy. 2015;26(8):498-505 and Chmielewski and Hinrich, Expert Opinion on Biological Therapy. 2015;15(8):1145-1154, whose disclosures are incorporated herein by reference in their entirety.

[0500] In some embodiments, one or more additional therapies are first-generation chimeric antigen receptors (CARs). In some embodiments, the chimeric antigen receptor is a second-generation CAR. In some embodiments, the chimeric antigen receptor is a third-generation CAR. In some embodiments, the chimeric antigen receptor is a fourth-generation CAR or a T cell redirected for universal cytokine death (TRUCK).

[0501] In some embodiments, the chimeric antigen receptor (CAR) comprises an antigen-binding domain, a transmembrane domain, and an extracellular domain including a cytoplasmic domain. In some embodiments, the CAR is entirely human. In some embodiments, the antigen-binding domain of the CAR is specific to one or more antigens. In some embodiments, a “spacer” domain or “hinge” domain is located between the extracellular domain (including the antigen-binding domain) and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. A “spacer domain” refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or the cytoplasmic domain in the polypeptide chain. A “hinge domain” refers to any oligopeptide or polypeptide that functions to confer flexibility to the CAR or its domain, or to prevent steric hindrance to the CAR or its domain. In some embodiments, the spacer domain or hinge domain may contain up to 300 amino acids (e.g., 10 to 100 amino acids, or 5 to 20 amino acids). In some embodiments, one or more spacer domains may be located in other regions of the CAR.

[0502] In some embodiments, the CARs of this disclosure include an antigen-binding domain, for example, a single-chain Fv(scFv) specific to a tumor antigen. The selection of the binding domain depends on the type and number of ligands that delimit the surface of the target cell. For example, the antigen-binding domain may be selected to recognize ligands that act as cell surface markers in target cells associated with a specific disease condition, e.g., cancer or autoimmune disease. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain in the CARs of this disclosure include those associated with cancer cells and / or other forms of diseased cells. In some embodiments, the CAR is engineered to target a target tumor antigen by manipulating a desired antigen-binding domain that specifically binds to the antigen in tumor cells encoded by an engineered nucleic acid, as provided herein.

[0503] An antigen-binding domain (e.g., scFv) that "specifically binds" to a target or epitope is a term understood in the art, and methods for determining such specific binding are also known in the art. A molecule is said to exhibit "specific binding" when it reacts or associates more frequently and rapidly with a longer duration and / or higher affinity to a particular target antigen than with an alternative target. An antigen-binding domain (e.g., scFv) that specifically binds to a first target antigen may or may not specifically bind to a second target antigen. Therefore, "specific binding" does not necessarily require (but may include) exclusive binding.

[0504] In some embodiments, immune cells expressing CARs are genetically modified to recognize multiple targets or antigens, enabling recognition of specific target or antigen expression patterns in tumor cells. Examples of CARs capable of binding to multiple targets include “split-signal CARs” that limit complete immune cell activation against tumors expressing multiple antigens; “tandem CARs” (TanCARs) containing an ectodomain with two scFvs; and “universal ectodomain CARs” incorporating avidin or fluorescein isothiocyanate (FITC) specific scFvs to recognize tumor cells incubated with tagged monoclonal antibodies (Mab).

[0505] A CAR is considered "bispecific" when it recognizes two different antigens (having two different antigen-recognition domains). In some embodiments, a bispecific CAR consists of two different antigen-recognition domains present in tandem in a single transgenic receptor (referred to as TanCAR; see, for example, Grada Z et al. Molecular Therapy Nucleic Acids 2013;2:e105, the whole of which is incorporated herein by reference). Thus, in some embodiments, the method involves delivering to a tumor a combination comprising a non-pleomorphic, crystalline or crystalline salt form of compound 1 and an immunotherapy agent, in co-administration with a checkpoint inhibitor, wherein the immunotherapy agent is an engineered nucleic acid encoding an antigen, or an engineered nucleic acid that induces the expression of an autoantigen, and delivering to the tumor immune cells expressing a bispecific CAR that binds to two antigens, one of which is encoded by the engineered nucleic acid.

[0506] In some embodiments, the CAR is an antigen-specific inhibitory CAR (iCAR), which may be used, for example, to avoid off-tumor toxicity (Fedorov, VD et al., published online December 11, 2013, is incorporated herein by reference in its entirety). The iCAR contains an antigen-specific inhibitory receptor and blocks nonspecific immunosuppression that may result from, for example, extratumor target expression. The iCAR may be based on, for example, the inhibitory molecule CTLA-4 or PD-1. In some embodiments, these iCARs block the T cell response from T cells activated by either their endogenous T cell receptor or the activating CAR. In some embodiments, this inhibitory effect is transient.

[0507] In some embodiments, CARs may be used in adoptive cell transplantation, in which immune cells are removed from a subject and modified to express receptors specific to an antigen, such as a tumor-specific antigen. The modified immune cells are then capable of recognizing and killing cancer cells and are reintroduced into the subject (each of which is incorporated herein by reference in whole: Pule, et al., Cytotherapy. 2003;5(3):211-226, Maude et al., Blood. 2015;125(26):4017-4023).

[0508] In other embodiments of this disclosure, the tumor antigen component in the vaccine of the present invention is any natural or synthetic tumor-associated protein or peptide, or a combination of tumor-associated proteins and / or peptides or glycoproteins or glycopeptides. In yet another embodiment, the antigen component may be patient-specific or common to many or most patients with a particular type of cancer. In one embodiment, the antigen component consists of cell lysates derived from tumor tissue isolated from the patient being treated. In another embodiment, the lysates may be manipulated or synthesized from exosomes derived from tumor tissue. In yet another embodiment, the antigen component consists of cell lysates derived from tumor tissue extracted from one or more unrelated individuals or from tumor cell lines.

[0509] In various embodiments, tumor-associated antigen components of a vaccine can be produced by any of a variety of well-known techniques. For individual protein components, the antigen protein is isolated from tumor tissue or tumor cell lines by standard chromatographic means, e.g., high-pressure liquid chromatography or affinity chromatography, or alternatively, synthesized by a suitable expression system, e.g., standard recombinant DNA techniques in E. coli, yeast, or plants. The tumor-associated antigen protein is then purified from the expression system by standard chromatographic means. In the case of peptide antigen components, these are generally prepared by standard automated synthesis. Proteins and peptides can be modified by the addition of amino acids, lipids, and other agents to improve their integration into vaccine delivery systems (such as multilayer liposomes). For tumor-associated antigen components or cell lines derived from a patient's own tumor or from a tumor of another individual, tumor tissue or a single-cell suspension derived from tumor tissue is typically homogenized in a suitable buffer. The homogenate can also be fractionated, for example, by centrifugation to isolate specific cellular components, such as cell membranes or soluble materials. Tumor material may be used directly, or tumor-associated antigens may be extracted for incorporation into a vaccine using a buffer containing a low concentration of a suitable agent, such as a washing agent. An example of a suitable washing agent for extracting antigenic proteins from tumor tissue, tumor cells, and tumor cell membranes is diheptanoylphosphatidylcholine. Exosomes derived from tumor tissue or tumor cells, whether autologous or heterologous to the patient, may be used as antigenic components for incorporation into a vaccine, or as starting material for the extraction of tumor-associated antigens.

[0510] In some embodiments of this disclosure, one or more additional therapies are cancer vaccine immunotherapies administered concurrently with checkpoint inhibitors. In various examples, the cancer vaccine comprises at least one tumor-associated antigen, at least one immunostimulant, and optionally, at least one cell-based immunotherapy agent. In some embodiments, the immunostimulant component in the cancer vaccine of this disclosure is any biological response modifier (BRM) having the ability to enhance the efficacy of the therapeutic cancer vaccine and induce humoral and cellular immune responses against cancer cells in a patient. According to one embodiment, the immunostimulant is a cytokine or a combination of cytokines. Examples of such cytokines include interferons, e.g., IFN-gamma; interleukins, e.g., IL-2, IL-15, and IL-23; colony-stimulating factors, e.g., M-CSF and GM-CSF; and tumor necrosis factor. In another embodiment, the immunostimulatory component of the disclosed cancer vaccine comprises one or more adjuvant-type immunostimulatory agents, with or without immunostimulatory cytokines, e.g., APC Toll-like receptor agonists or costimulatory / cell adhesion membrane proteins. Examples of Toll-like receptor agonists include lipids A and CpG, as well as costimulatory / adhesion proteins, e.g., CD80, CD86, and ICAM-1.

[0511] In some embodiments, one or more additional therapies are immunostimulants selected from the group consisting of IFN-gamma (IFN-γ), IL-2, IL-15, IL-23, M-CSF, GM-CSF, tumor necrosis factor, lipid A, CpG, CD80, CD86, and ICAM-1, or combinations thereof. According to other embodiments, cell-based immunotherapeutic agents are selected from the group consisting of dendritic cells, tumor-infiltrating T lymphocytes, chimeric antigen receptor-modified T effector cells directed toward the patient's tumor type, B lymphocytes, natural killer cells, myeloid cells, and any other cells of the patient's immune system, or combinations thereof. In one embodiment, the cancer vaccine immunostimulator comprises one or more cytokines, e.g., interleukin-2 (IL-2), GM-CSF, M-CSF, and interferon-gamma (IFN-γ); one or more Toll-like receptor agonists and / or adjuvants, e.g., monophosphoryl lipid A, lipid A, muramyl dipeptide (MDP) lipid conjugate, and double-stranded RNA; or one or more costimulatory membrane proteins and / or cell adhesion proteins, e.g., CD80, CD86, and ICAM-1; or any combination thereof. In one embodiment, the cancer vaccine comprises an immunostimulator which is a cytokine selected from the group consisting of interleukin-2 (IL-2), GM-CSF, M-CSF, and interferon-gamma (IFN-γ). In another embodiment, the cancer vaccine comprises an immunostimulator which is a Toll-like receptor agonist and / or adjuvant selected from the group consisting of monophosphoryl lipid A, lipid A, and muramyl dipeptide (MDP) lipid conjugate, and double-stranded RNA. In another embodiment, the cancer vaccine includes an immunostimulator which is a co-stimulatory membrane protein and / or cell adhesion protein selected from the group consisting of CD80, CD86, and ICAM-1.

