Method for inducing anti-cancer immune response

Abstract

The method, or preparing immunologically primed cancer cells using cancer cells collected from a patient, comprises treating the collected cancer cells ex vivo with a toxic concentration of a compound that alters sphingolipid metabolism, the toxic concentration being sufficient to induce immunogenic cell death in the cancer cells. In embodiments, the compound is 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide compound or a pharmaceutically acceptable salt thereof. In embodiments, the immunologically primed cancer cells overexpress calreticulin on their surface. In embodiments, the cancer cells are solid tumor cells. In embodiments, the cancer cells are circulating tumor cells. In embodiments, the method further comprises harvesting at least a portion of the immunologically primed cancer cells and suspending the cells in phosphate-buffered saline. In embodiments, the method further comprises transporting at least a portion of the immunologically primed cancer cells to a site of patient care.

Description

Background Art

[0001] Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells. There are many different types of cancer treatments, including traditional therapies (such as surgery, chemotherapy, and radiation therapy), newer forms of treatment (targeted therapy), and complementary and alternative therapies. It is becoming increasingly clear that cancer depends on multiple altered molecular pathways and can develop diverse resistance mechanisms to single-agent therapies. Therefore, combination regimens may offer the best hope for effective therapies with durable effects.

Summary of the Invention

[0002] According to aspects presented herein, an immunogenic cell death (ICD) inducer comprising a selective inhibitor of the toxic concentration of sphingosine kinase-2 (SK2) is disclosed. In embodiments, the selective inhibitor of SK2 is 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl) amide compound or a pharmaceutically acceptable salt thereof. In embodiments, the toxic concentration of the 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide compound or a pharmaceutically acceptable salt thereof is from about 35 μM to about 45 μM. In embodiments, in vitro, patient-derived cancer cells are treated with a 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide compound or a pharmaceutically acceptable salt thereof at about 35 μM to about 45 μM to result in sufficient immunogenic cell death (ICD) in the cancer cells. These primed cancer cells are used as cancer immunotherapy and can be readministered to the patient. In embodiments, these newly implanted primed cancer cells can cause large-scale ICD in untreated cancer cells within the patient.

[0003] According to the aspects shown in this specification, there is disclosed a method for preparing immunologically primed cancer cells using cancer cells collected from a patient, which comprises treating the cancer cells ex vivo with a compound at a toxic concentration that modifies sphingolipid metabolism, wherein the toxic concentration is sufficient to induce immunogenic cell death in the cancer cells. In embodiments, the compound that modifies sphingolipid metabolism is an inhibitor of sphingosine kinase. In embodiments, the compound that is an inhibitor of sphingosine kinase is a selective inhibitor of sphingosine kinase-2 (SK2). In embodiments, the selective inhibitor of SK2 is 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide or a pharmaceutically acceptable salt thereof. In embodiments, the collected cancer cells are treated for at least 24 hours. In embodiments, the toxic concentration of the selective inhibitor of SK2 is from about 20 μM to about 60 μM. In embodiments, the immunologically primed cancer cells overexpress calreticulin on their surfaces. In embodiments, the cancer cells are immune cells. In embodiments, the immune cells include T cells, natural killer (NK) cells, or dendritic cells. In embodiments, the cancer cells are blood cancer cells. In embodiments, the blood cancer cells are leukemia cells. In embodiments, the cancer cells are solid tumor cells. In embodiments, the cancer cells are circulating tumor cells. In embodiments, the method further comprises harvesting at least a portion of the immunologically primed cancer cells and suspending the cells in phosphate buffered saline. In embodiments, the method further comprises transporting at least a portion of the immunologically primed cancer cells to the patient's care location. In embodiments, the patient's care location is a hospital. In embodiments, the patient's care location is a cancer center. In embodiments, the method further comprises administering to the patient at least a portion of the transported immunologically primed cancer cells to induce an immune response. In embodiments, the immune response delays or halts the growth of cancer in the patient. In embodiments, the immune response halts cancer metastasis in the patient. In embodiments, the immune response makes the patient's immune system more efficient at killing cancer cells.In an embodiment, the method further comprises administering an effective amount of at least one checkpoint inhibitor.

[0004] According to aspects presented herein, a method of inducing an anti-cancer immune response in a patient, comprising removing cancer cells from the patient and treating the cells ex vivo with a pharmaceutically acceptable salt of 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide compound incorporated in a pharmaceutically acceptable formulation in an amount sufficient to induce, enhance, or promote immunogenic cell death in the cancer cells, and then administering the treated cells back to the original patient to elicit an immune response against the patient's cancer, is disclosed. In an embodiment, the patient's cells are blood cancer cells such as leukemia cells. In an embodiment, the patient's cells are solid tumor cells obtained from a biopsy or circulating tumor cells isolated from the patient's blood.

[0005] According to the aspects shown in this specification, an effective amount of an inhibitor of sphingosine kinase for use in a method of treating a target cancer, and an effective amount of at least one checkpoint inhibitor selected from the group consisting of a CTLA-4 receptor inhibitor, a PD-1 receptor inhibitor, a PD-L1 ligand inhibitor, a PD-L2 ligand inhibitor, a LAG-3 receptor inhibitor, a TIM-3 receptor inhibitor, a BTLA receptor inhibitor, a KIR receptor inhibitor, or any combination of the aforementioned checkpoint inhibitors are disclosed. In an embodiment, the checkpoint inhibitor is an inhibitor of the PD-L1 / PD-1 pathway. In an embodiment, the checkpoint inhibitor is an inhibitor of CTLA-4. In an embodiment, the cancer is a chemotherapy- or radiation-resistant cancer. In an embodiment, the inhibitor of sphingosine kinase is administered, and then the other inhibitor is administered within a suitable period. In an embodiment, the inhibitor of sphingosine kinase is an inhibitor of sphingosine-kinase-2. In an embodiment, the inhibitor of sphingosine-kinase-2 is 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide (ABC294640). In an embodiment, the inhibitor of the PD-L1 / PD-1 pathway is an anti-PD-L1 antibody, an anti-PD-1 antibody, or a combination thereof. In an embodiment, the anti-PD-L1 antibody or anti-PD-1 antibody is a monoclonal antibody. In an embodiment, the monoclonal antibody is a human antibody or a humanized antibody. In an embodiment, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody. In an embodiment, the anti-CTLA-4 antibody is a monoclonal antibody. In an embodiment, the monoclonal antibody is a human antibody or a humanized antibody.

[0006] According to the aspects shown in this specification, a method of treating cancer in a subject is disclosed, which includes administering an inhibitor of sphingosine kinase (SK) in an effective amount and an inhibitor of a checkpoint inhibitor in an effective amount to the subject. In an embodiment, the checkpoint inhibitor can be an antibody against CTLA4 (e.g., ipilimumab), or an antibody against PD-1 (e.g., pembrolizumab or nivolumab), or an antibody against PD-L1 (e.g., atezolizumab or durvalumab). Other antibodies or chemical inhibitors targeting these pathways are also within the scope of the present invention. For example, further inhibitors of the PD-L1 pathway include BMS-936559, MPDL3280A, BMS-936558, MK-3475, CT-011, or MEDI4736.

[0007] According to the aspects shown in this specification, a method of treating cancer in a patient is disclosed, which includes administering an inhibitor of sphingosine kinase in an effective amount and at least one of an inhibitor of the PD-L1 / PD-1 pathway or an inhibitor of CTLA-4. In an embodiment, the inhibitor of sphingosine kinase is an inhibitor of sphingosine-kinase-2. In an embodiment, the inhibitor of sphingosine-kinase-2 is 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide (ABC294640). In an embodiment, the inhibitor of the PD-L1 / PD-1 pathway is an anti-PD-L1 antibody, an anti-PD-1 antibody, or a combination thereof. In an embodiment, the anti-PD-L1 antibody or anti-PD-1 antibody is a monoclonal antibody. In an embodiment, the monoclonal antibody is a human antibody or a humanized antibody. In an embodiment, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody. In an embodiment, the anti-CTLA-4 antibody is a monoclonal antibody. In an embodiment, the monoclonal antibody is a human antibody or a humanized antibody.

[0008] According to the aspects presented in this specification, there is disclosed a method of treating melanoma in a patient in need thereof, comprising administering to the patient an effective amount of an inhibitor of sphingosine kinase and a CTLA-4 inhibitor. In embodiments, the inhibitor of sphingosine kinase is an inhibitor of sphingosine-kinase-2. In embodiments, the inhibitor of sphingosine-kinase-2 is 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide (ABC294640). In embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody. In embodiments, the anti-CTLA-4 antibody is a monoclonal antibody. In embodiments, the monoclonal antibody is a human antibody or a humanized antibody. In embodiments, treating melanoma is further defined as reducing the size of the tumor or inhibiting the growth of the tumor. In embodiments, the inhibitor is administered to a patient in need thereof at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In embodiments, a second cancer therapy is further administered to the patient. In embodiments, the second cancer therapy includes surgery, radiation therapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, or gene therapy. In embodiments, the melanoma is chemotherapy- or radiation-resistant melanoma.

[0009] According to the aspects shown herein, a method of treating melanoma in a patient in need of treatment of melanoma is disclosed, which includes administering to the patient an effective amount of an inhibitor of sphingosine kinase and an inhibitor of the PD-L1 / PD-1 pathway. In an embodiment, the inhibitor of sphingosine kinase is an inhibitor of sphingosine-kinase-2. In an embodiment, the inhibitor of sphingosine-kinase-2 is 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide (ABC294640). In an embodiment, the inhibitor of the PD-L1 / PD-1 pathway is an anti-PD-L1 antibody, an anti-PD-1 antibody, or a combination thereof. In an embodiment, the anti-PD-L1 antibody or the anti-PD-1 antibody is a monoclonal antibody. In an embodiment, the monoclonal antibody is a human antibody or a humanized antibody. In an embodiment, treating melanoma is further defined as reducing the size of the tumor or inhibiting the growth of the tumor. In an embodiment, the inhibitor is administered at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In an embodiment, the patient is further administered a second cancer therapy. In an embodiment, the second cancer therapy includes surgery, radiation therapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, or gene therapy. In an embodiment, the melanoma is chemotherapy- or radiation-resistant melanoma.

[0010] According to the aspects shown herein, a method of treating melanoma in a patient in need of treatment of melanoma, the method comprising administering an effective amount of 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide (ABC294640) and an inhibitor of the PD-L1 pathway is disclosed.

[0011] According to the aspects presented in this specification, a method for treating a patient suffering from lung cancer, comprising administering to a patient in need thereof, in combination with an inhibitor of sphingosine kinase administered orally, a therapeutically effective amount of an anti-cancer agent that is an antibody or an antigen-binding portion thereof that specifically binds to the PD-1 receptor and inhibits PD-1 activity (the "anti-PD-1 antibody or an antigen-binding portion thereof") by infusion for less than 60 minutes, is disclosed. In an embodiment, the inhibitor of sphingosine kinase is an inhibitor of sphingosine-kinase-2. In an embodiment, the inhibitor of sphingosine-kinase-2 is 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide (ABC294640).

[0012] According to the aspects presented in this specification, a method for treating a patient suffering from lung cancer, comprising administering to a patient in need thereof, in combination with a therapeutically effective amount of an inhibitor of sphingosine kinase administered orally, a flat dose of a therapeutically effective amount of an anti-cancer agent that is an antibody or an antigen-binding portion thereof that specifically binds to the PD-1 receptor and inhibits PD-1 activity, is disclosed. In an embodiment, the inhibitor of sphingosine kinase is an inhibitor of sphingosine-kinase-2. In an embodiment, the inhibitor of sphingosine-kinase-2 is 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide (ABC294640).

[0013] According to the aspects shown in this specification, a method for treating lung cancer in a patient, comprising administering to a patient in need thereof an effective amount of an inhibitor of sphingosine kinase and an inhibitor of anti-CTLA-4, is disclosed. In an embodiment, the inhibitor of sphingosine kinase is an inhibitor of sphingosine-kinase-2. In an embodiment, the inhibitor of sphingosine-kinase-2 is 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide (ABC294640). In an embodiment, the inhibitor of anti-CTLA-4 is an anti-CTLA-4 antibody. In an embodiment, the anti-CTLA-4 antibody is a monoclonal antibody. In an embodiment, the monoclonal antibody is a human antibody or a humanized antibody. In an embodiment, the anti-CTLA-4 monoclonal antibody is ipilimumab. In an embodiment, treating lung cancer is further defined as reducing the size of the tumor or inhibiting the growth of the tumor. In an embodiment, the inhibitor is administered at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In an embodiment, a second cancer therapy is further administered to the patient. In an embodiment, the second cancer therapy includes surgery, radiation therapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, or gene therapy.

[0014] According to the aspects shown in this specification, a method for treating lung cancer in a patient, comprising administering to the patient an effective amount of 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide (ABC294640) and an inhibitor of anti-CTLA-4, is disclosed. In an embodiment, the inhibitor of anti-CTLA-4 is an anti-CTLA-4 antibody. In an embodiment, the anti-CTLA-4 antibody is a monoclonal antibody. In an embodiment, the anti-CTLA-4 monoclonal antibody is ipilimumab.