[0512] In various embodiments, one or more additional therapies may include a cancer vaccine, which may incorporate any tumor antigen that can potentially be used to construct a fusion protein according to the present invention, and in particular the following: (a) NY-ESO-1, SSX2, SCP1 and RAGE, BAGE, GAGE ​​and MAGE family polypeptides, e.g., GAGE-1, GAGE-2, MAGE-1, which can be used to address melanoma, lung, head and neck, NSCLC, milk, gastrointestinal, and bladder tumors. (b) cancer-testicular antigens including MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12; (c) mutant antigens including p53 associated with various solid tumors, e.g., colorectal, lung, and head and neck cancers; e.g., p21 / Ras associated with melanoma, pancreatic cancer, and colorectal cancer; e.g., CDK4 associated with melanoma; e.g., MUM1 associated with melanoma; e.g., caspase-8 associated with head and neck cancer; e.g., CIA0205 associated with bladder cancer; e.g., HLA-A2-R1701, betacatenin associated with melanoma; e.g., TCR associated with T-cell non-Hodgkin lymphoma; e.g., BCR-abl associated with chronic myeloid leukemia; triose phosphate isomerase; KIA0205; CDC-27, and LDLR-FUT; (c) overexpression antigens including galectin 4 associated with colorectal cancer; e.g., Hodgkin (d) Galectin 9 associated with cancer; for example, proteinase 3 associated with chronic myeloid leukemia; for example, WT1 associated with various leukemias; for example, carbonic anhydrase associated with renal cancer; for example, aldolase A associated with lung cancer; for example, PRAME associated with melanoma; for example, HER-2 / neu associated with breast, colon, lung, and ovarian cancers; for example, mammoglobin and alpha-fetoprotein associated with liver cancer; for example, KSA associated with colorectal cancer; for example, gastrin associated with pancreatic and gastric cancers; for example, telomerase catalytic protein and MUC-1 associated with breast and ovarian cancers; for example, G-250 associated with renal cell carcinoma; for example, p53 associated with breast and colon cancers; and, for example, carcinoembryonic antigens associated with breast cancer, lung cancer, and gastrointestinal cancers, such as colorectal cancer; (d) Melanoma-melanocyte differentiation antigens, for example, MART-1 / Melan A; gpl00; MC1R; melanocyte-stimulating hormone receptor; tyrosinase;(e) For example, covalent antigens including tyrosinase-related protein-1 / TRP1 and tyrosinase-related protein-2 / TRP2 associated with melanoma; (f) prostate-related antigens including PAP, PSA, PSMA, PSH-P1, PSM-P1, and PSM-P2 associated with prostate cancer; (g) immunoglobulin idiotypes associated with myeloma and B-cell lymphoma. In certain embodiments, one or more TAAs include human papillomavirus (HPV) antigens such as pi5, Hom / Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigen, TSP-180, pl85erbB2, pl80erbB-3, c-met, mn-23H1, TAG-72-4, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, pi6, TAGE, PSCA, CT7, and 43-9. F, 5T4, 791Tgp72, beta-HCG, BCA225, BTAA, CA125, CA15-3 (CA27.29\BCAA), CA195, CA242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophyllin C-related protein), TAAL6, TAG72, TLP, TPS, or any combination thereof may be selected.

[0513] In some embodiments, one or more additional therapies may include a tumor antigen comprising a whole amino acid sequence, a portion thereof, or a specific immunogenic epitope of a human protein.

[0514] In various embodiments, one or more additional therapies may include mRNA that is operable to encode one or more of the aforementioned cancer antigens useful for synthesizing a cancer vaccine. In some exemplary embodiments, mRNA-based cancer vaccines may have one or more of the following characteristics: a) mRNA encoding each cancer antigen is interspersed by cleavage-selective sites; b) mRNA encoding each cancer antigen is directly linked to one another without the use of linkers; c) mRNA encoding each cancer antigen is linked to one another by a single nucleotide linker; d) Each cancer antigen contains 20 to 40 amino acids and includes a centrally located SNP variant; e) At least 40% of the cancer antigens have the highest affinity for class I MHC molecules from the subject; f) At least 40% of the cancer antigens have the highest affinity for class II MHC molecules from the subject; g) At least 40% of the cancer antigens have a predicted binding affinity of IC > 500 nM for HLA-A, HLA-B and / or DRB1; h) The mRNA encodes 1 to 15 cancer antigens; i) 10 to 60% of the cancer antigens have binding affinity for class I MHC and 10 to 60% of the cancer antigens have binding affinity for class II j) mRNA having binding affinity to MHC; and / or j) being arranged such that the cancer antigen is ordered to minimize pseudoepitopes.

[0515] In various embodiments, one or more additional therapies are RNA vaccines comprising at least one RNA polynucleotide having an open reading frame encoding at least one antigen polypeptide or its immunogenic fragment, thereby in combination with administering compound 1 in a non-polymorphic, crystalline, or crystalline salt form in either the same composition or separate compositions administered simultaneously or sequentially, thereby inducing an immune response in a subject specific to the antigen polypeptide or its immunogenic fragment, and the anti-antigen polypeptide antibody titer in the subject increases after vaccination compared to the anti-antigen polypeptide antibody titer in a subject who has been prophylactically effective with a conventional vaccine against cancer. "Anti-antigen polypeptide antibody" is a serum antibody that specifically binds to an antigen polypeptide.

[0516] A prophylactically effective dose is a therapeutically effective dose that prevents cancer progression at a clinically acceptable level. In some embodiments, the therapeutically effective dose is the dose listed in the vaccine's prescribing information. Conventional vaccines, as used herein, refer to vaccines other than the mRNA vaccine of the present invention. For example, conventional vaccines include, but are not limited to, vaccines of live microorganisms, vaccines of dead microorganisms, subunit vaccines, protein antigen vaccines, DNA vaccines, and the like. In exemplary embodiments, a conventional vaccine is a vaccine approved by a regulatory authority and / or registered with a national drug regulatory agency, such as the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA).

[0517] In some embodiments, the anti-antigen polypeptide antibody titer in subjects increases by 1 log to 10 log after vaccination compared to the anti-antigen polypeptide antibody titer in subjects who received a conventional cancer vaccine at a prophylactically effective dose. In some embodiments, the anti-antigen polypeptide antibody titer in subjects increases by 1 log after vaccination compared to the anti-antigen polypeptide antibody titer in subjects who received a conventional cancer vaccine at a prophylactically effective dose. In some embodiments, the anti-antigen polypeptide antibody titer in subjects increases by 2 log after vaccination compared to the anti-antigen polypeptide antibody titer in subjects who received a conventional cancer vaccine at a prophylactically effective dose.

[0518] Aspects of the present invention provide a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigen polypeptide, wherein the RNA polynucleotides are present in a formulation for in vivo administration to a host, thereby resulting in an antibody titer that is better than the standard for serological protection against the first antigen for an acceptable percentage of human subjects. In some embodiments, the antibody titer produced by the mRNA vaccine of the present invention is a neutralizing antibody titer. In some embodiments, the neutralizing antibody titer is greater than that of a protein vaccine. In other embodiments, the neutralizing antibody titer produced by the mRNA vaccine of the present invention is greater than that of an adjuvant-containing protein vaccine. In other embodiments, the neutralizing antibody titers produced by the mRNA vaccine of the present invention are 1,000-10,000, 1,200-10,000, 1,400-10,000, 1,500-10,000, 1,000-5,000, 1,000-4,000, 1,800-10,000, 2,000-10,000, 2,000-5,000, 2,000-3,000, 2,000-4,000, 3,000-5,000, 3,000-4,000, or 2,000-2,500. The neutralizing titer is typically expressed as the maximum serum dilution required to achieve a 50% reduction in plaque number.

[0519] In a preferred embodiment, the RNA vaccine immunotherapy agent of this disclosure (e.g., mRNA vaccine) produces antigen-specific antibodies in the blood or serum of a vaccinated subject at levels, concentrations, and / or titers that are prophylactically and / or therapeutically effective. As defined herein, the term antibody titer refers to the amount of antigen-specific antibodies produced in a subject, e.g., a human subject. In exemplary embodiments, antibody titer is expressed as the inverse of the maximum dilution (in serial dilution) that still yields a positive result. In exemplary embodiments, antibody titer is determined or measured by enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments, antibody titer is determined or measured by a neutralization assay, e.g., a microneutralization assay. In certain embodiments, antibody titer measurement is expressed as a ratio, e.g., 1:40, 1:100.

[0520] In exemplary embodiments of the present invention, an effective vaccine produces antibody titers greater than 1:40, greater than 1:100, greater than 1:400, greater than 1:1000, greater than 1:2000, greater than 1:3000, greater than 1:4000, greater than 1:500, greater than 1:6000, greater than 1:7500, and greater than 1:10000. In exemplary embodiments, antibody titers are produced or reached 10 days, 20 days, 30 days, 40 days, or 50 days or more after vaccination. In exemplary embodiments, titers are produced or reached after a single dose of the vaccine administered to the subject. In other embodiments, titers are produced or reached after multiple doses, for example, after the first and second doses (e.g., booster doses). In exemplary embodiments of the present invention, antigen-specific antibodies are measured in units of g / ml, or in units of IU / L (International Units per Liter) or mIU / ml (Milli-International Units per ml). In exemplary embodiments of the present invention, an effective vaccine produces >0.5 μg / mL, >0.1 μg / mL, >0.2 μg / mL, >0.35 μg / mL, >0.5 μg / mL, >1 μg / mL, >2 μg / mL, >5 μg / mL, or >10 μg / mL. In exemplary embodiments of the present invention, an effective vaccine produces >10 mIU / mL, >20 mIU / mL, >50 mIU / mL, >100 mIU / mL, >200 mIU / mL, >500 mIU / mL, or >1000 mIU / mL. In exemplary embodiments, antibody levels or concentrations are produced or reached 10, 20, 30, 40, or 50 days or more after vaccination. In exemplary embodiments, levels or concentrations are produced or reached after a single dose of the vaccine administered to the subject. In other embodiments, levels or concentrations are produced or reached after multiple doses, for example, after the first and second doses (e.g., booster doses). In exemplary embodiments, antibody levels or concentrations are determined or measured by enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments, antibody levels or concentrations are determined or measured by a neutralization assay, for example, a microneutralization assay.The present invention also provides a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigen polypeptide or chain-like polypeptide, wherein the RNA polynucleotide is formulated with a stabilizing element or adjuvant to produce a higher antibody titer that lasts longer than that produced by an mRNA vaccine encoding the first antigen polypeptide, and is present in a formulation for in vivo administration to a host. In some embodiments, the RNA polynucleotide is formulated to produce neutralizing antibodies within one week of a single dose. In some embodiments, the adjuvant is selected from cationic peptides and immunostimulatory nucleic acids. In some embodiments, the cationic peptide is a protamine.

[0521] An immunotherapy agent comprising a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification or optionally not comprising a nucleotide modification, wherein the open reading frame encodes a first antigen polypeptide or a chain polypeptide, and the RNA polynucleotide is present in a formulation for in vivo administration to the host such that the level of antigen expression in the host significantly exceeds the level of antigen expression produced by an mRNA vaccine encoding the first antigen polypeptide, which is formulated with a stabilizing element or an adjuvant.

[0522] Other embodiments provide a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification or optionally not comprising a nucleotide modification, wherein the open reading frame encodes a first antigen polypeptide or linear polypeptide, and the vaccine has at least 10 times fewer RNA polynucleotides than would be required for an unmodified mRNA vaccine to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotides are present in doses of 25 to 100 micrograms.

[0523] Aspects of the present invention also provide vaccine use units formulated for delivery to human subjects, comprising 10 μg to 400 μg of one or more RNA polynucleotides having an open reading frame comprising at least one chemical modification or optionally not comprising a nucleotide modification, wherein the open reading frame encodes a first antigen polypeptide or a chain polypeptide, and a pharmaceutically acceptable excipient. In some embodiments, the vaccine further comprises cationic lipid nanoparticles.

[0524] Aspects of the present invention also provide a method for creating, maintaining, or restoring antigenic memory against a tumor in an individual or population of individuals, comprising administering an antigenic memory booster nucleic acid vaccine to the individual or population, the vaccine comprising: (a) at least one RNA polynucleotide, the polynucleotide comprising at least one chemical modification, or optionally without nucleotide modifications, and comprising two or more codon-optimized open reading frames, the open reading frames encoding a series of reference antigenic polypeptides; and (b) optionally pharmaceutically acceptable excipients. In some embodiments, the vaccine is administered to the individual via a route selected from the group consisting of intramuscular, intradermal, and subcutaneous administration. In some embodiments, the administration step comprises bringing the subject's muscle tissue into contact with a device suitable for injecting the composition. In some embodiments, the administration step comprises bringing the subject's muscle tissue into contact with a device suitable for injecting the composition in combination with electroporation.

[0525] Aspects of the present invention provide a method for vaccinating a subject, comprising administering to the subject a single dose of a nucleic acid vaccine in a useful amount for vaccinating the subject, comprising administering to the subject a nucleic acid vaccine in a useful amount for vaccinating the subject, comprising one or more RNA polynucleotides having an open reading frame encoding a first antigen polypeptide or a chain-like polypeptide, in a dose of 25 μg / kg to 400 μg / kg.

[0526] Another embodiment provides a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame having at least one chemical modification, wherein the open reading frame encodes a first antigen polypeptide or linear polypeptide, and the vaccine has at least 10 times fewer RNA polynucleotides than would be required for an unmodified mRNA vaccine to produce an equivalent antibody titer. In some embodiments, the RNA polynucleotides are present in doses of 25 to 100 micrograms.

[0527] In some embodiments, one or more additional therapies, non-polymorphic, crystalline, or crystalline salt forms of compound 1, may be bispecific antibody immunotherapeutic agents. The bispecific antibody may comprise a protein construct having a first antigen-binding site and a second antigen-binding site that binds to cytotoxic immune cells. The first antigen-binding site may bind to a tumor antigen that is specifically treated by the combination of the present invention. For example, the first antigen-binding site may bind to tumor antigens of non-limiting examples selected from, among others, EGFR, HGFR, Her2, Ep-CAM, CD20, CD30, CD33, CD47, CD52, CD133, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin αVβ3, integrin α5β1, MUC1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and tenascin. In some embodiments, the first antigen-binding site has specificity for proteins or peptides that are overexpressed in tumor cells compared to corresponding non-tumor cells. Where used herein, "corresponding non-tumor cells" refers to non-tumor cells of the same cell type as the tumor cells. Note that such proteins are not necessarily different from tumor antigens.Non-limiting examples include carcinoembryonic antigen (CEA), which is overexpressed in most colorectal, rectal, breast, lung, pancreatic, and gastrointestinal cancers; heregulin receptor (HER-2, neu, or c-erbB-2), which is frequently overexpressed in breast, ovarian, colorectal, lung, prostate, and cervical cancers; epidermal growth factor receptor (EGFR), which is highly expressed in a range of solid tumors, including those of the breast, head and neck, non-small cell lung, and prostate; asialoglycoprotein receptor; transferrin receptor; serpin enzyme complex receptor, which is expressed in hepatocytes; fibroblast growth factor receptor (FGFR), which is overexpressed in pancreatic ductal adenocarcinoma cells; vascular endothelial growth factor receptor (VEGFR), which is used for anti-angiogenic gene therapy; folate receptor, which is selectively overexpressed in 90% of non-mucinous ovarian cancers; cell surface glycocalyx; carbohydrate receptor; and polymer immunoglobulin receptor.

[0528] The second antigen-binding moiety is any molecule that specifically binds to an antigen, protein, or polypeptide expressed on the surface of cytotoxic immune cells (CIK cells). Exemplary non-limiting antigens expressed on the surface of cytotoxic immune cells suitable for use in this disclosure may include CD2, CD3, CD4, CD5, CD8, CD11a, CD11b, CD14, CD16a, CD27, CD28, CD45, CD45RA, CD56, CD62L, Fc receptor, LFA, LFA-1, TCRαβ, CCR7, macrophage inflammatory protein 1a, perforin, PD-1, PD-L1, PD-L2, or CTLA-4, LAG-3, OX40, 41BB, LIGHT, CD40, GITR, TGF-beta, TIM-3, SIRP-alpha, TIGIT, VSIG8, BTLA, SIGLEC7, SIGLEC9, ICOS, B7H3, B7H4, FAS, BTNL2, CD27, and Fas ligand. In some embodiments, the second antigen-binding moiety binds to CD3 on cytotoxic immune cells, e.g., CIK cells. In some embodiments, the second antigen-binding moiety binds to CD56 on cytotoxic immune cells. In some embodiments, the second antigen-binding moiety binds to the Fc receptor on cytotoxic immune cells. In some embodiments, the Fc region of the bispecific antibody binds to the Fc receptor on cytotoxic immune cells. In some embodiments, the second antigen-binding moiety is any molecule that specifically binds to an antigen expressed on the surface of cytotoxic immune cells (e.g., CIK cells). The second antigen-binding moiety is specific to the antigen on cytotoxic immune cells. Examples of exemplary cytotoxic immune cells, but not limited to, include CIK cells, T cells, CD8+ T cells, activated T cells, monocytes, natural killer (NK) cells, NK T cells, lymphokine-activated killer (LAK) cells, macrophages, and dendritic cells. The second antigen-binding moiety specifically binds to an antigen expressed on the surface of cytotoxic immune cells.Exemplary non-limiting antigens expressed on the surface of cytotoxic immune cells suitable for modulation according to this disclosure may include CD2, CD3, CD4, CD5, CD8, CD11a, CD11b, CD14, CD16a, CD27, CD28, CD45, CD45RA, CD56, CD62L, Fc receptor, LFA, LFA-1, TCRαβ, CCR7, macrophage inflammatory protein 1a, perforin, PD-1, PD-L1, PD-L2, or CTLA-4, LAG-3, OX40, 41BB, LIGHT, CD40, GITR, TGF-beta, TIM-3, SIRP-alpha, TIGIT, VSIG8, BTLA, SIGLEC7, SIGLEC9, ICOS, B7H3, B7H4, FAS, BTNL2, CD27, and Fas ligand. In other embodiments, the bispecific antibody modifier is an activator of a costimulatory molecule (e.g., an OX40 agonist). In one embodiment, the OX40 agonist is a bispecific antibody molecule against OX40 and another tumor antigen or costimulatory antigen. The OX40 agonist may be administered alone or in combination with other immunomodulators, for example, inhibitors of PD-1, PD-L1, CTLA-4, CEACAM (e.g., CEACAM-1, -3 and / or -5), TIM-3, or LAG-3 (e.g., antibody constructs). In some embodiments, the anti-OX40 antibody molecule is a bispecific antibody that binds to GITR and PD-1, PD-L1, CTLA-4, CEACAM (e.g., CEACAM-1, -3 and / or -5), TIM-3, or LAG-3. In one exemplary embodiment, the OX40 antibody molecule is administered in combination with an anti-PD-1 antibody molecule (e.g., an anti-PD-1 molecule as described herein). The OX40 antibody molecule and the anti-PD-1 antibody molecule may be in the form of separate antibody compositions or as bispecific antibody molecules.In other embodiments, the OX40 agonist may be administered in combination with agonists of other co-stimulatory molecules, such as GITR, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligands. In some embodiments, the second antigen-binding moiety binds to Fc receptors in cytotoxic immune cells, such as CIK cells.

[0529] In some embodiments, bispecific antibody immunotherapy agents are specific to tumor antigens and CIK cells, leading to the elimination of tumor cells through the antitumor cytotoxicity of CIK cells by bringing tumor antigens expressed on tumor cells into very close proximity with CIK cells. In some embodiments, the bispecific antibody is specific to tumor antigens but not to CIK cells; however, the Fc region of the bispecific antibody can bind to the Fc receptor on CIK cells, and then, by bringing tumor cells into very close proximity with CIK cells, leads to the elimination of tumor cells through the antitumor cytotoxicity of CIK cells. In some embodiments, the bispecific antibody is specific to CIK cells but not to tumor cells; however, the Fc region of the bispecific antibody can bind to the Fc receptor on tumor cells, and then, by bringing tumor cells into very close proximity with CIK cells, leads to the elimination of tumor cells through the antitumor cytotoxicity of CIK cells.

[0530] In some embodiments, one or more additional therapies are immune cell-engagement multivalent antibody / fusion protein / construct immunotherapeutics administered concurrently with checkpoint inhibitors. In various embodiments, one or more additional therapies may include immune cell-engagement multivalent antibody / fusion protein / constructs that may contain recombinant structures, e.g., all engineered antibodies that do not mimic the original IgG structure. Here, different strategies for multimerizing antibody fragments are utilized. For example, shortening of the peptide linker between V domains forces scFv to self-associate into a dimer (diabody; 55 kDa). Bispecific diabodies are formed by the non-covalent association of two VHA-VLB and VHB-VLA fragments expressed in the same cell. This leads to the formation of a heterodimer with two different binding sites. Single-stranded diabodies (sc-diabodies) are bispecific molecules in which the VHA-VLB and VHB-VLA fragments are linked together by an additional third linker. A tandem-diabody (Tandab) is a tetravalent, bispecific antibody produced by two sc-diabodies.

[0531] This also includes di-diabodies known in the art. This 130kDa molecule is formed by the fusion of a diabody to the N-terminus of the CH3 domain of IgG, resulting in an IgG-like structure. Further diabody derivatives are triabodies and tetrabodies, which fold into trimer and tetramer fragments by shortening the linker to <5 or 0-2 residues. The (scFv)2 construct known as a "bispecific T cell engager" (BITE) is also exemplified. A BITE is a bispecific single-chain antibody consisting of two scFv antibody fragments linked via a flexible linker, directed towards a surface antigen in target cells and CD3 in T cells. Bivalent (Fab)2 and trivalent (Fab)3 antibody formats are also exemplified. Minibodies and trimer bodies generated from scFvs are also exemplified. Exemplary constructs useful for targeting tumor antigens may include one or more of the following: diabody, single-stranded (sc)-diabody (scFv)2, mini-antibodies, mini-bodies, barnasebulster, scFv-Fc, sc(Fab)2, trimer antibody constructs, triabody antibody constructs, trimerbody antibody constructs, triabody antibody constructs, collabodi antibody constructs, (scFv-TNFa)3, F(ab)3 / DNL. Exemplary examples of cytotoxic immune cells include, but are not limited to, CIK cells, T cells, CD8+ T cells, activated T cells, monocytes, natural killer (NK) cells, NK T cells, lymphokine-activated killer (LAK) cells, macrophages, and dendritic cells.

[0532] In some embodiments, one or more additional therapies are radioconjugates.