[0015] According to the aspects shown in this specification, a kit for preparing cells for immunogenic cell death is disclosed. The kit contains a compound at a toxic concentration that modifies sphingolipid metabolism, the toxic concentration being sufficient to induce immunogenic cell death in cancer cells, and includes a set of instructions for use. In an embodiment, the compound that modifies sphingolipid metabolism is an inhibitor of sphingosine kinase. In an embodiment, the compound that is an inhibitor of sphingosine kinase is a selective inhibitor of sphingosine kinase-2 (SK2). In an embodiment, the selective inhibitor of SK2 is 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl) amide or a pharmaceutically acceptable salt thereof. In an embodiment, the toxic concentration of the selective inhibitor of SK2 is from about 20 μM to about 60 μM.

[0016] According to the aspects presented in this specification, a kit for treating tumors is disclosed. The kit can include at least one checkpoint inhibitor compound, 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide or a pharmaceutically acceptable salt thereof, and a set of instructions for use. In an embodiment, the instructions for use include a label indicating a method of administering the inhibitor, including the route of administration, the dosage, and the duration of administration. In some forms of the kit, 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide or a pharmaceutically acceptable salt thereof is stored in a container separate from at least one immune checkpoint inhibitor compound. In some forms of the kit, at least one immune checkpoint inhibitor compound is a CTLA-4 receptor inhibitor, a PD-1 receptor inhibitor, a PD-L1 inhibitor, or a PD-L2 inhibitor, an LAG-3 receptor inhibitor, a TIM-3 receptor inhibitor, a BTLA receptor inhibitor, a KIR receptor inhibitor, or any combination of the aforementioned immune checkpoint inhibitor compounds. In some forms of the kit, the immune checkpoint inhibitor compound is an antibody or an antibody fragment. In some forms of the kit, at least one immune checkpoint inhibitor compound is an anti-CTLA-4 receptor antibody, an anti-PD-1 receptor antibody, an anti-LAG-3 receptor antibody, an anti-TIM-3 receptor antibody, an anti-BTLA receptor antibody, an anti-KIR receptor antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody, or any combination of the aforementioned antibodies. In some forms of the kit, at least one immune checkpoint inhibitor compound is in the form of a lyophilized solid. In some forms, the kit further includes an aqueous reconstitution solvent. In some forms of the kit, at least one immune checkpoint inhibitor compound is incorporated into a first pharmaceutically acceptable formulation, and 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide is incorporated into a second pharmaceutically acceptable formulation.

[0017] According to the aspects presented in this specification, a kit for treating a subject suffering from lung cancer, comprising an antibody or an antigen-binding portion thereof that specifically binds to the PD-1 receptor and inhibits PD-1 activity, with a flat dosage of at least about 240 mg, an inhibitor of sphingosine kinase in a dosage, and instructions for using the anti-PD-1 antibody or its antigen-binding portion and the inhibitor of sphingosine kinase in the methods of the present disclosure, is disclosed. In an embodiment, the instructions for use include a label indicating a method of administering the anti-PD-1 antibody or its antigen-binding portion and the inhibitor of sphingosine kinase, including the route of administration, dosage, and duration of administration. In an embodiment, the kit further comprises a dosage of another anti-cancer agent that is a dosage in the range of 0.1 to 10 mg / kg body weight of an antibody or an antigen-binding portion thereof that specifically binds to CTLA-4 and inhibits CTLA-4, and instructions further describing the method of using the anti-CTLA-4 antibody or its antigen-binding fragment.

[0018] According to the aspects presented in this specification, a kit for treating a subject suffering from lung cancer, comprising a dosage in the range of 0.1 to 10 mg / kg body weight of an anti-cancer agent that is an antibody or an antigen-binding portion thereof that specifically binds to the PD-1 receptor and inhibits PD-1 activity, a dosage of an inhibitor of sphingosine kinase, and instructions for using the anti-PD-1 antibody or its antigen-binding portion and the inhibitor of sphingosine kinase in the methods of the present disclosure, is disclosed. In an embodiment, the kit further comprises a dosage of another anti-cancer agent that is a dosage in the range of 0.1 to 10 mg / kg body weight of an antibody or an antigen-binding portion thereof that specifically binds to CTLA-4 and inhibits CTLA-4, and instructions further describing the method of using the anti-CTLA-4 antibody or its antigen-binding fragment.

[0019] According to aspects of the present disclosure, the cancer to be treated is selected from the group consisting of melanoma, cutaneous T-cell lymphoma, non-Hodgkin lymphoma, mycosis fungoides, Pagetoid reticulosis, Sézary syndrome, granulomatous slack skin, lymphomatoid papulosis, pityriasis lichenoides chronica, pityriasis lichenoides et varioliformis acuta, CD30+ cutaneous T-cell lymphoma, secondary cutaneous CD30+ large cell lymphoma, non-mycosis fungoides CD30 cutaneous large T-cell lymphoma, polymorphic T-cell lymphoma, Lennert lymphoma, subcutaneous T-cell lymphoma, angiocentric lymphoma, blastic NK-cell lymphoma, B-cell lymphoma, Hodgkin lymphoma (HL), head and neck tumors, squamous cell carcinoma, rhabdomyocarcoma, non-small cell lung cancer, small cell lung cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, renal cell carcinoma (RCC), colorectal cancer (CRC), acute myeloid leukemia (AML), breast cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, urothelial cancer, bladder cancer, gastric cancer, prostate small cell neuroendocrine carcinoma (SCNC), liver cancer, sarcoma, glioblastoma, liver cancer, oral squamous cell carcinoma, pancreatic cancer, kidney cancer, papillary thyroid cancer, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, bone cancer, metastasis, and nasopharyngeal cancer. In an embodiment, treating the cancer is further defined as reducing the size of the tumor or inhibiting the growth of the tumor.

[0020] According to aspects of the present disclosure, the inhibitor can be administered to a subject in need thereof at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In an embodiment of the present disclosure, a second cancer therapy is further administered to a subject in need thereof. In an embodiment, the second cancer therapy includes surgery, radiation therapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, or gene therapy. In an embodiment, the cancer is chemotherapy-resistant cancer or radiation-resistant cancer.

Brief Description of the Drawings

[0021] [Fig. 1]Administration of B16 melanoma cells treated with ABC294640 induces immunity against subsequently injected untreated B16 tumor cells, demonstrating that ABC294640 induces immunogenic cell death in tumor cells. In the "box & whisker" plots shown in FIGS. 1, 2, and 3A - 3C, the median tumor volume is indicated by the horizontal line within each bar. The bar range indicates the interquartile range, and the whiskers indicate the range between the smallest and largest tumors for each treatment group. [Fig. 2] Administration of Neuro - 2a neuroblastoma cells treated with ABC294640 induces immunity against subsequently injected untreated Neuro - 2a tumor cells, providing further evidence that ABC294640 induces immunogenic cell death in tumor cells. [Fig. 3A] Administration of Lewis lung carcinoma (LLC) cells treated with ABC294640 induces immunity against subsequently injected untreated LLC tumor cells. Tumor sizes are shown on day 15 (FIG. 3A), day 17 (FIG. 3B), and day 20 (FIG. 3C), demonstrating that administration of ABC294640 - treated cells causes a continuous suppression of tumor growth by untreated tumor cells. This provides further evidence that ABC294640 induces immunogenic cell death in tumor cells. [Fig. 3B] Administration of Lewis lung carcinoma (LLC) cells treated with ABC294640 induces immunity against subsequently injected untreated LLC tumor cells. Tumor sizes are shown on day 15 (FIG. 3A), day 17 (FIG. 3B), and day 20 (FIG. 3C), demonstrating that administration of ABC294640 - treated cells causes a continuous suppression of tumor growth by untreated tumor cells. This provides further evidence that ABC294640 induces immunogenic cell death in tumor cells. [Fig. 3C]Administration of Lewis lung carcinoma (LLC) cells treated with ABC294640 induces immunity against subsequently injected untreated LLC tumor cells. Tumor sizes are shown on day 15 (Figure 3A), day 17 (Figure 3B), and day 20 (Figure 3C), demonstrating that administration of ABC294640-treated cells causes a continuous suppression of tumor growth by untreated tumor cells. This is further evidence that ABC294640 induces immunogenic cell death in tumor cells. [Fig. 4] Administration of B16 melanoma or Lewis lung carcinoma (LLC) cells treated with ABC294640 induces immunity against subsequently injected untreated B16 tumor cells. This is evidence that ABC294640 induces cross-immunity. [Fig. 5] Administration of B16 melanoma or Lewis lung carcinoma (LLC) cells treated with ABC294640 induces immunity against subsequently injected untreated LLC tumor cells. This is further evidence that ABC294640 induces cross-immunity. [Fig. 6] Treatment of mice with either ABC294640 (ABC) alone or anti-PD-1 antibody alone shows that the growth of B16 melanoma tumors is partially inhibited. Treatment of mice with the combination of ABC294640 and anti-PD-1 antibody significantly increased the suppression of tumor growth. Symbols indicate mean tumor volume, and error bars indicate the standard error of the mean for each treatment group at the indicated time. [Fig. 7] Treatment of mice with either ABC294640 (ABC) alone or anti-PD-1 antibody alone shows that the survival of mice with B16 melanoma tumors is slightly extended. Treatment of mice with the combination of ABC294640 and anti-PD-1 antibody significantly increased the survival period of mice with tumors. [Fig. 8]Treatment of mice with either ABC294640 (ABC) alone or anti-CTLA4 antibody alone shows that the growth of LLC lung tumors is partially inhibited. Treatment of mice with a combination of ABC294640 and anti-CTLA4 antibody significantly increased the suppression of tumor growth. [Fig. 9] Treatment of mice with either ABC294640 (ABC) alone or anti-CTLA4 antibody alone shows that the survival of mice with LLC tumors is slightly prolonged. Treatment of mice with a combination of ABC294640 and anti-CTLA4 antibody significantly increased the survival period of mice with tumors.

Mode for Carrying Out the Invention

[0022] The sphingosine kinase (SK) inhibitors of the present disclosure are used in combination with one or more other anti-cancer therapies. Such other drugs may be administered simultaneously or sequentially with the SK inhibitors of the present invention via a certain route and in the amounts generally used therefor. When the SK inhibitors of the present invention are used simultaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the SK inhibitors of the present invention is preferred. Thus, the pharmaceutical compositions of the present invention include those that also contain one or more other active ingredients or therapeutic agents in addition to the SK inhibitors of the present invention. Examples of other therapeutic agents that may be combined with the SK inhibitors of the present invention, either administered separately or in the same pharmaceutical composition, include, but are not limited to, antibodies against CTLA-4, PD1, or PD-L1. The weight ratio of the SK inhibitor of the present invention to the second active ingredient may vary and will depend on the effective dosage of each component. Generally, the effective dosage of each will be used. The combination of the SK inhibitor of the present invention with other active ingredients will generally be within the ranges described above, but in each case, the effective dosage of each active ingredient needs to be used. In embodiments, the SK inhibitor is used in combination with a checkpoint inhibitor. In embodiments, the SK inhibitor is CTLA-4 (CD152), PD-1 (CD279), PDL-1 (CD274), TIM-3, LAG-3 (CD223), VISTA, KIR, NKG2A, BTLA, PD-1H, TIGIT, CD96, 4-1BB (CD137), 4-1BBL (CD137L), GARP, CSF-1R, A2AR, CD73, CD47, tryptophan 2,3-dioxygenase (TDO), or indoleamine 2,3-dioxygenase (IDO) in combination with one or more of the compounds that block the activity of.

[0023] The term To make the present disclosure more readily understood, certain terms are first defined. Unless otherwise explicitly provided herein, each of the following terms, as used in this application, shall have the meaning set forth below. Additional definitions are set forth throughout the present application.

[0024] As used herein, the terms "a", "an", "the", "at least one", and "one or more" are used interchangeably.

[0025] "Administering" refers to physically introducing a composition comprising an inhibitor of the invention to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Preferred routes of administration of the anti-PD-1 antibody include intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, or other parenteral routes of administration, such as injection or infusion. As used herein, the phrase "parenteral administration" means a mode of administration other than enteral and topical administration usually by injection and includes, but is not limited to, intravenous, intramuscular, arterial, intrathecal, lymphatic, intralesional, intracranial, intraorbital, intracardiac, intradermal, intraperitoneal, transcutaneous tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, epidural and intrasternal injection and infusion, as well as in vivo electroporation. The SK inhibitors of the present invention are typically administered by a non-parenteral route and preferably by oral administration. Other non-parenteral routes include topical, epidermal, or mucosal routes, such as intranasal, intravaginal, rectal, sublingual, or topical. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods.

[0026] As used in the present invention, the term "agent" refers to a compound having a pharmacological activity or effect on a patient. The terms "agent", "compound", and "drug" are used interchangeably herein.