[0533] In various embodiments, the radioactive conjugate is a small or large molecule (referred to herein as “cell targeting agent”), for example, a polypeptide, an antibody or an antibody fragment thereof, which is coupled to or otherwise immobilized with a radionuclide or multiple radionuclides, and as a result, the binding of the radioactive conjugate to its target (a protein or molecule on or within a cancer cell) leads to the death or pathological state of the cancer cell. In various embodiments, the radioactive conjugate may be a cell targeting agent labeled with a radionuclide, or the cell targeting agent may be coupled to or otherwise immobilized with particles, microparticles, or nanoparticles containing multiple radionuclides, the radionuclides may be the same or different. Methods for synthesizing radioactive conjugates are known in the art and may include a class of immunoglobulins or their antigen-binding moieties conjugated with toxic radionuclides.

[0534] In some embodiments, one or more additional therapies may be molecules that bind to cancer cells, which may be known as “cell-targeting agents.” As used herein, exemplary cell-targeting agents may enable drug-containing nanoparticles or radionuclides to target a particular type of target cell. Examples of cell-targeting agents include, but are not limited to, small molecules (e.g., folic acid, adenosine, purines) and large molecules (e.g., peptides or antibodies) that bind to or target tumor-associated antigens. Examples of tumor-associated antigens include, but are not limited to, adenosine receptors, alpha-v-beta 3, aminopeptidase P, alpha-fetoprotein, cancer antigen 125, carcinoembryonic antigen, c-caveolin-1, chemokine receptor, clatherin, carcinoembryonic antigen, CD20, epithelial tumor antigen, melanoma-associated antigen, Ras, p53, Her2 / Neu, ErbB2, ErbB3, ErbB4, folate receptor, prostate-specific membrane antigen, prostate-specific antigen, purine receptor, radiation-induced cell surface receptor, serpin B3, serpin B4, squamous cell carcinoma antigen, thrombospondin, tumor antigen 4, tumor-associated glycoprotein 72, thiosinase, and tyrosine kinase. In some embodiments, the cell targeting agent is folate or a folate derivative that specifically binds to the folate receptor (FR). In some embodiments, the cell targeting agent is an antibody, a bispecific antibody, a trispecific antibody, or an antigen-binding construct thereof, which specifically binds to cancer antigens selected from, among others, EGFR, HGFR, Her2, Ep-CAM, CD20, CD30, CD33, CD47, CD52, CD133, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin αVβ3, integrin α5β1, MUC1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and tenascin.

[0535] The use of folic acid as a targeting agent in radioactive conjugates also allows for the targeting and destruction of both tumor cells and regulatory T (Treg) cells. It is widely accepted that numerous Treg cells suppress tumor immunity. Specifically, Treg cells suppress (heterogeneous and auto) reactive T cells without killing them through contact-dependent or cytokine (e.g., IL-10, TGF-beta) secretion. FR4 is selectively upregulated in Treg cells. Antibody blockade of FR4 has been shown to deplete Treg cells and induce tumor immunity in tumor-carrying mice. Therefore, folic acid-coated PBM nanoparticles with cytotoxic properties take up FR-expressing cells for their destruction, inhibiting tumor progression both directly (i.e., BrCa cells) and indirectly (i.e., associated mammary tumor and surrounding Treg cells).

[0536] In another further embodiment, the targeting agent is an antibody or peptide capable of binding to tumor-associated antigens, or an immune cell-engagement multivalent antibody / fusion protein / construct, which may include, but are not limited to, adenosine receptors, alpha v beta 3, aminopeptidase P, alpha fetoprotein, cancer antigen 125, carcinoembryonic antigen, caveolin-1, chemokine receptor, clatherin, carcinoembryonic antigen, CD20, human growth factor receptor (HGFR), epithelial tumor antigen, melanoma-associated antigen, MUC1, Ras, p53, Her2 / Neu, ErbB2, ErbB3, ErbB4, folate receptor, prostate-specific membrane antigen, prostate-specific antigen, purine receptor, radiation-induced cell surface receptor, serpin B3, serpin B4, squamous cell carcinoma antigen, thrombospondin, tumor antigen 4, tumor-associated glycoprotein 72, tyrosinase, tyrosine kinase, etc.

[0537] In some embodiments, one or more additional therapies are vaccination protocols. In some embodiments, the vaccine may include one used to stimulate an immune response to a cancer antigen.

[0538] The amounts of both the non-polymorphic, crystalline, or crystalline salt form of Compound 1 as disclosed herein, and one or more additional therapeutic agents (in those compositions containing such additional therapeutic agents), which can be combined with excipient materials to create a single-dose formulation, vary depending on the host being treated and the specific mode of administration. In certain embodiments, the compositions of the present invention are formulated so that those of the present invention can be administered in doses of 0.01 to 100 mg / kg body weight / day.

[0539] Additional therapeutic agents may act synergistically. Therefore, the amount of additional therapeutic agent in such a composition may be less than that required in monotherapy using only that agent, or, given that lower doses are used, side effects on the patient may be less. In certain embodiments, an additional therapeutic agent may be administered in such a composition at a dose of 0.01 to 10,000 μg / kg body weight / day.

[0540] In some embodiments, one or more additional therapies are kinase inhibitors selected from: Akt1, Akt2, Akt3, TGF-βR, PKA, PKG, PKC, CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2, HER3, HER4, 1NS-R, IGF-1R, IR-R, PDGFαR, PDGFβ / R, CSFIR, KIT, FLK-II, KDR / F LK-1, FLK-4, flt-1, FGFR1, FGFR2, FGFR3, FGFR4, Ron, Sea, TRKA, TRKB, TRKC, FLT3, VEGFR / Flt2, Flt4, EphA1, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYR, FRK, JAK, ABL, ALK, CDK7, CDK12, CDK13, KRAS, and B-Raf. In some embodiments, one or more additional therapies are inhibitors of CD47 and MALT1 proteins.

[0541] In some embodiments, one or more additional therapies are poly-ADP-ribose polymerase (PARP) inhibitors. Exemplary PARP inhibitors include, but are not limited to, olaparib (Lynparza®), lucapriv (Rubraca®), niraparib (Zejula®), talazoparib (Talzenna®), and TPST-1120.

[0542] In some embodiments, one or more additional therapies are kinase inhibitors. Exemplary kinase inhibitors include imatinib, baricitinib, gefitinib, erlotinib, sorafenib, dasatinib, sunitinib, lapatinib, nilotinib, pirfenidone, zanubrutinib, upadacitinib, fedratinib, entrectinib, alpelisib, pazopanib, crizotinib, vemurafenib, vandetanib, ruxolitinib, axitinib, bosutinib, regorafenib, tofacitinib, cabozantinib, ponatinib, trametinib, dabrafenib, and afatinib. This includes ibrutinib, ceritinib, idelalisib, nintedanib, palbociclib, lenvatinib, cobimetinib, abemaciclib, acalabrutinib, alectinib, binimetinib, brigatinib, encorafenib, erdafitinib, everolimus, fostamatinib, glitter, lalotrectinib, lorlatinib, netalusdil, osimertinib, pexidartinib, ribociclib, temsirolimus, XL-147, XL-765, XL-499, and XL-880. In some embodiments, the kinase inhibitor is an HSP90 inhibitor (e.g., XL888), a liver X receptor (LXR) modulator, a retinoid-related orphan receptor gamma (RORy) modulator, a CK1 inhibitor, a CK1-α inhibitor, a Wnt pathway inhibitor (e.g., SST-215), or a mineralocorticoid receptor inhibitor (e.g., esaxerenone or XL-550), for the treatment of diseases disclosed herein, such as cancer.

[0543] In some embodiments, one or more additional therapies are polatuzumab vedotin.

[0544] The pharmaceutical compositions containing a non-polymorph, crystalline, or crystalline salt form of Compound 1 according to this disclosure typically comprise an effective amount of a non-polymorph, crystalline, or crystalline salt form of Compound 1, an immunotherapeutic agent, and / or both, dispersed in a pharmaceutically acceptable excipient. The phrase "pharmaceutically acceptable" means, where necessary, molecular entities and compositions that do not produce harmful, allergic, or other adverse reactions when administered to animals, such as humans. Preparation of pharmaceutical compositions containing a non-polymorph, crystalline, or crystalline salt form of Compound 1 is described in Remington's Pharmaceutical Sciences, 21 st As illustrated by Ed., (Lippincott, Williams and Wilkins, Philadelphia, PA, 2006), this is known to those skilled in the art in light of this disclosure. Furthermore, it is understood that, in the case of administration to animals (e.g., humans), the preparation should meet sterility, pyrogenicity, general safety and purity standards. Specific examples of pharmacologically acceptable excipients for combination compositions containing a non-polymorph, crystalline or crystalline salt form of Compound 1 in mixture with the immunotherapeutic agents described herein are borate buffer or sterile saline solution (0.9% NaCl).

[0545] Immunotherapy agents, for example, formulations of immune checkpoint modulator antibodies used in this disclosure, are antibodies having a desired degree of purity, according to Remington's Pharmaceutical Sciences 21. stThe compounds may be prepared for storage in the form of lyophilized formulations, aqueous solutions, and / or suspensions by mixing with optional pharmaceutically acceptable excipients or stabilizers as fully described and explained in Ed. (Lippincott, Williams and Wilkins, Philadelphia, PA, 2006). Acceptable excipients, buffers, or stabilizers are nontoxic to the recipient at the doses and concentrations used and include suitable aqueous and / or non-aqueous excipients that may be used in the pharmaceutical compositions of this disclosure, e.g., water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Adequate fluidity may be maintained, for example, by the use of coating materials (e.g., lecithin), by maintaining the required particle size in the case of dispersions, and by the use of surfactants, buffers (e.g., phosphates, citrates, and other organic acids). Antioxidants, for example, (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.; preservatives (for example, octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens, such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); may also contain low molecular weight (less than about 10 residues).Other exemplary pharmaceutically acceptable excipients may include polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0546] In one exemplary embodiment, the pharmaceutical composition may optionally contain pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers and toxicity modifiers, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate, as required to approximate physiological conditions. In some embodiments, the checkpoint inhibitor antibodies or antigen-binding fragments of the present disclosure may be formulated for storage and reconstituted in excipients suitable for lyophilization for storage before use by lyophilization and reconstitution techniques known in the art. In one exemplary pharmaceutical composition containing one or more checkpoint inhibitor antibodies or antigen-binding fragments, the composition is formulated as a sterile, preservative-free solution of one or more checkpoint inhibitor antibodies or antigen-binding fragments for intravenous or subcutaneous administration. The formulation may be supplied as either a disposable, disposable pre-filled pen containing, for example, about 1 mL of pre-filled glass syringe, or a disposable facility-use vial. Preferably, the pharmaceutical composition containing a checkpoint inhibitor antibody or its antigen-binding fragment is clear and colorless, with a pH in the range of about 6.9 to 5.0, preferably 6.5 to 5.0, and more preferably 6.0 to 5.0. In various embodiments, the formulation containing the pharmaceutical composition, when reconstituted and administered to a subject, may contain about 500 mg to about 10 mg, or about 400 mg to about 20 mg, or about 300 mg to about 30 mg, or about 200 mg to about 50 mg of the checkpoint inhibitor antibody or its antigen-binding fragment per mL of solution. Exemplary injectable or infusion excipients for parenteral administration, e.g., intravenous, intramuscular, intraperitoneal, or subcutaneous administration, may include mannitol, citrate monohydrate, dibasic sodium phosphate dihydrate, monobasic sodium phosphate dihydrate, polysorbate 80, sodium chloride, sodium citrate, and water.

[0547] In another exemplary embodiment, one or more immunotherapeutic agents, or their antigen-binding fragments, are formulated for intravenous or subcutaneous administration as a sterile aqueous solution containing an antibody in a concentration of 1 to 75 mg / mL, more preferably about 5 to 60 mg / mL, even more preferably about 10 to 50 mg / mL, or even more preferably about 10 to 40 mg / mL, at a pH in the range of about 5 to 6, together with sodium acetate, polysorbate 80, and sodium chloride. Preferably, the intravenous or subcutaneous formulation is a sterile aqueous solution containing an immunotherapeutic agent in a concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg / mL, such as an immune checkpoint inhibitor antibody or its antigen-binding fragment, at pH 5.5, together with 20 mM sodium acetate, 0.2 mg / mL polysorbate 80, and 140 mM sodium chloride. Furthermore, solutions containing checkpoint inhibitor antibodies or their antigen-binding fragments may include, among many other compounds, histidine, mannitol, sucrose, trehalose, glycine, poly(ethylene) glycol, EDTA, methionine, and any combination thereof, as well as many other compounds known in the relevant art.

[0548] In one embodiment, the pharmaceutical composition of the present disclosure, along with a non-polymorphic, crystalline, or crystalline salt form of compound 1, comprises the following components at pH 5.8: 5 to 500 mg of the immunotherapy agent of the present disclosure or its antigen-binding fragment, 10 mM histidine, 5% sucrose, and 0.01% polysorbate 80. This composition may be provided as a lyophilized powder. When the powder is reconstituted in its entire volume, the composition retains the same formulation. Alternatively, the powder may be reconstituted in half volume, in which case the composition comprises, at pH 5.8, 10 to 500 mg of the immunotherapy agent of the present disclosure or its antigen-binding fragment, 20 mM histidine, 10% sucrose, and 0.02% polysorbate 80.

[0549] In one embodiment, a portion of the dose is administered by intravenous bolus, and the remainder by infusion of the immunotherapy agent. For example, intravenous infusion of an immunotherapy agent or its antigen-binding fragment in amounts of about 0.001 to about 200 mg / kg, e.g., about 0.001 mg / kg to about 100 mg / kg, or about 0.001 mg / kg to about 50 mg / kg, or about 0.001 mg / kg to about 10 mg / kg may be given as a bolus, and the remainder of the antibody dose may be administered by intravenous infusion. The predetermined dose of the immunotherapy agent or its antigen-binding fragment may be administered over a period of, for example, 1 to 2 to 5 hours.

[0550] In further embodiments, a portion of the dose is administered by subcutaneous injection and / or infusion in the form of a bolus, and the remainder by infusion of the immunotherapy formulation. In some exemplary doses, the immunotherapy formulation may be administered subcutaneously in doses of the immunotherapy agent or its antigen-binding fragment for intravenous injection, ranging from about 0.001 to about 200 mg / kg, for example, about 0.001 mg / kg to about 100 mg / kg, or about 0.001 mg / kg to about 50 mg / kg, or about 0.001 mg / kg to about 10 mg / kg. In some embodiments, the dose may be given as a bolus, and the remaining immunotherapy dose may be administered by subcutaneous or intravenous injection. A predetermined dose of the immunotherapy agent or its antigen-binding fragment may be administered over a period of, for example, 1 to 2 to 5 hours.

[0551] The formulations described herein may also contain more than one active compound, preferably compounds having complementary activity that does not adversely affect each other, as needed for the specific indication being treated. For example, it may be desirable to provide one or more immunotherapeutic agents having other specificities. Alternatively, or in addition, the composition may include anti-inflammatory agents, chemotherapeutic agents, cytotoxic agents, cytokines, growth inhibitors, and / or small molecule antagonists. Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0552] Preparations used for in vivo administration should be sterile or nearly sterile. This can be easily achieved by filtration through a sterile filtration membrane.

[0553] In various embodiments, exemplary formulations of the pharmaceutical compositions described herein may be prepared using methods widely known in the field of pharmaceutical formulations. Generally, such preparation methods may include the steps of combining the active ingredient with excipients or one or more other adjuncts, and then, if desired, packaging the product into desired single or multi-dose units.

[0554] In some embodiments, compositions comprising a non-polymorphic, crystalline, or crystalline salt form of compound 1 may also be delivered by vesicle, and the immunotherapy agent may be delivered in the same liposomal formulation or in separate formulations compatible with the liposomal formulation containing a non-polymorphic, crystalline, or crystalline salt form of compound 1. In some exemplary examples, liposomes containing one or more liposome surface portions, e.g., polyethylene glycol, antibodies, and antibody fragments targeting desired tumor surface antigens, receptors, growth factors, glycoproteins, glycolipids, or neoantigens, are selectively delivered to specific cells or organs, thereby enhancing the delivery of the targeted drug.

[0555] In another embodiment, the non-polymorphic, crystalline, or crystalline salt form of compound 1 may be delivered by vesicles, particularly liposomes (see Langer, Science 249:1527-1533 (1990), Treat et al., in LIPOSOMES IN THE THERAPY OF INFECTIOUS DISEASE AND CANCER, Lopez-Berestein and Fidler (eds.), Liss, NY, pp.353-365 (1989), Lopez-Berestein, ibid., pp.317-327; generally see the above sections).

[0556] In another embodiment, a composition containing a non-polymorph, crystalline, or crystalline salt form of compound 1, or a combination thereof, or a composition containing an immunotherapy agent, may be delivered by a controlled-release system. In one embodiment, a pump may be used (see Langer, above; Sefton, CRC Crit.Ref.Biomed.Eng.14:201(1987), Buchwald et al., Surgery 88:507(1980), Saudek et al., N.Engl.J.Med.321:574(1989)). In another embodiment, the controlled release of a non-polymorph, crystalline, or crystalline salt form of compound 1 may include a polymer material that imparts sustained, intermediate, pulsed, or alternating release (MEDICAL APPLICATIONS OF CONTROLLED RELEASE, Langer and Wise(eds.), CRC Pres., Boca See Raton, Fla. (1974), CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN AND PERFORMANCE, Smolen and Ball (eds.), Wiley, New York (1984), Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989), Howard et al. See also al., J. Neurosurg. 71:105 (1989). Other controlled emission systems discussed in the review by Langer (Science 249:1527-1533 (1990)) may be used.

[0557] The optimal concentration of the active ingredient(s) in the selected medium can be determined empirically according to procedures well known to those skilled in the art, and depends on the desired final pharmaceutical formulation and the intended use.

[0558] The present disclosure also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the pharmaceutical compositions of the present disclosure, minimally comprising a non-polymorphic form, a crystalline form or a crystalline salt form of Compound 1 described herein, and one or more checkpoint inhibitor antibodies or antigen-binding fragments thereof. In other embodiments, the kit may contain one or more additional containers providing pharmaceutically acceptable excipients, such as diluents. In one embodiment, the kit may comprise at least one container, which may contain a non-polymorphic form, a crystalline form or a crystalline salt form of Compound 1 of the present disclosure, a checkpoint inhibitor antibody or an antigen-binding fragment thereof. The kit may also include a series of instructions for preparing the final pharmaceutical composition and administering it to a subject in need thereof for the treatment of a checkpoint molecule-mediated disease or disorder.

[0559] Labeled Compounds and Assay Methods Another aspect relates to the labeled non-polymorphic forms, crystalline forms or crystalline salt forms of the present invention (radioactive labels, fluorescent labels, etc.), which are useful not only in imaging techniques but also in both in vitro and in vivo assays for localizing and quantifying TAM kinase in tissue samples, including human, and for identifying TAM kinase ligands by inhibitory binding of the labeled compounds. Accordingly, the present invention includes a TAM kinase assay containing such labeled compounds.

[0560] The present invention further includes the isotopically labeled non-polymorphic forms, crystalline forms or crystalline salt forms of the present invention. An "isotopically" or "radioactively labeled" compound is a crystalline form or a crystalline salt form of the present invention, in which one or more atoms are replaced or substituted by atoms having an atomic mass or mass number different from that typically found in nature (i.e., naturally occurring). Suitable radionuclides that can be incorporated into the non-polymorphic forms, crystalline forms or crystalline salt forms of the present invention include, but are not limited to, 2 H (also denoted as D for deuterium), 3 H (also denoted as T for tritium),11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 18 F, 35 S, 36 Cl, 82 Br, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, and 131 I is included. The radionuclides incorporated into immediately radiolabeled compounds depend on the specific application of that radiolabeled compound. For example, in in vitro metalloprotease labeling and competitive assays, 3 H, 14 C, 82 Br, 125 I, 131 I, or 35 Compounds incorporating sulfur are generally the most useful. For radioactive imaging applications, 11 C, 18 F, 125 I, 123 I, 124 I, 131 I, 75 Br, 76 Br or 77 Br will generally be the most useful. In some embodiments, non-polymorphic, crystalline, or crystalline salt forms described herein are used in which one or more hydrogens, such as hydrogens bonded to carbon atoms, are replaced by deuterium. Such compounds exhibit increased resistance to metabolism and are therefore useful for increasing the half-life of any compound when administered to mammals, particularly humans.

[0561] It is understood that a “radioactive label” or “labeled compound” is a compound incorporating at least one radionuclide. In some embodiments, the radionuclide is: 3 H, 14 C, 125 I, 35 S, and 82Selected from the group consisting of Br.

[0562] The present invention may further include a synthetic method for incorporating radioactive isotopes into the non-polymorphic, crystalline, or crystalline salt forms of the present invention. Synthetic methods for incorporating radioactive isotopes into organic compounds are well known in the art, and those skilled in the art will readily recognize methods applicable to the compounds of the present invention.

[0563] The labeled compounds of the present invention can be used in screening assays for identifying / evaluating compounds. For example, a newly synthesized or identified compound to be labeled (i.e., a test compound) can be evaluated for its ability to bind to TAM by monitoring its concentration changes upon contact with TAM kinase through tracking of the label. For example, a test compound (to be labeled) can be evaluated for its ability to reduce the binding of another compound known to bind to TAM kinase (i.e., a standard compound). Thus, the ability of a test compound to compete with a standard compound for binding to TAM kinase directly correlates to its binding affinity. Conversely, in some other screening assays, the standard compound is labeled and the test compound is not. Thus, the concentration of the labeled standard compound is monitored to assess competition between the labeled compound and the test compound, and thus the relative binding affinity of the test compound is confirmed.

[0564] Preparations and Examples General experimental techniques Aqueous slurry experiment: A salt of compound 1, which was determined to have an aqueous solubility of less than 1 mg / mL, was slurryed in 20 mL of water at ambient temperature for 1 day. The solid was then collected by vacuum filtration and analyzed by XRPD.

[0565] Crush Cooling (CC): Concentrated solutions of compound 1 and various counterions were prepared in MeOH under high temperature stirring. Capped vials containing the high-temperature solutions were transferred to a freezer (approximately -20°C) and rapidly cooled. The formed solid was collected. If no solid was present, additional crystallization techniques were used.