[0027] "Alleviating" refers to any reduction in the scope, severity, frequency, and / or likelihood of the symptoms or clinical sign characteristics of a particular condition.

[0028] The "cancer cells treated with an SK inhibitor" or "cancer cells treated with ABC296460" of the present invention will have increased expression of calreticulin on the surface of the treated cells. Without being bound by theory, the overexpression of calreticulin is thought to act as at least one of the neoantigens that promote the immune response. The surface expression of calreticulin can be measured by flow cytometry of the cells prior to injection, and the cells can be sorted into subsets with high expression of calreticulin in order to optimize the immune response. The cancer cells treated with an SK inhibitor or the cancer cells treated with ABC294640 prepared by the methods disclosed herein are particularly effective in inducing an anti-cancer agent immune response.

[0029] Calreticulin, also known as calregulin, CRP55, CaBP3, calsecuestrin-like protein, and endoplasmic reticulum resident protein 60 (ERp60), is a multifunctional soluble protein that binds to Ca 2+ ions (a second messenger in signal transduction) and inactivates it.

[0030] An "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen and includes at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain includes a heavy chain variable region (abbreviated herein as Y H and a heavy chain constant region. The heavy chain constant region includes three constant domains, Cm, Cm, and m-. Each light chain includes a light chain variable region (abbreviated herein as YL) and a light chain constant region. The light chain constant region includes one constant domain CL. V # and the YL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) that are interspersed among more conserved regions called framework regions (FRs). Each Y HAnd YL comprises three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0031] "Antibody fragment" refers to a sub - portion of an antibody that retains at least a part of the binding function of the parental antibody to a ligand.

[0032] "Antibody derivative" refers to a chemically modified form of an antibody or an antibody fragment. Some examples of derivatives include the attachment of other functional molecules such as PEG groups, peptides, proteins, or other antibodies.

[0033] The term "immunogenic cell death" ("ICD") refers to any type of cell death that elicits an immune response. ICD is accompanied by changes in the composition of the cell surface.

[0034] "Known to cause immunogenic cell death in vitro" means the toxic amount of a compound, such as an SK inhibitor like compound ABC294640, selected to cause the death of at least 75% of tumor cells. In embodiments, the concentration of an SK inhibitor, such as ABC294640, known to cause cell death in vitro is from about 10 μM to about 100 μM, from about 20 μM to about 60 μM, from about 25 μM to about 55 μM, from about 30 μM to about 50 μM, from about 35 μM to about 45 μM. In embodiments, the concentration of an SK inhibitor, such as ABC294640, known to cause immunogenic cell death in vitro is 40 μM.

[0035] The "ex vivo" method disclosed herein means treating cells collected from a patient with a compound primed to be able to modify sphingolipid metabolism in vitro and then returned to the patient's body.

[0036] The term "monoclonal antibody" ("mAh") refers to a preparation of antibody molecules of a single molecular composition, i.e., an unnatural preparation of antibody molecules that have essentially the same primary sequence and exhibit a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are examples of isolated antibodies. MAbs can be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0037] Monoclonal antibodies, antibody fragments, and antibody derivatives for blocking immune checkpoint pathways can be prepared by any of several methods known to those skilled in the art, including but not limited to somatic cell hybridization techniques and hybridoma methods. Hybridoma generation is described in Antibodies, A Laboratory Manual, Harlow and Lane, 1988, Cold Spring Harbor Publications, New York. Human monoclonal antibodies can be identified and isolated, for example, by screening phage display libraries of human immunoglobulin genes by the methods described in U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,698, 6,582,915, and 6,593,081. Monoclonal antibodies can be prepared using the general methods described in U.S. Patent No. 6,331,415 (Cabilly).

[0038] "Neoantigen" is a unique molecule or protein that helps immune cells identify and fight cancer cells. In embodiments, treatment of patient-derived cancer cells in vitro with an SK inhibitor, e.g., at a toxic concentration of ABC294640, results in overexpression of calreticulin on the surface of the treated cancer cells. These treated ("primed") cancer cells can then be administered to a patient to help fight / treat the cancer.

[0039] A "human" antibody (HuMAb) refers to an antibody having a variable region in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Further, when the antibody includes a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random mutagenesis or site-directed mutagenesis in vitro, or somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used synonymously. As an example, human monoclonal antibodies can be prepared using XenoMouse™ (Abgenix, Freemont, Calif.) or hybridomas of B cells from XenoMouse. XenoMouse is a mouse host having functional human immunoglobulin genes as described in U.S. Patent No. 6,162,963 (Kucherlapati).

[0040] A "humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody have been replaced with the corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions are unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are tolerated as long as they do not interfere with the ability of the antibody to bind to a particular antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody.

[0041] A "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0042] An "anti-antigen" antibody refers to an antibody that specifically binds to an antigen. For example, an anti-PD-1 antibody specifically binds to PD-1, and an anti-CTLA-4 antibody specifically binds to CTLA-4.

[0043] "Block", "blocking", "blockade", and their variants have the same meaning as "inhibit", "inhibiting", "inhibition", and their variants. The term "blockade" is meant to encompass both partial blockade and complete blockade.

[0044] "Cell-mediated immune activity" refers to a biological activity that is considered part of a cell-mediated immune response, such as, for example, an increase in the production of at least one T HI cytokine.

[0045] A "checkpoint inhibitor" or "immune checkpoint inhibitor" includes any agent that enhances the immune system or immune response. Such inhibitors can include small molecules, peptides, polypeptides, proteins, antibodies, antibody fragments or their antigen-binding fragments that bind to and block or inhibit an immune checkpoint receptor, or antibodies that bind to and block or inhibit an immune checkpoint receptor ligand. Exemplary checkpoint molecules that can be targeted for blockade or inhibition include CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (belonging to the CD2 molecule family, all NK, γδ, and memory CD8 +(expressed in (αβ)), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR and various B-7 family ligands, including but not limited to these. Examples of B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7. Checkpoint inhibitors include antibodies or antigen-binding fragments thereof, other binding proteins, biological therapeutics or small molecules that bind to one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049 and block or inhibit their activity. Exemplary immune checkpoint inhibitors include tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-Hl; MEDI4736), MK-3475 (PD-1 blocker), nivolumab (anti-PD1 antibody), CT-011 (anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody), MSB0010718C (anti-PDL1 antibody), and Yervoy / ipilimumab (anti-CTLA-4 checkpoint inhibitor). Checkpoint protein ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.

[0046] "Immune cells" refers to cells that are directly or indirectly involved in the immune system, i.e., the generation or maintenance of an immune response, regardless of whether the immune response is natural, acquired, humoral, or cell-mediated.

[0047] "Presumed" and its variations refer to any measurable increase in cell activity. For example, induction of an immune response can include, for example, production of cytokines, activation, proliferation, or maturation of immune cell populations, and / or other indicators of increased immune function.

[0048] As used herein, the terms "subject" or "patient" are synonymous and refer to human adults, children, or infants.

[0049] Programmed death-1 (PD-1) is an important immune checkpoint receptor that is expressed by activated T cells and B cells and mediates immunosuppression. PD-1 is a member of the CD28 family of receptors, including CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1, programmed cell death ligand-1 (PD-L1, CD274, B7-H1) and programmed cell death ligand-2 (PD-L2, CD273, B7-DC), have been identified. PD-L1 and PD-L2 have been shown to downregulate T cell activation and cytokine secretion when bound to PD-1, which is expressed on antigen-presenting cells and many human cancers. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found at GenBank accession number U64863. Inhibition of the PD-1 / PD-L1 interaction mediates potent antitumor activity in preclinical models (U.S. Pat. Nos. 8,008,449 and 7,943,743), and the use of antibody inhibitors of the PD-1 / PD-L1 interaction for treating cancer is in clinical trials (Brahmer et al, 2010, Topalian et al, 2012a, Topalian et al, 2014, Hamid et al., 2013, Brahmer et al, 2012, Flies et al, 2011, Pardoll, 2012, Hamid and Carvajal, 2013).

[0050] Blocking of PD-1 / PD-L1 ligation using antibodies against PD-L1 has been shown to restore and enhance T cell activation in many systems. Patients with progressive cancer effects benefit from treatment with monoclonal antibodies against PD-L1. Preclinical animal models of tumors and chronic infections have shown that blockade of the PD-1 / PD-L1 pathway by monoclonal antibodies can enhance the immune response and control tumor rejection or infection. Anti-tumor immunotherapy by blockade of PD-1 / PD-L1 can enhance the therapeutic immune response against many histologically different tumors.

[0051] Examples of PD-1 / PD-L1 inhibitors currently marketed in the United States include pembrolizumab (Keytruda®, Merck), nivolumab (Opdivo®, Bristol-Myers Squibb), atezolizumab (Tecentriq®, Roche), avelumab (Bavencio®, EMD and Pfizer), and durvalumab (Imfinzi®, AstraZeneca). Any of these PD-1 / PD-L1 inhibitors can be used in combination with the SK inhibitor of the present invention.

[0052] Nivolumab (formerly named 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitory antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking downregulation of anti-tumor T cell function (U.S. Patent No. 8,008,449, Wang et al., 2014). Nivolumab has shown activity in various advanced solid tumors, including renal cell carcinoma (renal adenocarcinoma or adrenal tumor), melanoma, and non-small cell lung cancer (NSCLC) (Topalian et al, 2012a, Topalian et al., 2014, Drake et al, 2013, International Publication No. 2013 / 173223).

[0053] Ipilimumab (YERVOY®) is the first checkpoint antibody approved by the FDA in 2011 and, as disclosed in U.S. Patent No. 6,984,720, blocks the binding of CTLA-4 to B7 ligands, thereby stimulating T cell activation and improving the overall survival (OS) of patients with advanced melanoma (Hodi et al, 2010). It is a fully human IgG1 monoclonal antibody. Ipilimumab is approved for the treatment of melanoma at a dose of 3 mg / kg given intravenously every 3 weeks for 4 doses. Thus, in a preferred form, 3 mg / kg is the maximum dose of ipilimumab used in combination with an anti-PD-1 antibody. However, in certain forms, anti-CTLA-4 antibodies such as ipilimumab can be administered in the range of about 0.3 to 10 mg / kg body weight every 2 or 3 weeks when combined with nivolumab. Doses of ipilimumab that are significantly less than the approved 3 mg / kg every 3 weeks, for example, 0.3 mg / kg or less every 3 or 4 weeks, are considered sub-therapeutic. The combination dose of 3 mg / kg nivolumab and 3 mg / kg ipilimumab exceeds the MTD in the melanoma population, but the combinations of 1 mg / kg nivolumab + 3 mg / kg ipilimumab or 3 mg / kg nivolumab + 1 mg / kg ipilimumab have been shown to be tolerable in melanoma patients (Wolchok et al., 2013). Thus, nivolumab is tolerated up to a maximum of 10 mg / kg given intravenously every 2 weeks, but in a preferred form, the dose of the anti-PD-1 antibody does not exceed 3 mg / kg when combined with ipilimumab. In certain forms, based on risk-benefit and PK-PD evaluations, the doses used include the combinations of 1 mg / kg nivolumab + 3 mg / kg ipilimumab, 3 mg / kg nivolumab + 1 mg / kg ipilimumab, or 3 mg / kg nivolumab + 3 mg / kg ipilimumab, each administered at a dosing frequency of once every 2 to 4 weeks, preferably once every 3 weeks.In certain other embodiments, nivolumab is administered at a dosage of 0.1, 0.3, 1, 2, 3, or 5 mg / kg in combination with ipilimumab administered at a dosage of 0.1, 0.3, 1, 2, 3, or 5 mg / kg, once every two weeks, once every three weeks, or once every four weeks.

[0054] As used herein, the term "PD-1 antibody" refers to an antibody that antagonizes lymphocyte activity and / or proliferation by antagonizing PD-1. The term "antagonize activity" relates to a decrease (or reduction) in lymphocyte proliferation activity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. The term "antagonize" can be used interchangeably with the terms "inhibitory" and "inhibit". PD-1 mediated activity can be quantitatively determined using the T cell proliferation assay described herein.

[0055] A "pharmaceutically acceptable formulation" is capable of delivering a therapeutically effective amount of a compound of the present disclosure to a subject by a selected route of administration and generally is acceptable to the subject and can have an acceptable toxicity profile (preferably being non-toxic to minimally toxic at the administered dosage). Suitable pharmaceutically acceptable formulations are described in Remington’s Pharmaceutical Sciences, 18 th Edition (1990), Mack Publishing Co. and can be readily selected by one of ordinary skill in the art.

[0056] "Pharmaceutically acceptable salts" refers to derivatives of a compound modified by converting at least one acidic or basic group in the compound into a non-toxic salt form. Examples of "pharmaceutically acceptable salts" are described in Berge in Journal of Pharmaceutical Science (1977), 66, pages 1-19 and include acid addition salts and base addition salts. Acid addition salts include mineral or organic acid salts of basic moieties (such as amine groups) in the compound. Suitable acid addition salts include, for example, those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid. Suitable acid addition salts derived from organic acids include monocarboxylic and dicarboxylic acids (such as acetic acid, propionic acid), hydroxyalkanoic acids (such as citric acid, tartaric acid), aromatic acids (such as benzoic acid, xinofoic acid, pamoic acid), aliphatic and aromatic sulfonic acids (such as para-toluenesulfonic acid). Base addition salts include alkaline earth mineral salts and organic amine salts of acidic moieties (such as carboxylic acid groups) in the compound. Suitable base addition salts include sodium, potassium, magnesium, calcium salts, etc. Additional suitable base addition salts include non-toxic organic amines such as choline, ethylenediamine, etc.

[0057] When used in the present invention, the term "suitable period" refers to the period that begins when a subject starts treatment for the diagnosis of cancer using the methods of the present disclosure and continues through the treatment until the subject stops treatment. In embodiments, the suitable period is one week. In embodiments, the suitable period is from one week to two weeks. In embodiments, the suitable period is two weeks. In embodiments, the suitable period is from two weeks to three weeks. In embodiments, the suitable period is three weeks. In embodiments, the suitable period is from three weeks to four weeks. In embodiments, the suitable period is four weeks. In embodiments, the suitable period is from four weeks to five weeks. In embodiments, the suitable period is five weeks. In embodiments, the suitable period is from five weeks to six weeks. In embodiments, the suitable period is six weeks. In embodiments, the suitable period is from six weeks to seven weeks. In embodiments, the suitable period is seven weeks. In embodiments, the suitable period is from seven weeks to eight weeks. In embodiments, the suitable period is eight weeks. In embodiments, the suitable period is at least two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months. In embodiments, the suitable period is at least one year.

[0058] As used herein, the term "synergistic" refers to the modulated or correlated action of two or more agents of the present invention such that the combined action is greater than the sum of the individual actions acting separately. In embodiments, the agents of the present invention, when administered together as part of a treatment regimen, provide a therapeutic synergistic effect without concomitant synergistic side effects (e.g., cross-reactive agents, by way of non-limiting example).

[0059] "Therapeutically effective amount" or "therapeutically effective dosage" means an amount that alleviates at least one symptom or clinical sign of a tumor. Alleviation of at least one symptom or clinical sign of a tumor can include a decrease in tumor size, stabilization of tumor size or growth, a decrease in the rate of tumor growth, an increase in tumor necrosis, changes in tumor structure such as disintegration, changes in biochemical markers associated with a decrease in tumor formation, a decrease in tumor progression, or a decrease in tumor survival.

[0060] As used in the present invention, "treating cancer", "treatment", and "treat" include, but are not limited to, preventing or reducing the development of cancer, reducing the symptoms of cancer, suppressing or inhibiting the growth of established cancer, preventing the metastasis and / or invasion of existing cancer, promoting or inducing the regression of cancer, inhibiting or suppressing the proliferation of cancerous cells, reducing angiogenesis, killing malignant or cancerous tumor cells, or increasing the amount of apoptotic cancer cells.

[0061] "A patient in need of treatment", as used herein, means a patient identified as being in need of treatment. For example, a patient in need of cancer treatment is a patient identified as having cancer or at risk of developing cancer. A patient can be diagnosed as being in need of treatment by a healthcare professional and / or by performing one or more diagnostic assays. For example, a patient in need of cancer treatment can be a patient diagnosed with cancer or diagnosed as being at risk of cancer by a healthcare professional. Diagnostic assays for assessing whether a patient has cancer or is at risk of developing cancer are known in the art.

[0062] The use of the term "flat dose" with respect to the methods and dosages of the present invention means a dosage administered to a patient without consideration of the patient's body weight or body surface area (BSA). Thus, a flat dose is provided as an absolute amount of the drug (e.g., an anti-PD-1 antibody) rather than as a mg / kg dosage. For example, a 60 kg human and a 100 kg human would receive the same dosage of antibody (e.g., 240 mg of anti-PD1 antibody).

[0063] As used herein, the term "weight-based dosage" means that the dosage administered to a patient is calculated based on the patient's weight. For example, if a patient weighing 60 kg requires an anti-PD-1 antibody at 3 mg / kg, the appropriate amount of the anti-PD-1 antibody for administration (i.e., 180 mg) can be calculated and used.

[0064] An increase in at least one cell-mediated immune response of a cell population containing tumor cells refers to an increase in at least one biochemical marker, histological marker, or immunological marker associated with an improvement in the immunological profile of the tumor microenvironment. Markers whose increased amounts are associated with an improvement in the immunological profile of the tumor microenvironment include chemokines such as interferon α, interferon γ, interferon-inducible protein, TNF-α, CCL2, CCL3, CCL4, CXCL2, activated T cells, activated B cells, activated NK-cells, tumor-specific T cells, activated tumor-associated macrophages, chemokine receptors such as CCR6, or tumor-associated lymphocyte aggregates, but are not limited thereto.

[0065] Markers associated with the tumor microenvironment can be determined, for example, by analysis of a biopsy (e.g., needle biopsy) from a tumor, local tumor region, or lymph nodes in the tumor draining region. Analysis of the markers can be performed using standard techniques such as histology (H&E staining), flow cytometry, gene expression assays (quantitative PCR), immunochemical techniques, and other techniques well known to those skilled in the art.

[0066] As used herein, the term "in vitro" refers to procedures performed in an artificial environment, for example, but not limited to, in a test tube or cell culture system. One of ordinary skill in the art will understand, for example, that an isolated SK enzyme can be contacted with a modulator in an in vitro environment. Alternatively, isolated cells can be contacted with a modulator in an in vitro environment.

[0067] As used herein, the term "in vivo" refers to procedures that occur within a living organism, including, but not limited to, humans, monkeys, mice, rats, rabbits, cows, horses, pigs, dogs, cats, or primates.

[0068] The active ingredients or agents useful in the present invention include those described herein in any of their pharmaceutically acceptable forms, including their isomers, salts, solvates, and polymorphs, as well as racemic mixtures and prodrugs.

[0069] Inhibitors of sphingosine kinase of the present disclosure Sphingosine kinase (SK) is an oncogenic sphingolipid metabolic enzyme that reduces apoptosis-promoting ceramide and catalyzes the formation of the mitogenic second messenger sphingosine-1-phosphate (SIP). Thus, SK is an attractive target for cancer therapy because blockade of SIP results in inhibition of proliferation and induction of apoptosis in cancer cells. The present disclosure provides aryladamantane compounds that inhibit SK. In embodiments, the SK inhibitor is a selective inhibitor of sphingosine kinase-1 (SKI). In embodiments, the SK inhibitor is a selective inhibitor of sphingosine kinase-2 (SK2). In embodiments, the SK inhibitor is a dual inhibitor of sphingosine kinase (inhibiting both sphingosine kinase-1 and sphingosine kinase-2).

[0070] Examples of the aryladamantane compounds of the present invention that are inhibitors of SK are generally represented by Formula 1 shown below:

[0071]

Chemical formula

[0072] The aryladamantane compound of formula 1 is a compound of formula I - 1,

[0073] [Chemical formula] and pharmaceutically acceptable salts thereof, wherein, R1 is H, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, heteroalkyl, aryl, alkylaryl, alkenylaryl, heterocyclyl, heteroaryl, alkylheteroaryl, heterocycloalkyl, alkyl - heterocycloalkyl, acyl, aroyl, halogen, haloalkyl, alkoxy, haloalkyl, hydroxyalkyl, alkanoyl, -COOH, -OH, -SH, -S-alkyl, -CN, -NO2, -NH2, -CO2(alkyl), -OC(O)alkyl, carbamoyl, mono- or dialkylaminocarbamoyl, mono- or dialkylcarbamoyl, mono- or dialkylamulno, aminoalkyl, mono- or dialkylamulnoalkyl, thiocarbamoyl, or mono- or dialkylthiocarbamoyl, and R2 is H, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, heteroalkyl, aryl, alkylaryl, alkenylaryl, heterocyclyl, heteroaryl, alkylheteroaryl, heterocycloalkyl, alkyl - heterocycloalkyl, acyl, aroyl, halogen, haloalkyl, alkoxy, haloalkyl, hydroxyalkyl, alkanoyl, -COOH, -OH, -SH, -S-alkyl, -CN, -NO2, -NH2, -CO2(alkyl), -OC(O)alkyl, carbamoyl, mono- or dialkylaminocarbamoyl, mono- or dialkylcarbamoyl, mono- or dialkylamulno, aminoalkyl, mono- or dialkylamulnoalkyl, thiocarbamoyl, mono- or dialkylthiocarbamoyl, alkyl-S-alkyl, -heteroaryl-aryl, -alkyl-heteroaryl-aryl, -NH-aryl, -alkenyl-heteroaryl, -heteroaryl, -NH-alkyl, -NH-cycloalkyl, or -alkenyl-heteroaryl-aryl, and Each alkyl and cyclic moiety of the above R1 and R2 groups is independently selected from up to five groups which are optionally substituted with (C1-C6) alkyl, halogen, haloalkyl, -OC(O)(C1-C6 alkyl), -C(O)O(C1-C6 alkyl), -CONR’R”, OC(O)NR’R’, -NR’C(O)R”, -CF3, -OCF3, -OH, C1-C6 alkoxy, hydroxyalkyl, -CN, -CO2H, -SH, -S-alkyl, -SOR’R”, -SO2R’, -NO2, or NR’R”, where R’ and R” are independently H or (C1-C6) alkyl, and each alkyl moiety of the substituents is further optionally substituted with one, two or three groups independently selected from halogen, CN, OH and NH2.

[0074] The aryladamantane compound of formula I is a compound of formula II

[0075]

Chemical formula

[0076] The compound of formula II is wherein Y is -C(R4,R5)- or -N(R4)-, R1 is H, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, heteroalkyl, aryl, alkylaryl, alkenylaryl, heterocyclyl, heteroaryl, alkylheteroaryl, heterocycloalkyl, alkyl-heterocycloalkyl, acyl, aroyl, halogen, haloalkyl, alkoxy, haloalkyl, hydroxyalkyl, alkanoyl, -COOH, -OH, -SH, -S-alkyl, -CN, -NO2, -NH2, -CO2(alkyl), -OC(O)alkyl, carbamoyl, mono- or dialkylcarbamoyl, mono- or dialkylthiocarbamoyl, mono- or dialkylamino, aminoalkyl, mono- or dialkylaminoalkyl, thiocarbamoyl, or mono- or dialkylthiocarbamoyl, R2 is H, alkyl, cycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, heteroalkyl, aryl, alkylaryl, alkenylaryl, heterocyclyl, heteroaryl, alkylheteroaryl, heterocycloalkyl, alkyl - heterocycloalkyl, acyl, aroyl, halogen, haloalkyl, alkoxy, haloalkoxy, hydroxyalkyl, alkanoyl, -COOH, -OH, -SH, -S-alkyl, -CN, -NO2, -NH2, -CO2.(alkyl), -OC(O)alkyl, carbamoyl, mono - or dialkylaminocarbamoyl, mono - or dialkylcarbamoyl, mono - or dialkylamino, aminoalkyl, mono - or dialkylaminoalkyl, thiocarbamoyl, mono - or dialkylthiocarbamoyl, alkyl - S - alkyl, -heteroaryl - aryl, -alkyl - heteroaryl - aryl, -C(O)-NH-aryl, -alkenyl - heteroaryl, -C(O)-heteroaryl, or -alkenyl - heteroaryl - aryl, and each alkyl and ring moiety of each of the above R1 and R2 groups is independently optionally substituted with up to 5 groups which are (C1 - C6)alkyl, halogen, haloalkyl - OC(O)(C1 - C6 alkyl), C(O)O(C1 - C6 alkyl), CONR4R5, -OC(O)NR4R5 - NR4C(O)R5, -CF3, -OCF3, -OH, C1 - C6 alkoxy, hydroxyalkyl, -CN, -CO2H, -SH, -S-alkyl, -SOR4R5, -SO2R4R5, -NO2, or NR4R5, and each alkyl moiety of the substituents is further optionally substituted with 1, 2, or 3 groups independently selected from halogen, CN, OH, NH2, R3 is H, alkyl, or oxo(=O), and R4 and R5 are independently H or (C1 - C6)alkyl, and includes a compound.

[0077] Particularly preferred aryladamantane SK inhibitor compounds of the present invention are exemplified below and are referred to as ABC294640 [3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide].

[0078] [Chem.]

[0079] The exact amount of the SK inhibitor incorporated into a particular method or therapeutic combination of the present disclosure can vary according to factors known in the art, such as, for example, the physical and clinical condition of the subject, the method of administration, the content of the formulation, the intended dosing regimen or sequence, etc. Therefore, it is not practical to specifically describe the amounts constituting a therapeutically effective amount of the SK inhibitor for all possible uses. However, those skilled in the art can readily determine the appropriate amount by considering such factors.