[0566] Crush precipitation (CP): Clear solutions of compound 1 and coformer were prepared at RT in various solvents. Aliquots of various antisolvents were slowly added to the solution while gently stirring until the solids crushed from the solution. The mixture was stirred for a specified period of time. The formed solids were collected by positive pressure filtration.

[0567] Rapid Cooling (FC): Concentrated solutions of compound 1 and various counterions were prepared in acetone or MeOH with stirring at high temperature. Capped vials containing the high-temperature solutions were transferred to a benchtop at ambient temperature. The formed solid was collected. If no solid was present, additional crystallization techniques were used.

[0568] Fast evaporation (FE): Clear solutions of compound 1 and coformer were prepared in various solvents. The vials were left uncapped and the solvent was evaporated under ambient conditions.

[0569] Interconversion slurry: A slurry of compound 1 form A was prepared by adding a sufficient amount of solid to a given solvent system under ambient conditions so that undissolved solids were present. The mixture was then stirred for an extended period to ensure saturation. The solid of the desired form was then added to an aliquot of a saturated solution (filtered through a 0.2 μm nylon filter) so that undissolved solids were present. The mixture was then stirred for an extended period at ambient temperature to isolate the solid.

[0570] Isolation techniques: Generally, before isolating the solid, non-ambient samples were removed from their respective temperature-controlled devices to minimize equilibrium to ambient temperature, and then isolation was performed rapidly.

[0571] Decantation of the liquid phase: Some of the solids isolated from the solution-based crystallization technique were collected by (if necessary) centrifugation of the suspension to discard the liquid phase and allowing the moist solids to stand. The solids were dried for a short period of time (e.g., by air drying or drying under nitrogen) unless otherwise specified herein as “analytical wet material”.

[0572] Positive pressure filtration: The solid was collected on a 0.2 μm nylon or PTFE filter by pressing the slurry through a syringe and Swinnex filter holder assembly. Generally, the solid was dried for a short time by blowing 20 mL of air from a syringe onto the filter. Where referred to as "analytical wet material" herein, the solid was left to remain moist with the mother liquor. Some samples were further dried for a short time under a gentle stream of nitrogen gas before analysis.

[0573] Vacuum filtration: The solid was collected in a paper or nylon filter by vacuum filtration, air-dried briefly in the filter under reduced pressure, and then transferred to a vial.

[0574] Reaction crystallization (RC): Compound 1 and a mixture of various coformers were combined in a high-temperature acetone slurry so that the molar concentration of the coformer was twice that of the API. The solution was stirred for a given period of time. When a clear solution was observed, additional crystallization techniques were used.

[0575] Stability test: Various compound salts were placed in open vials in a 75% RH chamber (saturated sodium chloride solution (solutiona)). The RH chamber was placed in a 40°C oven for 15-16 days. Samples were analyzed by PLM and XRPD at the end of the period.

[0576] Slow cooling (SC): Concentrated solutions of compound 1 and various coformers were prepared in various solvents with stirring at high temperatures. The vials were capped in a heated sample block, the hot plate was turned off, and the vials were gradually cooled to ambient temperature in the heated vial block. The clear solutions were further cooled in a refrigerator (5-7°C) and / or freezer (approximately -20°C) during the cooling to ambient temperature. If no solid was present, additional crystallization techniques were used.

[0577] Slow evaporation: Solutions were prepared in various solvents with stirring and typically filtered through a 0.2 μm nylon or PTFE filter. Each solution was evaporated under ambient conditions (e.g., loosely capped or covered with perforated aluminum foil) from a covered vial, unless otherwise specified. The solutions were evaporated to dryness unless otherwise indicated as partial evaporation (a solid containing a small amount of residual solvent), in which case the solid was isolated as described herein.

[0578] Solubility estimation: Aliquots of various solvents were added to the measured amount of compound 1 while stirring (typically by sonication) at the stated temperature until complete dissolution was achieved, as determined by visual observation. If dissolution occurred after the addition of the first aliquot, the value was reported as ">". If dissolution did not occur, the value was reported as "<".

[0579] Estimation of aqueous solubility: Aliquots of water were added to various single salts of different compounds under sonication.

[0580] Slurry Experiment: Saturated solutions of compound 1 and various coformers were prepared in various solvents and solvent mixtures. The mixtures were stirred at ambient temperature and high temperature for the noted durations. The solids were collected using the techniques described, with additional crystallization techniques used where appropriate.

[0581] Desolvation in a vacuum oven: Desolvation was attempted for the salt of compound 1, which had been determined to be a solvate by various analytical methods. The sample was placed in a vacuum oven at a temperature ranging from ambient temperature to 80°C for a given period of time. The sample was analyzed by XRPD and / or TGA to determine the success of desolvation.

[0582] Vapor diffusion: Concentrated solutions were prepared in various solvents and typically filtered through a 0.2 μm nylon or PTFE filter. The filtered solutions were dispensed into smaller vials and then placed in larger vials containing the antisolvent. The smaller vials were left uncapped, while the larger vials were capped to allow vapor diffusion to occur. Any solids present were isolated as described herein.

[0583] Vapor stress: A selected solid was transferred to a smaller vial, which was then placed inside a larger vial containing a solvent. The smaller vial was left uncapped, while the larger vial was capped, and vapor stress was generated at the temperatures described.

[0584] A coformer means one or more pharmaceutically acceptable bases and / or pharmaceutically acceptable acids disclosed herein that associate with compound 1. Exemplary coformers as used herein include fumaric acid, HCl, and phosphoric acid.

[0585] Instrument-based technology: Differential Scanning Calorimetry (DSC): DSC was performed using a Mettler-Toledo DSC3+ differential scanning calorimeter. Temperature calibration was performed using adamantane, phenyl salicylate, indium, tin, and zinc. Samples were placed in sealed or open aluminum DSC pans and their weights were accurately recorded. The weighed aluminum pans configured as sample pans were placed on the reference side of the cell. Samples were analyzed from -30 to 250°C at a ramp rate of 10°C / min. Thermograms were plotted against the reference temperature (x-axis), but results are reported according to the sample temperature.

[0586] Dynamic vapor adsorption (DVS) a. VTI: Automatic vapor sorption (VS) data was collected using a VTI SGA-100 vapor sorption analyzer. NaCl and PVP were used as calibration standards. Samples were dried before analysis. Adsorption and desorption data were collected under nitrogen purging in 10% RH increments over the range of 5% to 95% RH. The equilibrium standard used for analysis was a weight change of less than 0.0100% in 5 minutes, using a maximum equilibrium time of 3 hours. Data were not corrected for the initial water content of the samples.

[0587] b. Intrinsic: Automatic vapor sorption (VS) data was collected using a Surface Measurement System DVS Intrinsic instrument. Samples were not dried before analysis. Adsorption and desorption data were collected under nitrogen purging in 10% RH increments over the range of 5% to 95% RH. The equilibrium criterion used for analysis was a weight change of less than 0.0100% in 5 minutes, using a maximum equilibrium time of 3 hours. Data were not corrected for the initial water content of the samples.

[0588] Hot Stage Microscopy (HSM): Hot stage microscopy was performed using a Linkam hot stage (FTIR600) mounted on a Leica DM LP microscope equipped with a SPOT Insight® color digital camera. Temperature calibration was performed using the USP melting point standard. The sample was placed on a coverslip, and a second coverslip was placed on top of the sample. As the stage heated, each sample was visually observed using a 20x objective lens with a cross polarizer and primary red compensator. Images were captured using SPOT software (version 4.5.9).

[0589] Optical microscopy: Samples were observed under a Motic or Wolfe optical microscope with a cross-polarizer, or under a Leica stereomicroscope with a primary red compensator with a cross-polarizer.

[0590] Determination of pKa and logP: The determination of pKa and logP was performed by Pion Inc. / Sirius Analytical Instruments Ltd. in East Sussex, United Kingdom.

[0591] Solution proton nuclear magnetic resonance spectroscopy ( 1 HNMR):Solution 1 1H NMR spectra were obtained using Spectral Data Services of Champaign, IL. Samples were prepared by dissolving approximately 5-10 mg of the sample in DMSO-d6.

[0592] Thermogravimetric Analysis (TGA): Thermogravimetric analysis was performed using a Mettler Toledo TGA / DSC3+ analyzer. Temperature calibration was performed using phenyl salicylate, indium, tin, and zinc. Samples were placed in aluminum pans. Open pans were inserted into the TG furnace. The furnace was heated under nitrogen. Each sample was heated from ambient temperature to 350°C at a ramp rate of 2, 5, or 10°C / min. Thermograms were plotted against reference temperature (x-axis), but results are reported according to sample temperature.

[0593] X-ray powder diffraction (XRPD) a. Reflection: XRPD patterns were collected using a PANalytical X'Pert PRO MPD diffractometer with incident beam Cu Kα radiation generated at room temperature (298 Kelvin) using a long fine focus source and nickel filter. The diffractometer was constructed using symmetric Bragg-Brentano geometry. Prior to analysis, a silicon sample (NIST SRM 640e) was analyzed to demonstrate that the observed position of the Si 111 peak matched the NIST-certified position. The sample specimen was filled into wells. A scattering prevention slit (SS) was used to minimize background generated by air. Solar slits were used for both the incident and diffracted beams to minimize spreading from axial divergence. Diffraction patterns were collected using a scanning position-sensing detector (X'Celerator) located 240 mm from the sample, and Data Collector software version 2.2b.

[0594] b. Transmission: XRPD patterns were collected using a PANalytical X'Pert PRO MPD diffractometer with Cu radiation from an incident beam generated at room temperature (298 Kelvin) using an Optix long fine focus source. Cu Kα X-rays were focused onto the detector through the specimen using an elliptically gradually changing multilayer mirror. Prior to analysis, a silicon specimen (NIST SRM 640e) was analyzed to demonstrate that the observed position of the Si 111 peak coincided with the NIST-certified position. The sample specimen was sandwiched between 3 μm thick films and analyzed by transmission geometry. Background caused by air was minimized using a beam stop, short anti-scattering extension, and anti-scattering knife edge. Solar slits were used for the incident and diffracted beams to minimize spreading from axial divergence. Diffraction patterns were collected using a scanning position-sensing detector (X'Celerator) located 240 mm from the specimen, and Data Collector software version 2.2b.

[0595] XRPD Index Indexing and structural refinement are performed by computer calculations. In the referenced figure for a given indexed XRPD pattern, the agreement between the acceptable peak positions marked by bars and the observed peaks indicates a consistent unit cell determination. Successful indexing of the pattern indicates that the sample is composed primarily of single-crystal phases, unless otherwise noted. The assigned extinction symbols, unit cell parameters, and corresponding space groups are shown in the table.