[0080] Anti-PD-1 antibody Human monoclonal antibodies that bind specifically to PD-1 with high affinity are disclosed in U.S. Patent No. 8,008,449. Other anti-PD-1 monoclonal antibodies are described, for example, in U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, and International Publication No. 2012 / 145493. Each of the anti-PD-1 human monoclonal antibodies disclosed in U.S. Patent No. 8,008,449 has been demonstrated to exhibit one of the following characteristics: (a) When determined by surface plasmon resonance using a Biacore biosensor system, 1×10” 7Binding to human PD-1 with a KD of less than M, (b) not substantially binding to human CD28, CTLA-4 or ICOS, (c) increasing T cell proliferation in a mixed lymphocyte reaction (MLR) assay, (d) increasing interferon γ production in an MLR assay, (e) increasing IL-2 secretion in an MLR assay, (f) binding to human PD-1 and cynomolgus PD-1, (g) inhibiting the binding of PD-L1 and / or PD-L2 to PD-1, (h) stimulating an antigen-specific memory response, (i) stimulating an antibody response, and (j) inhibiting tumor cell growth in vivo. Anti-PD-1 antibodies that can be used in the methods of the present invention include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one, preferably at least five, of the aforementioned properties. A preferred anti-PD-1 antibody is nivolumab. Another preferred anti-PD-1 antibody is pembrolizumab.

[0081] Anti-PD-1 antibodies that can be used in the methods of the present disclosure also include isolated antibodies that specifically bind to human PD-1 and cross-compete with nivolumab for binding to human PD-1 (see, e.g., U.S. Patent No. 8,008,449; International Publication No. 2013 / 173223). The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically inhibit the binding of other cross-competing antibodies to that specific epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of nivolumab by binding to the same epitope region of PD-1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays such as Biacore analysis, ELISA assays, or flow cytometry (see, e.g., International Publication No. 2013 / 173223).

[0082] In certain embodiments, an antibody that cross-competes with nivolumab for binding to human PD-1 or that binds to the same epitope region of human PD-1 as nivolumab is a monoclonal antibody. For administration to a human subject, these cross-competing antibodies are preferably chimeric antibodies, or more preferably humanized antibodies or human antibodies. Such chimeric antibodies, humanized antibodies or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0083] The anti-PD-1 antibodies that can be used in the methods of the invention disclosed herein also include the antigen-binding portions of the above antibodies. It has been well demonstrated that the antigen-binding function of an antibody can be exerted by fragments of the full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include (i) L , H , C L and a Fab fragment that is a monovalent fragment consisting of the Cm domain; (ii) an F(ab')2 fragment that is a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region, (iii) an Fd fragment consisting of a V # and C domain, and (iv) an Fv fragment consisting of the Vi and V # domains of a single arm of the antibody.

[0084] The anti-CTLA-4 antibodies of the invention bind to human CTLA-4 and interfere with the interaction between CTLA-4 and the human B7 receptor. Since the interaction between CTLA-4 and B7 transduces signals that result in the inactivation of T cells having the CTLA-4 receptor, interference with this interaction effectively induces, enhances or prolongs the activation of such T cells, thereby inducing, enhancing or prolonging the immune response.

[0085] Human monoclonal antibodies that specifically bind to CTLA-4 with high affinity are disclosed in U.S. Patent Nos. 6,984,720 and 7,605,238. Other anti-PD-1 monoclonal antibodies are described, for example, in U.S. Patent Nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121. The anti-PD-1 human monoclonal antibodies disclosed in U.S. Patent Nos. 6,984,720 and 7,605,238 have been demonstrated to exhibit one or more of the following properties: (a) when determined by Biacore analysis, at least about 10 7 M’ 1 or about 10 9 M’ 1 or about 10 10 M” 1 ~10 11 M’ 1 or greater equilibrium association constant (K a )-reflected binding affinity to specifically bind to human CTLA-4, (b) at least about 10 3 , about 10 4 or about 10 5 m’ 1 s’ 1 kinetic association constant (ka), (c) at least about 10 3 , about 10 4 or about 10 5 m” 1 s” 1 kinetic dissociation constant (k ^ ), and (d) inhibiting the binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86). Anti-CTLA-4 antibodies that can be used in the present invention include monoclonal antibodies that specifically bind to human CTLA-4 and exhibit at least one, preferably at least three, of the aforementioned characteristics.

[0086] Examples of anti-CTLA-4 antibodies that can be used in the disclosed method include isolated antibodies that specifically bind to human PD-1 and cross-compete with ipilimumab or tremelimumab for binding to human CTLA-4, or bind to the same epitope region of human CTLA-4 as ipilimumab or tremelimumab. In certain preferred embodiments, antibodies that cross-compete with ipilimumab or tremelimumab for binding to human CTLA-4, or bind to the same epitope region of human PD-1 as ipilimumab or tremelimumab, are antibodies that include a heavy chain of the human IgG1 isotype. For administration to a human subject, these cross-competing antibodies are preferably chimeric antibodies, or more preferably humanized antibodies or human antibodies. Also included as anti-CTLA-4 antibodies that can be used are antigen-binding portions of the above antibodies, such as Fab, F(ab’)2, Fd or Fv fragments.

[0087] In certain embodiments, the invention includes the use of certain classes of checkpoint inhibitor drugs that inhibit the activity of cytotoxic T lymphocyte antigen-4 (CTLA-4). Suitable anti-CTLA4 antagonist agents for use in the methods of the invention include anti-CTLA4 antibodies, human anti-CTLA4 antibodies, mouse anti-CTLA4 antibodies, mammalian anti-CTLA4 antibodies, humanized anti-CTLA4 antibodies, monoclonal anti-CTLA4 antibodies, polyclonal anti-CTLA4 antibodies, chimeric anti-CTLA4 antibodies, MDX-010 (ipilimumab), tremelimumab, anti-CD28 antibodies, anti-CTLA4 adnectins, anti-CTLA4 domain antibodies, single-chain anti-CTLA4 fragments, heavy-chain anti-CTLA4 fragments, light-chain anti-CTLA4 fragments, CTLA4 inhibitors that antagonize co-stimulatory pathways, antibodies disclosed in WO 2001 / 014424, antibodies disclosed in WO 2004 / 035607, antibodies disclosed in US 2005 / 0201994, and antibodies disclosed in EP 1212422 (B1), but are not limited thereto. Additional CTLA-4 antibodies are described in US Pat. Nos. 5,811,097, 5,855,887, 6,051,227, and 6,984,720, and in WO 01 / 14424 and WO 00 / 37504, and in US 2002 / 0039581 and US 2002 / 086014. Other anti-CTLA-4 antibodies that can be used in the methods of the invention include, for example, those described in WO 98 / 42752, US Pat. Nos. 6,682,736 and 6,207,156, Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17):10067-10071 (1998), Camacho et al., J. Clin. Oncology, 22(145):Abstract No. 2505 (2004) (antibody CP-675206), Mokyr et al., Cancer Res., 58:5301-5304 (1998), and US Pat. Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281.

[0088] Examples of additional anti-CTLA4 antagonists include, but are not limited to: any inhibitor that can generally interfere with the ability of the CD28 antigen to bind to its cognate ligand, thereby inhibiting the ability of CTLA4 to bind to its cognate ligand, enhancing the T cell response via the co-stimulatory pathway, interfering with the ability of B7 to bind to CD28 and / or CTLA4, interfering with the ability of B7 to activate the co-stimulatory pathway, interfering with the ability of CD80 to bind to CD28 and / or CTLA4, interfering with the ability of CD80 to activate the co-stimulatory pathway, interfering with the ability of CD86 to bind to CD28 and / or CTLA4, interfering with the ability of CD86 to activate the co-stimulatory pathway, and interfering with the co-stimulatory pathway. This includes, among other members of the co-stimulatory pathway, small molecule inhibitors of CD28, CD80, CD86, CTLA4, antibodies against CD28, CD80, CD86, CTLA4 among other members of the co-stimulatory pathway, antisense molecules against CD28, CD80, CD86, CTLA4 among other members of the co-stimulatory pathway, adnectins against CD28, CD80, CD86, CTLA4 among other members of the co-stimulatory pathway, and RNAi inhibitors (both single-stranded and double-stranded) of CD28, CD80, CD86, CTLA4 among other anti-CTLA4 antagonist agents, as needed.

[0089] In some embodiments of the present disclosure, an immune checkpoint inhibitor compound inhibits the signaling interaction between an immune checkpoint receptor and its corresponding ligand. The immune checkpoint inhibitor compound can act by inhibiting (antagonizing) the immune checkpoint receptor or by inhibiting the ligand of the immune checkpoint receptor (some examples of ligands include PD-L1 and PD-L2), thereby blocking the activation of the immune checkpoint pathway (some examples of receptors include CTLA-4, PD-1, LAG-3, TIM-3, BTLA, and KIR). In such embodiments, the effect of the immune checkpoint inhibitory compound is to reduce or eliminate the downregulation of a particular aspect of the immune system anti-tumor response in the tumor microenvironment.

[0090] The immune checkpoint receptor cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is expressed on T cells and is involved in a signaling pathway that reduces the level of T cell activation. CTLA-4 is thought to be able to downregulate T cell activation through competitive binding and dissociation of CD80 and CD86. In addition, CTLA-4 has been shown to be involved in enhancing the immunosuppressive activity of T Reg cells.

[0091] In some embodiments of the present disclosure, the immune checkpoint inhibitor compound is a small organic molecule (molecular weight less than 1000 daltons), a peptide, a polypeptide, a protein, an antibody, an antibody fragment, or an antibody derivative. In some embodiments, the immune checkpoint inhibitor compound is an antibody. In some embodiments, the antibody is a monoclonal antibody, specifically, a human or humanized monoclonal antibody.

[0092] The preparation of immune checkpoint antibodies and methods for us are described in the following exemplary publications. The preparation and therapeutic use of anti-CTLA-4 antibodies are described in U.S. Patent No. 7,229,628 (Allison), U.S. Patent No. 7,311,910 (Linsley), and U.S. Patent No. 8,017,144 (Korman). The preparation and therapeutic use of anti-PD-1 antibodies are described in U.S. Patent No. 8,008,449 (Korman) and U.S. Patent No. 8,552,154 (Freeman). The preparation and therapeutic use of anti-PD-L1 antibodies are described in U.S. Patent No. 7,943,743 (Korman). The preparation and therapeutic use of anti-TIM-3 antibodies are described in U.S. Patent No. 8,101,176 (Kuchroo) and U.S. Patent No. 8,552,156 (Tagayanagi). The preparation and therapeutic use of anti-LAG-3 antibodies are described in U.S. Patent Application No. 2011 / 0150892 (Thudium) and International Publication No. 2014 / 008218 (Lonberg). The preparation and therapeutic use of anti-KIR antibodies are described in U.S. Patent No. 8,119,775 (Moretta). The preparation of an antibody that blocks the BTLA regulatory inhibitory pathway (anti-BTLA antibody) is described in U.S. Patent No. 8,563,694 (Mataraza).

[0093] In some forms of the present disclosure, the immune checkpoint inhibitor compound is a CTLA-4 receptor inhibitor, a PD-1 receptor inhibitor, a LAG-3 receptor inhibitor, a TIM-3 receptor inhibitor, a BTLA receptor inhibitor, or a KIR receptor inhibitor. In some forms, the immune checkpoint inhibitory compound is an inhibitor of PD-L1 or an inhibitor of PD-L2.

[0094] Any suitable daily dose of a checkpoint inhibitor is contemplated for use in the compositions, dosage forms, and methods disclosed herein. The daily dose of a checkpoint inhibitor depends on a plurality of factors and its determination is within the skill of the art. For example, the daily dose of a checkpoint inhibitor depends on the strength of the checkpoint inhibitor. A weak immune checkpoint inhibitor will require a higher daily dose than a moderate immune checkpoint inhibitor, and a moderate immune checkpoint inhibitor will require a higher daily dose than a potent immune checkpoint inhibitor. For example, Merck's pembrolizumab (Keytruda) is approved for 2 mg / kg intravenously (iv) (50 mg of lyophilized strength) over 30 minutes every 3 weeks. Nivolumab (OPDVO) is administered intravenously at 3 mg / kg over 60 minutes every 2 weeks (injectable dosage form: 40 mg / 4 mL and 100 mg / 10 / ml in single-use vials). Ipilimumab (YERVOY) is administered intravenously at 3 mg / kg over 90 minutes every 3 weeks for a total of 4 doses (dosage form: 50 mg / 10 ml, 200 mg / 40 ml).

[0095] Solid forms for oral administration may contain pharmaceutically acceptable binders, sweeteners, disintegrants, diluents, flavoring agents, coating agents, preservatives, lubricants, and / or retardants. Suitable binders include acacia gum, gelatin, corn starch, tragacanth gum, sodium alginate, carboxymethyl cellulose or polyethylene glycol (PEG). Suitable sweeteners include sucrose, lactose, glucose, aspartame, or saccharin. Suitable disintegrants include corn starch, methyl cellulose, polyvinyl pyrrolidone, xanthan gum, bentonite, alginic acid, or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate, or dicalcium phosphate. Suitable flavoring agents include peppermint oil, wintergreen, cherry, orange, or raspberry flavor oil. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid, and / or their esters, wax, fatty alcohol, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, α-tocopherol, ascorbic acid, methyl paraben, propyl paraben, or sodium bisulfate. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride, or talc. Suitable retardants include glyceryl monostearate or glyceryl distearate.

[0096] The compositions and methods of the present invention can include formulations of compound(s) that, when administered to a subject, result in a concentration of compound(s) that treat filovirus-mediated diseases. The compound(s) may be contained in any suitable amount in any suitable carrier substance and generally are present in an amount of 1 to 95% by weight of the total weight of the composition. The composition may be provided in a dosage form suitable for oral, parenteral (e.g., intravenous or intramuscular), rectal, dermatological, cutaneous, nasal, intravaginal, inhalation, transdermal (patch), intraocular, intrathecal, or intracranial routes of administration. Thus, the composition may be, for example, in the form of tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, patches, lotions, osmotic delivery devices, suppositories, enemas, injections, implants, sprays, or aerosols. The pharmaceutical composition may be formulated according to conventional pharmaceutical practices.

[0097] The pharmaceutical compositions according to the present invention or used in the methods of the present invention may be formulated to release the active compound immediately after administration or at any predetermined time or period after administration. The latter type of compositions are generally known as controlled release formulations and include (i) formulations that produce a substantially constant concentration of the agent(s) of the present invention over a long period of time, (ii) formulations that produce a substantially constant concentration of the agent(s) of the present invention in the body over a long period of time after a predetermined delay time, (iii) formulations that maintain a relatively constant effective level of the agent(s) in the body while simultaneously minimizing undesirable side effects associated with fluctuations (sawtooth dynamic patterns) in the plasma levels of the agent(s) to sustain the action of the agent(s) over a predetermined period, (iv) formulations that localize the action of the agent(s), e.g., the spatial placement of the controlled release composition adjacent to or in the diseased tissue or organ, (v) formulations that achieve convenience of administration, e.g., administration of the composition once a week or once every two weeks, and (vi) formulations that target the action of the agent(s) using a carrier or chemical derivative to deliver the combination to specific target cell types.

[0098] To obtain controlled release that exceeds the metabolic rate of the compound for which the rate of release is a problem, any of a number of strategies can be pursued. In one example, controlled release can be obtained by appropriate selection of various formulation parameters and components, including, for example, various types of controlled release compositions and coatings. Thus, the compound(s) is / are formulated with appropriate excipients in a pharmaceutical composition that releases the compound(s) in a controlled manner upon administration. Examples include single or multiple unit tablet or capsule compositions, oils, suspensions, emulsions, microcapsules, molecular complexes, microspheres, nanoparticles, patches, and liposomes.

[0099] The administration of the compounds is not intended to be limited to a single formulation and delivery method for all compounds of the combination. The combination can be administered, for example, using any of the above formulations and methods, using separate formulations and / or delivery methods for each compound of the combination. In one example, the first agent is delivered orally and the second agent is delivered intravenously.

[0100] The dosage of the compound or combination of compounds depends on several factors, including the route of administration, the type of disease being treated, the severity of the infection, whether it is administered initially in the early or late stages of the infection, and the age, weight, and health of the patient being treated. For combinations containing synergistic pairs of agents identified herein, the recommended dosage of the antiviral agent can be below the recommended dosage shown in the Physician’s Desk Reference, 69 th Edition (2015).

[0101] As described above, the compound(s) in question may be administered orally in the form of tablets, capsules, elixirs or syrups, or in the form of suppositories. Parenteral administration of the compound is preferably carried out, for example, in the form of an aqueous physiological saline solution or together with the compound(s) incorporated into liposomes. If the compound itself is not sufficiently soluble to dissolve, solubilizing agents such as ethanol can be applied. The correct dosage of the compound can be determined by examining the effectiveness of the compound in a viral replication assay and its toxicity in humans.

[0102] The agent of the present invention is also a useful tool for elucidating mechanism-of-action information regarding biological pathways involved in viral diseases. Such information can lead to the development of new combinations or single agents for treating, preventing or alleviating viral diseases. Methods known in the art for determining biological pathways can be used to determine the pathways or pathway networks affected by contacting virus-infected cells (e.g., primary macrophage cells) with the compounds of the present invention. Such methods include analyzing cell components that are expressed or re-pressurized after contact with the compounds of the present invention as compared to untreated, positive or negative control compounds, and / or new single agents and combinations, or analyzing some other activities of cells or viruses such as enzyme activity, nutrient uptake, and proliferation. The cell components to be analyzed can include gene transcripts and protein expression. Suitable methods can include standard biochemical techniques, radiolabeling the compounds of the present invention (e.g., 14 C or 3 H labeling), and gene expression profiling, for example, using 2D gels to observe compounds that bind to proteins. Once identified, such compounds can be used in in vivo models (e.g., knockout or transgenic mice) to further validate the tool or to develop new agents or strategies for treating viral diseases.

[0103] Kits and Packages The terms "kit" and "pharmaceutical kit" refer to a commercially available kit or package containing one or more pharmaceutical compositions and instructions for their use in one or more suitable containers. In one embodiment, a kit is provided that includes ABC294640 and instructions for its administration. In one embodiment, a kit is provided that includes ABC294640 in combination with one or more (e.g., one, two, three, one or two, or one to three) additional therapeutic agents and instructions for their administration.

[0104] In embodiments, the checkpoint inhibitors of the present disclosure are formulated into dosage units packaged in a single packaging. Single packagings include, but are not limited to, bottles, cold-resistant bottles, ampoules, and tubes. In embodiments, the checkpoint inhibitors of the present disclosure and optionally additional therapeutic agents are formulated into dosage units and individually packaged in a single packaging for each single dosage unit. Such individually packaged units may contain a pharmaceutical composition in any form, including, but not limited to, liquid form, solid form, powder form, granule form, effervescent powder or tablet, hard or soft capsule, emulsion, suspension, syrup, suppository, tablet, troche, lozenge, solution, buccal patch, thin film, oral gel, chewable tablet, chewing gum, and disposable syringe. Such individually packaged units may be combined in a package made of one or more of paper, cardboard, paperboard, metal foil, and plastic foil, such as a blister pack. One or more dosage units may be administered once or several times a day. One or more dosage units may be administered three times a day. One or more dosage units may be administered twice a day. One or more dosage units may be administered on the first day, and one or more dosage units may be administered on subsequent days.

Examples

[0105] The present invention can be better understood with reference to the following examples. These examples are intended to represent specific embodiments of the present invention and are not intended to limit the scope of the present invention.

[0106] Example 1 Synthesis method of 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide, ABC294640 As an example, the synthesis process of ABC294640 is described in Scheme 1. Adamantane-1-carboxylic acid (1) was directly brominated in the presence of aluminum chloride (AlCl3) to obtain the 3-bromide derivative (2) of 1, which was converted to (3) by the Friedel-Crafts reaction. 3 was reacted with thionyl chloride (SOCl2) to obtain 3-R-substituted-1-adamantanecarbonyl chloride 4. In THF, 4 was reacted with a substituted amine, for example, 4-aminomethylpyridine (5), to obtain (6, which is also represented as ABC294640) and related amide compounds.

[0107]

Chemical formula

[0108] More specifically, adamantane-1-carboxylic acid (1) (45 g, 0.25 mol) was added to a mixture of AlCh (45 g, 0.34 mol) and Bn (450 g) at 0 °C, stirred at 0-10 °C for 48 hours, held at about 20 °C for 5 hours, poured into 500 g of crushed ice, diluted with 300 ml of CHCl3, and decolorized with solid Na2S2O5. The aqueous phase was extracted with Et20 (50 ml x 2). The combined organic solutions were washed with H2O and extracted with 10% NaOH. The alkaline extract was acidified with 2N H2SO4 to obtain 49 g (yield = 75.7%) of 3-bromo-adamantane-1-carboxylic acid (2).

[0109] Over a period of 30 minutes, 3-bromo-adamantane-1-carboxylic acid (2) (16.0 g, 61.7 mmol) in 50 ml of dry chlorobenzene at -10 °C was added to 100 ml of dry chlorobenzene and 9.3 g of 70 mmol AlCl3. The mixture was then warmed to room temperature for 1 hour and then heated to 90 °C for 10 hours. The mixture was then poured onto 200 g of crushed ice and filtered to obtain 14.2 g (yield = 79.3%) of 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (3).

[0110] 3 was reacted with an equimolar amount of 1,1'-carbonyldiimidazole (CDI) to obtain the intermediate 3-R-substituted-1-adamantanecarbonylimidazole (4). By reacting 4 with a substituted amine, the corresponding adamantiamide was obtained.

[0111] For example, by reacting 3 with 4-aminomethylpyridine (5) in toluene, {3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide} (6, also designated as ABC294640) was produced in a yield of 92.6% and had a melting point of 128 - 130 °C. 1 HNMR(300MHz,CDCl3)δ1.72 - 2.25(m,12H,Admant-CH),4.44 - 4.46(d,J = 6Hz,2H,CH2-Py),6.18(m,1H,HN),7.13 - 7.15(d,J = 6Hz,2H,H-Py),7.15 - 7.30(m,4H,H-Ph),8.52 - 8.54(d,J = 6 Hz,2H,H-Py); 13 C NMR(300 MHz,CDCl3)δ28.98,35.73,36.71,38.77,42.18,42.37,44.88,122.38,125.30,126.57,128.56,129.26,148.39,150.20 177.76;MS m / z(relative intensity)381.50(MH + ,100),383.41(90),384.35(80).

[0112] Example 2 The second synthesis method of ABC294640 and the second synthesis method of related adamantiamides are described in Scheme 2. The 3-phenyl-substituted intermediate (3) was prepared as described above. By reacting 3 with 1,1'-carbonyldiimidazole (CDI), the 3-R-substituted-1-adamantanecarbonylimidazole intermediate (4) was obtained. In toluene, by reacting 4 with a substituted amine, for example, 4-aminomethylpyridine 5, 6 {3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide} was obtained.

[0113] [Chemical formula]

[0114] By condensing various aromatic compounds with 2, various sets of substituted aryladamantanes can be efficiently synthesized, and various such compounds are commercially available. Additionally, various coupling reagents and primary amine-containing compounds can be used to efficiently complete the amidation of 3. The following examples provide some representative examples of the products of this process. However, these methods can be adapted to produce many structurally related adamantiamides that are contemplated as the subject of the present invention. For a full disclosure of these methods, U.S. Patent No. 7,338,961 is incorporated herein by reference for its teachings.

[0115] Example 3 ABC294640 is an inhibitor of sphingosine kinase-2. One indicator that tumor cells undergoing immunogenic cell death (ICD) show increased expression of calreticulin on the surface of tumor cells is the increased expression of calreticulin on the cell surface. Calreticulin is normally expressed in the endoplasmic reticulum (ER) of cells. However, when cells are treated with agents that promote ER stress, calreticulin expression can be observed on the outer surface of the cell. Promotion of ER stress is a typical mechanism of action for inducing ICD, and thus measuring the surface expression of calreticulin is an established method for determining whether a compound causes ICD. The effect of ABC294640 on the surface expression of calreticulin in several different types of tumor cells was tested. In these experiments, the test tumor cells were incubated with ABC294640 at various concentrations, and the cells were harvested 4 to 24 hours after the addition of ABC294640. The cells were then incubated with a fluorescently labeled antibody that selectively binds to calreticulin, washed, and then analyzed by flow cytometry to quantify the amount of surface calreticulin on many individual cells. The resulting data were analyzed by determining the geometric mean of the fluorescence intensity of the cell population using algorithms well known in the field of flow cytometry. The data provided in Table 1 summarize the effect of treatment with ABC294640 on the surface expression of calreticulin in a tumor cell panel. For each cell type, samples were treated with dimethyl sulfoxide (DMSO, the solvent used to dissolve ABC294640) or 40 μM ABC294640 for 24 hours. The geometric mean of the fluorescence intensity of the cell samples treated with DMSO was represented as 1.0, and the data for the cell samples treated with ABC294640 were represented relative to the DMSO control. In all cases, treatment with ABC294640 caused an increase in the surface expression of calreticulin, and the responses were in the range of 1.46 to 3.64. It should be noted that this data was calculated on a logarithmic scale such that a geometric mean fluorescence of 2.0 indicates a 10-fold increase in the amount of surface calreticulin.In summary, the treatment with ABC294640 increased the surface calreticulin expression by approximately 3 - fold to over 400 - fold in pancreatic, prostate, neuroblastoma, breast, lung, and melanoma tumor cells. Therefore, ABC294640 increased ICD in a wide range of tumors.