[0596] PD-1 antibody The PD-1 antibody used in the examples was purchased from BioXcell catalog number BE0146, clone RPMI-14, lot 780120J3. [Examples]

[0597] Preparation Example 1: Synthesis of Compound 1 Step 1: N-(4-fluorophenyl)-N-(4-hydroxyphenyl)cyclopropane-1,1-dicarboxamide(4): [ka]

[0598] To a solution of compound 2 (10 g, 44.80 mmol, 1 equivalent) and compound 3 (5.87 g, 53.8 mmol, 1.2 equivalents) in dimethylacetamide (DMA) (60 mL), 3-(ethyliminomethyleneamino)-N,N-dimethyl-propane-1-amine hydrochloride (EDCI) (10.31 g, 53.8 mmol, 1.2 equivalents) was added. The mixture was vigorously stirred at 20°C until the reaction was complete. The mixture was poured into saturated aqueous NaHCO3 (aq) (400 mL) and extracted with SiO2 (4 × 100 mL). The combined organic phase was washed with saturated aqueous NaCl (100 mL), dried on anhydrous Na2SO4, and concentrated. Compound 4 (21 g, crude) (50% purity) was obtained. 1 H C 17 H 15 MS(EI) for FN2O3: Measured value 314.9 (MH+).

[0599] Step 2: Methyl 4-[4-[[1-[(4-fluorophenyl)carbamoyl]cyclopropane-carbonyl]amino]phenoxy]-7-methoxyquinoline-6-carboxylate(6): [ka]

[0600] A mixture of compound 4 (5.99 g, 9.5 mmol, 1.2 equivalents), compound 5 (2 g, 8.0 mmol, 1.0 equivalent), Pd(OAc)2 (89 mg, 397.4 μmol, 0.05 equivalents), rac-2-(di-tert-butylphosphino)-1,1'-binaphthyl (TrixiePhos, 316.71 mg, 794.7 μmol, 0.1 equivalents), and K3PO4 (2.53 g, 11.9 mmol, 1.5 equivalents) in anisole (50 mL) was stirred at 110 °C for 2 hours (h) under a nitrogen atmosphere. The mixture was filtered, and the filtrate was concentrated. The residue was purified by flash silica gel chromatography (1:1 petroleum ether:SiO to 20:1 SiO:MeOH). Compound 6 was obtained (2.6 g, 61.8% yield). 1 H NMR(400MHz, CDCl3)δ9.38(s, 1H), 8.80(s, 1H), 8.63(d, 2H), 7.64(d, 2H), 7.54-7.41(m, 3H), 7.18(d, 2H), 7.09-7.01(m, 2H), 6.43(d, 1H), 4.05(s, 3H), 3.97(s, 3H), 1.78-1.72(m, 2H), 1.69-1.63(m, 2H); C 29 H 24MS(EI) for FN3O6: Measured value 530.0 (MH+).

[0601] Step 3: 4-[4-[[1-[(4-fluorophenyl)carbamoyl]cyclopropane-carbonyl]amino]phenoxy]-7-methoxyquinoline-6-carboxylic acid (7) [ka]

[0602] Compound 6 (1.8 g, 3.4 mmol, 1 equivalent) was dissolved in tetrahydrofuran (THF) (15 mL) and MeOH (15 mL), to which 2 M NaOH aqueous solution (7 mL, 4.1 equivalents) was added. The mixture was stirred at 6-13°C for 4 hours. The mixture was adjusted to approximately pH 8 with 1 M HCl aqueous solution, concentrated, and the solvent was removed. Water (50 mL) was added, and the mixture was adjusted to approximately pH 6 with 1 M HCl aqueous solution. The resulting precipitate was filtered, washed with water (2 × 10 mL), and dried under vacuum. Compound 7 was obtained (1.7 g, yield 97.0%). 1 H NMR(400MHz, DMSO-d6)δ10.22(s, 1H), 10.08(s, 1H), 8.65(d, 1H), 8.48(s, 1H), 7.77(d, 2H), 7.64(dd, 2H)7.47(s, 1H), 7.25(d, 2H), 7.15(t, 2H), 6.45(d, 1H), 3.96(s, 3H), 1.47(s, 4H);C 28 H 22 MS(EI) for FN3O6: Measured value 516.1 (MH+).

[0603] Step 4: 1-N'-(4-fluorophenyl)-1-N-[4-[7-methoxy-6-(methylcarbamoyl)quinoline-4-yl]oxyphenyl]cyclopropane-1,1-dicarbboxamide(1) [ka]

[0604] A solution of compound 7 (300 mg, 582.0 μmol, 1 equivalent), HATU (332 mg, 873.2 μmol, 1.5 equivalents), and DIEA (301 mg, 2.3 mmol, 406 μL, 4 equivalents) in DMF (10 mL) was stirred at 6-10°C for 1 hour. Methaneamine hydrochloride (79 mg, 1.2 mmol, 2.0 equivalents) was added, and the mixture was stirred at 6-10°C for 17 hours. The mixture was filtered, and the resulting filtrate was purified by preparative HPLC (column: Waters Xbridge 150 mm*25 mm*5 μm, gradient: 33-63% of acetonitrile in 10 mM NH4HCO3 aqueous solution, flow rate: 25 mL / min). Compound 1 was obtained (105.4 mg, yield 34.3%). 1 H NMR(400MHz, DMSO-d6)δ10.20(s, 1H), 10.06(s, 1H), 8.65(d, 1H), 8.61(s, 1H), 8.42-8.33(m, 1H), 7.77(d, 2H), 7.68-7.61(m, 2H), 7.51 (s, 1H), 7.25 (d, 2H), 7.19-7.11 (m, 2H), 6.46 (d, 1H), 4.02 (s, 3H), 2.84 (d, 3H)1.47 (s, 4H) 29 H 25 MS(EI) for FN4O5: Measured value 529.1 (MH+).

[0605] Example 1: Preparation of Compound 1 fumarate form A One equivalent of fumaric acid in acetone was added to one equivalent of the free base of compound 1, and the resulting reddish slurry was stirred at approximately 50°C for 4 days. Next, the slurry was subjected to a scaling cycle up to RT, and stirred for an additional day to obtain a pink slurry. The solid was then removed by positive pressure filtration to obtain a mixture of fumarate form A and free base form A.

[0606] Example 2: Preparation of Compound 1 Hemifumarate Form B Two equivalents of fumaric acid in acetone were added to one equivalent of the free base of compound 1. The resulting reddish slurry was stirred at approximately 50°C for 6 days to obtain an off-white slurry. The solid was then removed by positive pressure filtration of the high-temperature solution to obtain hemi-fumarate form B.

[0607] Example 3: Preparation of Compound 1 HCl form A One equivalent of HCl was added to the free base of compound 1 in THF, and the resulting dark reddish slurry was stirred at RT for 3 days to obtain a thick off-white slurry. The solid was then removed by positive pressure filtration to obtain HCl form A.

[0608] Example 4: Preparation of Compound 1 HCl form B One equivalent of HCl was added to the free base of compound 1 in chloroform, and the resulting reddish slurry was stirred at approximately 50°C for 3 days to obtain a pale pink slurry. The solid was then removed by positive pressure filtration to obtain HCl form B.

[0609] Example 5: Preparation of Compound 1 HCl form C One equivalent of HCl was added to the free base of compound 1 in methanol at a temperature of approximately 60°C, resulting in a yellowish slurry. The solution was then subjected to CC to approximately -20°C and kept at a low temperature for about 2 days to obtain a clear orange solution. Partial FE yielded a clear red solution, to which 4 volumes of the antisolvent MTBE were added, and the solution was stirred at RT for 1 day to obtain an off-white solid compound 1 in HCl form C, separated by positive pressure filtration.

[0610] Example 6: Preparation of Compound 1 HCl form D Two equivalents of HCl were added to the free base of compound 1 at approximately 50°C, and the resulting pink slurry was stirred at 50°C for 5 days. The solid compound 1 HCl form D was separated by positive pressure filtration.

[0611] Example 7: Preparation of Compound 1 Form A Compound 1 form A is likely the most thermodynamically stable crystalline form of the free base of Compound 1. Therefore, multiple procedures may lead to the formation of this form. A list of some possible procedures for obtaining Compound 1 form A is enumerated in the table below. This list is not intended to be exclusive, and in fact, there may be many more further procedures that produce this form.

[0612] Selected procedure for preparing compound 1 form A [Table 7]

[0613] Example 8: Preparation of Compound 1 Form B Compound 1 was dissolved in AcOH and crystallized by VD using diethyl ether as the antisolvent.

[0614] Example 9: Preparation of Compound 1 Form C Compound 1 was dissolved in HFIPA and crystallized by CP using MTBE as the antisolvent.

[0615] Example 10: Preparation of Compound 1 Form D Compound 1 was dissolved in methanol and crystallized by carbon monoxide poisoning (CC). The mixture was then slurryed at 2-8°C to obtain form D.

[0616] Example 11: Preparation of Compound 1 Form E Method A: Compound 1 was dissolved in THF and crystallized by CC.

[0617] Method B: Compound 1 was dissolved in 90:10 THF:water and precipitated by CP.

[0618] Example 12: Preparation of Compound 1 Form F Method A: Compound 1 was dissolved in chloroform and crystallized by SE.

[0619] Method B: Compound 1 was slurryed in chloroform.

[0620] Example 13: Preparation of Compound 1 Form G Compound 1 was dissolved in chloroform, and the mixture was crystallized by placing it in a freezer.

[0621] Example 14: Preparation of Compound 1 Form H Morphology H was obtained by VS of amorphous compound 1 using DCM.

[0622] Example 15: Preparation of Compound 1 Form K Compound 1, form K, was prepared by desolvation of form F or form G, which are chloroform solvates.

[0623] Example 16: Preparation of Compound 1 Form O Compound form 1, O, was discovered when forming salts with various counterions in a TFE-containing solvent system, and it is highly likely that this is a TFE solvate.

[0624] Example 17: Preparation of phosphate form A of compound 1 One molar equivalent of phosphoric acid was added to a slurry of compound 1 in chloroform, and the resulting mixture was then slurryed at approximately 50°C for 3 days. The product was isolated by positive pressure filtration.

[0625] Example 18: Preparation of Compound 1 Form I Compound 1 in a 90:10 THF / water mixture was crush-precipitated with heptane, and then stirred at freezing temperature for 7 days.

[0626] Example 19: Preparation of Compound 1 Form J Compound 1 was slurryed in acetone for 14 days.

[0627] Example 20: Preparation of Compound Form 1 L Compound 1 was slurryed in chloroform for 14 days.

[0628] Example 21: Preparation of Compound 1 Form M Dehydration of compound form 1 E in a vacuum oven at approximately 77°C for one day.

[0629] Example 22: Preparation of Compound 1 Form N Compound 1 was slurryed in a 70:30 TFE / MTBE mixture at room temperature for 7 days.

[0630] Example 23: In vivo study of the effect of compound 1 on tumor angiogenesis Animals carrying MC38 tumors were treated for 5 days with various doses of compound 1 (PO, qd; 3 mg / kg, 10 mg / kg, and 30 mg / kg of compound 1). The tumors were analyzed for the presence of tumor microvessels by CD31 staining. Figures 1A and 1B compare tumor microvessels after administration of different doses of comparison 1 with those of the vehicle. The results indicate that compound 1 inhibits in vivo angiogenesis. Compared to the vehicle, the mean number of vessels was significantly reduced after treatment with 30 mg / kg of compound 1 (26.9 vs. 17.6). A dose-dependent reduction was also observed in the presence of tumor microvessels.