[0116]

Table 1

[0117] Example 4 In - vivo demonstration that ABC294640 induces immunogenic cell death in B16 tumor cells The ability of ABC294640 to induce ICD was evaluated using a syngeneic mouse model in which mouse melanoma B16 cells (ATCC CRL - 6322) were treated with ABC294640 in vitro and then transplanted subcutaneously into immunocompetent (C57BL / 6) mice. C57B1 / 6 mice (6 - 8 weeks old, male) were obtained from Jackson Laboratories and maintained under standard conditions with free access to food and water. Clinical - grade ABC294640 (Batch CHP110607) was manufactured under a GMP contract by ChemPacific Corporation (Baltimore, MD) and used in all studies. B16 cells were obtained from ATCC and cultured under standard conditions in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. B16 cells were treated in culture for 24 hours with a concentration of ABC294640 known to cause cell death to induce cell death - cells were treated with 40 μM of ABC294640. Then, the cells treated with ABC296460 were harvested by trypsinizing the culture and scraping the cells off the plate, suspended in phosphate - buffered saline (PBS), and injected subcutaneously into the left hind flank at a total volume of 0.1 ml (500,000 stained cells). The control group was injected with PBS only into the left hind leg. Seven days later, 100,000 untreated B16 cells were transplanted into the right hind flank of the mice in both groups (n = 10 / group). Tumor growth was measured 3 times a week with digital calipers, and the tumor volume was calculated using the formula (L×W 2 ) / 2. The tumor volume reached 3,000 mm3 Mice were euthanized when the above was reached. Figure 1 shows data on B16 tumor size on day 14 after transplantation into either mice pretreated with PBS (control) or mice pretreated with B16 cells treated with ABC294640 (immunized). Tumors in control mice reached an average size of 2344 ± 361 mm 3 on day 14. In contrast, the cells injected into immunized mice reached an average size of only 641 ± 210 mm 3 on day 14 (p = 0.0007). These data demonstrate that treatment of B16 tumor cells with ABC294640 induces ICD that significantly reduces tumor growth in subsequently challenged mice.

[0118] Example 5 In vivo demonstration that ABC294640 induces immunogenic cell death in Neuro-2a tumor cells In a second form of the experiment, the ability of ABC294640 to induce ICD was evaluated using a syngeneic mouse model in which mouse Neuro-2a neuroblastoma cells were treated in vitro with ABC294640 and then transplanted subcutaneously into immunocompetent (A / J) mice. The source of the mice, ABC294640, and the tumor cells were the same as those detailed in Example 4. Neuro-2a cells were treated in culture for 24 hours at a concentration of ABC294640 known to cause cell death to induce cell death - cells were treated with 40 μM ABC294640. Then, the cells treated with ABC294640 were collected by trypsinizing the culture and scraping the cells off the plate, suspended in phosphate-buffered saline (PBS), and subcutaneously injected into the left hind flank at a total volume of 0.1 ml (5,000,000 stained cells). The control group was injected with PBS only into the left hind leg. Seven days later, 1,000,000 untreated Neuro-2a cells were transplanted into the right hind leg of the mice in both groups (n = 4 - 5 / group). Tumor growth was measured, and tumor volume was calculated as described in Example 1. Figure 2 shows the data of Neuro-2a tumor size at day 22 after transplantation into either mice pretreated with PBS (control) or mice pretreated with ABC294640-treated Neuro-2a cells (immunized). The tumors in the control mice reached an average size of 1039 ± 450 mm 3 on day 22. In contrast, the cells injected into the immunized mice reached an average size of only 15 ± 15 mm 3 on day 22 (p = 0.085). All mice in the control group had tumors, and 75% of the immunized group had no measurable tumors on day 22. These data demonstrate that treating Neuro-2a tumor cells with ABC294640 causes ICD, which significantly reduces tumor growth in subsequently challenged mice.

[0119] Example 6 In Vivo Demonstration That ABC294640 Induces Immunogenic Cell Death in Lewis Lung Carcinoma (LLC) Tumor Cells In the third form of the experiment, the ability of ABC294640 to induce ICD was evaluated using an isogenic mouse model in which mouse LLC cells (ATCC CRL-1642) were treated in vitro with ABC294640 and then subcutaneously transplanted into immunocompetent (C57BL / 6) mice. The source of the mice, ABC294640, and the tumor cells were the same as those detailed in Example 4. LLC cells were treated in culture with 40 μM ABC294640 for 24 hours to induce cell death. The cells treated with ABC294640 were then harvested by trypsinizing the culture and scraping the cells off the plate, suspended in phosphate-buffered saline (PBS), and subcutaneously injected into the left posterior flank at a total volume of 0.1 ml (5,000,000 stained cells). The control group was injected with PBS only into the left hind leg. Seven days later, 1,000,000 untreated LLC cells were transplanted into the right hind leg of the mice in both groups (n = 10 / group). Tumor growth was measured, and the tumor volume was calculated as described in Example 1. Figures 3A - 3C show data on LLC tumor size on days 15, 17, and 20 after transplantation into either mice pretreated with PBS (control) or mice pretreated with ABC294640-treated LLC cells (immunized). The tumors in the control mice gradually increased as the experiment progressed, reaching an average size of 651 ± 114, 1190 ± 143, and 2263 ± 227 mm 3 on days 15, 17, and 20, respectively. In contrast, the cells injected into the immunized mice reached 209 ± 18 (p = 0.0012), 510 ± 94 (p = 0.0009), and 1220 ± 320 (p = 0.016) mm 3 on days 15, 17, and 20. These data demonstrate that treating LLC tumor cells with ABC294640 causes ICD, which significantly reduces tumor growth in mice challenged subsequently.

[0120] Example 7 In Vivo Demonstration that ABC294640 Induces Cross-Immunity The above examples demonstrated that treating multiple tumor cell lines in vitro using ABC294640 and subsequently administering the treated cells to normal mice suppresses the growth of subsequently administered untreated samples of the same tumor cells. In the following study, the hypothesis that administering one of the ABC294640-treated tumor cells not only suppresses the growth of the same type of tumor cells but also confers "cross" immunity to different types of tumor cells was tested. The source of the mice, ABC294640, and the tumor cells were the same as those detailed in the previous examples. Separately, B16 or LLC cells were treated in culture with 40 μM ABC294640 for 24 hours to induce cell death. The B16 or LLC cells treated with ABC294640 were then harvested by trypsinizing the culture and scraping the cells off the plate, suspended in phosphate-buffered saline (PBS), and subcutaneously injected into the left hind flank at a total volume of 0.1 ml (500,000 stained B16 cells or 5,000,000 stained LLC cells). Control mice were injected with PBS only into the left hind leg. Seven days later, the mice were randomized into four groups as summarized below and challenged in the right hind flank with either 100,000 live B16 cells or 1,000,000 live LLC cells to evaluate tumor growth. Thus, the "cross" test groups were group 3 consisting of mice immunized with lung cancer cells treated with ABC294640 and challenged with untreated melanoma cells, and group 6 consisting of mice immunized with melanoma cells treated with ABC294640 and challenged with untreated lung cancer cells.

[0121]

Table 2

[0122] Tumor growth was measured and tumor volume was calculated as described in Example 1. The tumor volume was 3,000 mm 3When the above was reached, the mice were euthanized. Figure 4 shows data on B16 tumor size on day 19 after transplantation into either mice pretreated with PBS (control), mice pretreated with B16 cells treated with ABC294640, or mice pretreated with LLC cells treated with ABC294640. Tumors in control mice reached an average size of 702 ± 144 mm 3 . In contrast, cells injected into B16-immunized mice reached an average size of 203 ± 15 mm 3 (p = 0.018). Cells injected into LLC-immunized mice reached an average size of 102 ± 51 mm 3 (p = 0.0009). Thus, vaccination with either melanoma or lung cancer cells treated with ABC294640 suppressed the subsequent growth of untreated melanoma cells. Figure 5 shows data on LLC tumor size on day 28 after transplantation into either mice pretreated with PBS (control), mice pretreated with B16 cells treated with ABC294640, or mice pretreated with LLC cells treated with ABC294640. Tumors in control mice reached an average size of 479 ± 113 mm 3 . In contrast, cells injected into B16-immunized mice reached an average size of 208 ± 74 mm 3 (p = 0.0003). Cells injected into LLC-immunized mice reached an average size of 177 ± 68 mm 3 (p < 0.001). Thus, vaccination with either melanoma or lung cancer cells treated with ABC294640 suppressed the subsequent growth of untreated lung cancer cells. These data demonstrate that treating tumor cells with ABC294640 in vitro promotes immunization against multiple tumor types in mice subsequently challenged.

[0123] Example 8 Drugs that induce ICD with anti-tumor activity in vivo of ABC294640 in combination with anti-PD-1 antibodies can enhance the anti-tumor activity of checkpoint antibodies. Since the above data clearly demonstrate that ABC294640 induces ICD in several types of cancer, the combined effect of treating tumor-bearing mice with ABC294640 and anti-PD-1 antibodies was examined in a B16 tumor model. Anti-mouse PD-1 (catalog number BE0146) antibody was purchased from BioXCell (West Lebanon, NH). For C57BL / 6 mice, 100,000 B16 cells suspended in PBS were subcutaneously injected on day 0 of the experiment. On day 3 of the experiment, the mice were randomized into the following four treatment groups (n = 10 / group): control (vehicle only); ABC294640 only; anti-PD-1 antibody only; and ABC294640 in combination with anti-PD-1 antibody. ABC294640 was suspended in vehicle (46.7% PEG, 46.7% saline and 6.6% ethanol) and administered by forced oral gavage at 50 mg / kg, 5 days / week (i.e., days 3 - 7, 10 - 14, 17 - 21, etc.) until sacrifice. The anti-PD-1 antibody was suspended in sterile PBS and administered by intraperitoneal (i.p.) injection at a dose of 200 μg / mouse on days 3, 6, and 10. Mice in the combination treatment group received both the antibody and ABC294640 treatment simultaneously on the days the antibody was scheduled. Mice in the control group received oral vehicle and / or sterile PBS i.p. on all days the treated mice received either ABC294640 or the antibody. Tumors were measured three times a week with digital calipers and the volume was calculated using the formula (L × W 2 ) / 2. Mice were euthanized when the tumor volume reached μ3,000 mm 3 . Figure 6 demonstrates the growth of B16 tumors in this experiment. Tumors in control mice grew very aggressively after about 10 days. On day 19, the mean tumor volumes of the control, ABC294640 only, anti-PD-1 antibody only, and combination treatment groups were 1702 ± 373, 892 ± 364, 783 ± 265, and 190 ± 114 mm 3It was the case. The tumor volumes of ABC294640 alone and anti-PD-1 antibody alone were not significantly different from those of the control group. However, the tumor volume in the ABC294640 + anti-PD-1 treatment group decreased very significantly compared to the control group (p = 0.0011). As directed by the IACUC protocol, each mouse was sacrificed when the tumor volume reached 3,000 mm 3 3 . Figure 7 shows the survival curves of the mice in this experiment. The mice in the control group had a median survival of 21 days, and all animals were sacrificed on day 29. Treatment with ABC294640 alone resulted in a median survival of 24 days, and 30% of the mice survived until day 56 when the experiment ended (p = 0.009). Similarly, treatment with anti-PD-1 alone increased the median survival until day 23 (p = 0.033), and 20% of the mice survived until day 56. The combination of ABC294640 and anti-PD-1 antibody significantly increased the median survival to 35 days, and 30% of these mice survived until day 56 (p < 0.0001). Therefore, by combining ABC294640 and PD-1 checkpoint antibodies, the anti-tumor activity is significantly improved, and the survival period is longer than that of either drug alone.

[0124] Example 9 In Vivo Anti-Tumor Activity of ABC294640 in Combination with Anti-CTLA4 Antibody The combinatorial effect of treating mice bearing tumors with ABC294640 and anti-CTLA4 antibody was examined in an LLC tumor model. Anti-mouse CTLA-4 (Catalog No. BE0131) antibody was purchased from BioXCell (West Lebanon, NH). For mice, 1,000,000 LLC cells suspended in PBS were subcutaneously injected on day 0 of the experiment. On day 3 of the experiment, the mice were randomized into the following four treatment groups (n = 5 / group): control (vehicle only); ABC294640 only; anti-CTLA4 antibody only; and ABC294640 in combination with anti-CTLA4 antibody. ABC294640 was suspended in vehicle (46.7% PEG, 46.7% saline, and 6.6% ethanol) and administered by forced oral gavage at 50 mg / kg, 5 days / week (i.e., on days 3 - 7, 10 - 14, 17 - 21, etc.) until sacrifice. The anti-CTLA4 antibody was suspended in dilution buffer (BioXCell, Catalog No. IP0070) and administered by intraperitoneal (i.p.) injection at a dose of 200 μg / mouse on days 3, 6, 10, 13, 17, and 20. Mice in the combination treatment group received both the antibody and ABC294640 treatment simultaneously on the days the antibody was scheduled. Mice in the control group received oral vehicle and / or sterile PBS i.p. on all days the treated mice received either ABC294640 or the antibody. Tumors were measured three times per week with digital calipers and the volume was calculated using the formula (L × W 2 ) / 2. Mice were euthanized when the tumor volume reached 3,000 mm 3 or greater. Figure 8 demonstrates the growth of LLC tumors in this experiment. Tumors in control mice gradually grew after approximately 7 days. On day 21, the mean tumor volumes of the control, ABC294640 only, anti-CTLA4 antibody only, and combination treatment groups were 4622 ± 548, 3197 ± 914, 3029 ± 675, and 1274 ± 336 mm 3It was the case. The tumor volumes of ABC294640 alone and the anti-CTLA4 antibody alone were not significantly different from the control group. However, the tumor volume in the ABC294640 + anti-CTLA4 treatment group decreased very significantly compared to the control group (p = 0.0008). As instructed by the IACUC protocol, each mouse was sacrificed when the tumor volume reached 3,000 mm 3 3 . Figure 9 shows the survival curves of the mice in this experiment. The mice in the control group had a median survival of 19 days, and all animals were sacrificed on day 21. Treatment with ABC294640 alone gave a median survival on day 22, and treatment with anti-CTLA4 had no effect on the median survival. The combination of ABC294640 and the anti-CTLA4 antibody increased the median survival beyond 26 days, and 60% of these mice survived until day 26. Therefore, by combining ABC294640 and the CTLA4 checkpoint antibody, the antitumor activity was significantly improved, and the survival period was longer than that of either drug alone.