[0631] Example 24: Effect of combination therapy with compound 1 on immune cells Animals carrying MC38 tumors were treated for 7 days with compound 1 (PO, qd; 10 mg / kg) and anti-PD-1 antibody (IP, days 1, 2, 4, and 6; 5 mg / kg). The tumors were analyzed for the presence of cytotoxic T cells by CD8 staining. The results indicate that the combination of compound 1 and PD-1 increased natural killer (NK) and NK-T cells within the tumors. Elevated levels of T cells and B cells in the blood were observed. Total macrophages and dendritic cells decreased, but g / mMDSC and M2 macrophages increased. It was also observed that g / mMDSC macrophages and dendritic cells decreased, but M2 macrophages in the blood increased.

[0632] Figures 2A and 2B compare the number of CD8+ cells after treatment with PD-1, compound 1+PD-1, and vehicle. (Compound 1+PD-1 at 10 mg / kg (0.5 mm) 2 The average number of CD8+ T cells after treatment with 96.1% per unit area was measured by the vehicle (0.5 mm). 2 (34.8 per unit) and PD-1 treatment (0.5 mm2 A significant increase was observed compared to 49.8%.

[0633] Example 25: Effect of combination therapy with compound 1 on tumor growth in the MC38 model Animals carrying MC38 tumors were treated with compound 1 (3 mg / kg) + PD-1 (5 mg / kg), compound 1 (3 mg / kg) + PD-L1 (5 mg / kg), and compound 1 (3 mg / kg) + CTLA-4 (5 mg / kg), and tumor volume was analyzed. Figures 3A-3C show the tumor volume after treatment with combination therapy. Compared with compound 1, PD-1, PD-L1, and CTLA-4, combination therapy significantly reduced tumor volume, significantly slowed tumor growth, or stopped tumor growth.

[0634] Example 26: Evaluation of the in vivo efficacy of Compound 1 after oral administration to female Balb / c mice carrying CT26 tumors. During cancer progression, the expression of TAM (TYRO3, AXL, MER) receptor tyrosine kinases (RTKs) across multiple cell types influences the range of exogenous cellular features in the tumor microenvironment (TME). Activation of these receptors on tumor cells leads to increased tumor growth, survival, and metastasis potential, while their activation on immune cell subtypes can result in resistance to immunosuppressive and chemotherapy regimens.

[0635] Compound 1 has been shown to be potent in vitro against MET, VEGFR2, and TAM RTK receptors, AXL, and MER. Apart from its effects on tumor cells, Compound 1 may also affect TAM RTK signaling in tumor-associated macrophages, where it inhibits efferocytosis and polarizes macrophages toward an immunopermissible M1 phenotype. Treatment with Compound 1 also resulted in reduced angiogenic capacity in TMEs, as evidenced by decreased angiogenesis and VEGFR2 inhibition. Overall, these effects have been shown to result in a significant improvement in tumor growth inhibition (TGI) when combined with an anti-PD-1 antibody.

[0636] This study investigated the inhibitory effects of compound 1, both as a monotherapy and in combination with an anti-PD-1 inhibitor, in Balb / c mice transplanted with CT26 colon cancer cells. CT26 cells inoculated into syngeneic mice are highly oncogenic (Brattain et al. 1980) and share molecular characteristics with undifferentiated invasive human colorectal cancer cells (Castle (et al. 2014). Therefore, the CT26 cell line can serve as an effective cell model for metastatic and poorly differentiated human colorectal cancer. Furthermore, this model has been reported to express significant levels of RTKs, indicating a potential dependence on these signaling pathways for maintaining the oncogenic phenotype in vivo (Pryzybyszewska et al. 2017).

[0637] method Cell line culture and maintenance CT26 colon cancer cells (ATCC, CRL-2638) were thawed from the Exelixis Pharmacological Cell Bank into T-75 flasks (Corning, 43064U) using fresh medium, i.e., RPMI-1640 (GIBCO, A384002) containing 10% fetal bovine serum. The cells were incubated at 37°C in a humidified incubator with 5% CO2 and grown to 80-90% confluence before transplantation.

[0638] transplant Mice were anesthetized with isoflurane during transplantation. 160 Balb / C mice were subcutaneously (sc) transplanted into the right posterior flank using a 25G needle attached to a 1 ml syringe, with 1 million CT26 cells in 0.1 ml of serum-free culture medium containing an equal volume of Matrigel (Corning, 354235, protein concentration 11.0 mg / ml, endotoxin level <1.5 units / ml). Cells were prepared in 50 ml tubes, stored on ice, and mixed before loading each syringe.

[0639] Randomization After randomization, 120 mice were selected using Studylog software to match an average size of 180 mm³. Mice were then selected for each treatment group. There were 5 mice per cage, and two cages were used per group.

[0640] Dosage administration Oral doses were administered via a 20ga, 1.5-inch stainless steel needle with a silicone tip (VWR, 20068-666). Mice were administered daily according to their body weight. Survival was monitored for up to 40 days. Compound 1 (EXEL-04621820), lot 11 from the Exelixis compound repository was used for administration as monotherapy or in combination with anti-PD-1 (BioXcell catalog number BE0146, clone RPMI-14, lot 780120J3).

[0641] observation Tumor volume and body weight were measured twice a week for all animals using Studylog software.

[0642] statistical methods Significance levels represent the difference compared to the vehicle or anti-PD-1 treatment group and were determined using the non-parametric Mann-Whitney U test. The significance levels depicted are *: p<.05, **: p<.01, ***: p<.001, and ****: p<.0001.

[0643] result Treatment of xenograft mice with compound 1 at doses of 1, 3, 10, and 30 mg / kg / day for 40 days resulted in delayed tumor growth. Further delay in tumor growth was observed when compound 1 treatment was combined with 5 mg / kg of anti-PD-1 (Figure 4). Survival benefits and dose-response were observed with compound 1 alone and in combination with PD-1. Statistical significance (P < 0.0001) was observed between compound 1 and the vehicle. Statistical significance (P < 0.0001) was also observed between the compound 1 + PD-1 combination and PD-1 alone. Compound 1 + PD-1 vs. compound 1 also showed statistical significance (P < 0.05). These results are shown in Figure 5.

[0644] Compound 1, when combined with PD-1 therapy, also improved conditional survival in CT26 xenografts compared to compound 1 alone or anti-PD-1 therapy. Figure 6A compares tumor growth after treatment with either the vehicle, 30 mg / kg of compound 1, 10 mg / kg of anti-PD-1, or both. Figure 6B shows Kaplan-Meier plots indicating conditional survival for CT26 tumor-bearing mice after treatment.

[0645] Example 27: In vitro effect of compound 1 on receptor tyrosine kinase inhibition The inhibitory activity of compound 1 was evaluated using a radioactive assay platform with multiple kinase catalytic activity assays. Compound 1 inhibited MET and VEGFR2, and IC2 50 The values ​​are 3.0 nM and 15 nM, respectively. Compound 1 also shows high potency against AXL and MER, and IC 50 The values ​​were 5.8 nM and 0.6 nM, respectively. All in vitro biochemical assays were performed at an ATP concentration of 10 μM.

[0646] Example 28: In vitro effect of compound 1 on inhibiting RTK autophosphorylation in human cell lines The activity of compound 1 was evaluated using cell-based assays to assess the mechanistic effects of the compound in tumor and normal cells. Consistent with biochemical data, compound 1 is a potent inhibitor of cellular RTKs, as assessed by analysis of receptor autophosphorylation measured by enzyme-linked immunosorbent assay. The assay was performed using ligand-stimulated cell lines (PC-3, HUVEC, and A-172) with their respective receptors to induce autophosphorylation (MET, VEGFR2, and AXL) or transfected cell lines (293A) with expression vectors encoding the target receptor (MER). We also investigated the inhibition of endogenous phosphorylated MET by compound 1 in Hs746T gastric cancer cells, which is attributed to the presence of a skip splice mutation in exon 14 of the MET gene. This alteration of MET exon 14 results in increased MET stability and oncogenic activation, including constitutive MET phosphorylation. This mutation is also known to occur in 3% of lung adenocarcinoma patients and is associated with sensitivity to MET-targeted therapy (Frampton et al 2015). Compound 1 is IC 50 It showed potent inhibition of MET autophosphorylation in Hs746T cells at a value of 26 nM, and therefore would be a potent therapeutic candidate for patients in this subset. 50 Table 1 shows an overview of the values. [Table 8]

[0647] Example 29: Tumor pharmacodynamics and efficacy response in a preclinical in vivo model Three human tumor cell lines, NCI-H441, MDA-MB-231, and SNU-5, were selected for use in tumor xenograft studies using athymic nude, NSG, and CB.17SCID mice, respectively. Pharmacodynamic modulation of MET phosphorylation was also measured in NCI-H441 and SNU-5 xenografts. Tumor-bearing mice were orally administered once daily for 14 days (qd×14), tumors were excised, and treated for MET phosphorylation analysis by Western blotting. In vivo, compound 1 potently inhibited MET phosphorylation in a dose-dependent manner (Table 2): [Table 9]

[0648] Compound 1 showed significant dose-dependent antitumor activity in all three xenograft models tested when administered orally daily for 14 days (Table 3), suggesting its potential for broad-spectrum antitumor activity. [Table 10]

[0649] Example 30: Absorption, Distribution, Metabolism and Excretion (ADME) The plasma pharmacokinetics (PK) of compound 1 were characterized in rats and beagle dogs. The toxicological (TK) of orally administered compound 1 was evaluated in repeated dose studies in rats and dogs. Plasma protein binding was determined in vitro in mouse, rat, dog, monkey, and human plasma. The biotransformation and metabolite profiles of compound 1 were investigated in vitro using liver microsomal fractions and hepatocytes from mice, rats, dogs, monkeys, and humans, and in vivo in rats and dogs. Transporter interactions between P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) were also tested, in vitro experiments were conducted to evaluate the induction and inhibition of cytochrome P450 (CYP) by compound 1, and the substrate specificity of compound 1 to CYP and glucuronosyltransferase (UGT) were also tested.

[0650] Pharmacokinetics (PK) and toxicological kinetics (TK) of Compound 1 in rats and dogs. PIB formulations in rats and dogs In rats and dogs, the PK and TK of compound 1 were evaluated after intravenous (IV; single dose PK only) and oral (PO) administration of compound 1 in a formulation prepared with polyethylene glycol 400 (PEG400):ethanol (EtOH):reverse osmosis water (RO water) (45:5:50 v / v / v).

[0651] The oral bioavailability (%F) of compound 1 was evaluated in rats (0.09–0.27 mg / kg) and dogs (0.2–0.6 mg / kg). The absolute oral bioavailability of compound 1 was 62–77% in rats and 47–70% in dogs. After oral administration of compound 1, the terminal phase half-life (t1 / 2, z) ranged from 4.5–5.3 hours in rats and 1–3 hours in dogs. After a single oral administration in rats and dogs, the exposure (C) of compound 1 was evaluated. max [Maximum plasma concentration] and AUC 0-inf The ...

Claims

[Claim 1] The invention described in the specification.

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