[0125] Example 10 In vivo antitumor activity of ABC294640 combined with an anti-PD-L1 antibody The combined effect of treating mice with tumors with ABC294640 and an anti-PD-L1 antibody was examined in a B16 tumor model. The anti-mouse PD-L1 (catalog number BE0101) antibody was purchased from BioXCell (West Lebanon, NH). Mice were subcutaneously injected with 100,000 B16 cells suspended in PBS on day 0 of the experiment. When the tumor reached 300 mm 3When the above volume was reached, the mice were randomized into the following four treatment groups (n = 5 - 6 / group): control (vehicle only); ABC294640 only; anti-PD-L1 antibody only, and ABC294640 combined with anti-PD-L1 antibody. The day of randomization was noted as day 1 of experiment for each mouse. ABC294640 was suspended in vehicle (46.7% PEG, 46.7% saline and 6.6% ethanol) and administered by forced oral gavage at 50 mg / kg, 5 days / week until sacrifice. The anti-PD-L1 antibody was suspended in dilution buffer (BioXCell, catalog number IP0070) and administered by intraperitoneal (i.p.) injection at a dose of 200 μg / mouse on days 1, 3, 5, and 7. Mice in the combination treatment group received both the antibody and ABC294640 treatment simultaneously on the days the antibody was scheduled. Mice in the control group received oral vehicle and / or sterile PBS i.p. on all days the treated mice received either ABC294640 or the antibody. Tumors were measured three times a week with digital calipers and the volume was calculated using the formula (L × W 2 ) / 2. Mice were euthanized when the tumor volume reached 3,000 mm 3 or greater. The following table shows the median survival of each treatment group.

[0126]

Table 3

[0127] Mice in the control group had a median survival of 8.5 days and all animals were sacrificed on day 12. Treatment with ABC294640 only or anti-PD-L1 only provided median survivals of 10 days and 10.5 days, respectively. The combination of ABC294640 and anti-PD-L1 antibody increased the median survival to 16 days (p = 0.0029, compared to control). Thus, combining ABC294640 with a PD-L1 checkpoint antibody significantly improves antitumor activity and prolongs the survival period compared to either agent alone.

[0128] Disclosed herein is a method for treating cancer in a subject, comprising administering to the subject an inhibitor of an effective amount of sphingosine kinase (SK) and an inhibitor of an effective amount of a checkpoint inhibitor. In embodiments, the checkpoint inhibitor can be an antibody against CTLA4 (e.g., ipilimumab), or an antibody against PD-1 (e.g., pembrolizumab or nivolumab), or an antibody against PD-L1 (e.g., atezolizumab or durvalumab). Other antibodies or chemical inhibitors targeting these pathways are also within the scope of the present invention. For example, further inhibitors of the PD-L1 pathway include BMS-936559, MPDL3280A, BMS-936558, MK-3475, CT-011, or MEDI4736.

[0129] In embodiments, tumor cells can be isolated from the blood or diseased tissue of a cancer patient and treated ex vivo with ABC294640 for about 24 hours. The treated cells can then be delivered into the patient's bloodstream to promote an immune response against the cancer.

[0130] In embodiments, the inhibitor of sphingosine kinase is a compound represented by Formula I

[0131]

Chemical formula

[0132] [Chemical formula]

[0133] In an embodiment, treating cancer is further defined as reducing the size of a tumor or inhibiting the growth of a tumor. In an embodiment, the inhibitor is administered at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In an embodiment, a second cancer therapy is further administered to the subject. In an embodiment, the second cancer therapy includes surgery, radiation therapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, or gene therapy. In an embodiment, the melanoma is a chemotherapy- or radiation-resistant melanoma. In an embodiment, the effective amount includes at least about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or 300 μg / kg or mg / kg per kg of the subject's body weight.

[0134] A method of preparing immunologically primed cancer cells using cancer cells collected from a patient involves treating the cancer cells ex vivo with a compound at a toxic concentration that modifies sphingolipid metabolism, the toxic concentration being sufficient to induce immunogenic cell death in the cancer cells. In embodiments, the compound that modifies sphingolipid metabolism is an inhibitor of sphingosine kinase. In embodiments, the compound that is an inhibitor of sphingosine kinase is a selective inhibitor of sphingosine kinase-2 (SK2). In embodiments, the selective inhibitor of SK2 is 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide or a pharmaceutically acceptable salt thereof. In embodiments, the collected cancer cells are treated for at least 24 hours. In embodiments, the toxic concentration of the selective inhibitor of SK2 is from about 20 μM to about 60 μM. In embodiments, the immunologically primed cancer cells overexpress calreticulin on their surface. In embodiments, the cancer cells are immune cells. In embodiments, the immunologically primed cancer cells express calreticulin on their surface at about 3-fold or more (e.g., about 3-fold to about 400-fold or more) than non-primed cancer cells. In some embodiments, the immunologically primed cancer cells express calreticulin on their surface at about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold or more than non-primed cancer cells. In some embodiments, the immunologically primed cancer cells express calreticulin on their surface at about 3-fold to about 10-fold, about 3-fold to about 50-fold, about 3-fold to about 100-fold, about 3-fold to about 150-fold, about 3-fold to about 200-fold, about 3-fold to about 250-fold, about 3-fold to about 300-fold, about 3-fold to about 350-fold, about 3-fold to about 400-fold or more than non-primed cancer cells.In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 10 to about 50 times, about 10 to about 100 times, about 10 to about 150 times, about 10 to about 200 times, about 10 to about 250 times, about 10 to about 300 times, about 10 to about 350 times, about 10 to about 400 times or more than non-primed cancer cells. In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 50 to about 100 times, about 50 to about 150 times, about 50 to about 200 times, about 50 to about 250 times, about 50 to about 300 times, about 50 to about 350 times, about 50 to about 400 times or more than non-primed cancer cells. In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 100 to about 150 times, about 100 to about 200 times, about 100 to about 250 times, about 100 to about 300 times, about 100 to about 350 times, about 100 to about 400 times or more than non-primed cancer cells. In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 150 to about 200 times, about 150 to about 250 times, about 150 to about 300 times, about 150 to about 350 times, about 150 to about 400 times or more than non-primed cancer cells. In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 200 to about 250 times, about 200 to about 300 times, about 200 to about 350 times, about 200 to about 400 times or more than non-primed cancer cells. In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 250 to about 300 times, about 250 to about 350 times, about 250 to about 400 times or more than non-primed cancer cells. In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 300 to about 350 times, about 300 to about 400 times or more than non-primed cancer cells. In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 350 to about 400 times or more than non-primed cancer cells.In an embodiment, the immune cells include T cells, natural killer (NK) cells, or dendritic cells. In an embodiment, the cancer cells are blood cancer cells. In an embodiment, the blood cancer cells are leukemia cells. In an embodiment, the cancer cells are solid tumor cells. In an embodiment, the cancer cells are circulating tumor cells. In an embodiment, the method further includes collecting at least a portion of the immunologically primed cancer cells and suspending the cells in phosphate buffered saline. In an embodiment, the method further includes transporting at least a portion of the immunologically primed cancer cells to a patient care location. In an embodiment, the patient care location is a hospital. In an embodiment, the patient care location is a cancer center. In an embodiment, the method further includes administering to the patient at least a portion of the transported immunologically primed cancer cells to induce an immune response. In an embodiment, the immune response delays or halts cancer growth in the patient. In an embodiment, the immune response halts cancer metastasis in the patient. In an embodiment, the immune response makes the patient's immune system more efficient in killing cancer cells. In an embodiment, the method further includes administering an effective amount of at least one checkpoint inhibitor.

[0135] A medicament for treating cancer, comprising immunologically primed cancer cells obtained by the method according to the present invention.

[0136] The following steps: receiving cancer cells collected from a patient; and treating the collected cancer cells ex vivo with a compound at a toxic concentration that modifies sphingolipid metabolism to prepare immunologically primed cancer cells, wherein the toxic concentration is sufficient to induce immunogenic cell death in the cancer cells, an immunologically primed cancer cell prepared by the method. In embodiments, the compound that modifies sphingolipid metabolism is an inhibitor of sphingosine kinase. In embodiments, the inhibitor of sphingosine kinase is a selective inhibitor of sphingosine kinase-2 (SK2). In embodiments, the selective inhibitor of SK2 is 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide or a pharmaceutically acceptable salt thereof. In embodiments, the collected cancer cells are treated for at least 24 hours. In embodiments, the toxic concentration of the selective inhibitor of SK2 is from about 20 μM to about 60 μM. In embodiments, the immunologically primed cancer cells overexpress calreticulin on their surface. In embodiments, the immunologically primed cancer cells express calreticulin on their surface at about 3-fold or more (e.g., about 3-fold to about 400-fold or more) than non-primed cancer cells. In some embodiments, the immunologically primed cancer cells express calreticulin on their surface at about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold or more than non-primed cancer cells. In some embodiments, the immunologically primed cancer cells express calreticulin on their surface at about 3-fold to about 10-fold, about 3-fold to about 50-fold, about 3-fold to about 100-fold, about 3-fold to about 150-fold, about 3-fold to about 200-fold, about 3-fold to about 250-fold, about 3-fold to about 300-fold, about 3-fold to about 350-fold, about 3-fold to about 400-fold or more than non-primed cancer cells.In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 10 to about 50 times, about 10 to about 100 times, about 10 to about 150 times, about 10 to about 200 times, about 10 to about 250 times, about 10 to about 300 times, about 10 to about 350 times, about 10 to about 400 times or more, compared to non-primed cancer cells. In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 50 to about 100 times, about 50 to about 150 times, about 50 to about 200 times, about 50 to about 250 times, about 50 to about 300 times, about 50 to about 350 times, about 50 to about 400 times or more, compared to non-primed cancer cells. In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 100 to about 150 times, about 100 to about 200 times, about 100 to about 250 times, about 100 to about 300 times, about 100 to about 350 times, about 100 to about 400 times or more, compared to non-primed cancer cells. In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 150 to about 200 times, about 150 to about 250 times, about 150 to about 300 times, about 150 to about 350 times, about 150 to about 400 times or more, compared to non-primed cancer cells. In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 200 to about 250 times, about 200 to about 300 times, about 200 to about 350 times, about 200 to about 400 times or more, compared to non-primed cancer cells. In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 250 to about 300 times, about 250 to about 350 times, about 250 to about 400 times or more, compared to non-primed cancer cells. In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 300 to about 350 times, about 300 to about 400 times or more, compared to non-primed cancer cells. In some embodiments, immunologically primed cancer cells express calreticulin on their surface at about 350 to about 400 times or more, compared to non-primed cancer cells.In an embodiment, the cancer cells are immune cells. In an embodiment, the immune cells include T cells, natural killer (NK) cells or dendritic cells. In an embodiment, the cancer cells are blood cancer cells. In an embodiment, the blood cancer cells are leukemia cells. In an embodiment, the cancer cells are solid tumor cells. In an embodiment, the cancer cells are circulating tumor cells. In an embodiment, the pharmaceutical composition includes the above immunologically primed cancer cells. In an embodiment, the pharmaceutical composition further includes an effective amount of at least one checkpoint inhibitor. In an embodiment, the use of the above immunologically primed cancer cells in the preparation of a pharmaceutical composition for promoting an immune response in a patient is disclosed. In an embodiment, the immune response delays or halts the growth of cancer in the patient. In an embodiment, the immune response halts cancer metastasis in the patient. In an embodiment, the immune response makes the patient's immune system more efficient in killing cancer cells.

[0137] Description 1: An ex vivo method of immunologically priming cancer cells collected from a patient, the method comprising treating the cancer cells collected from the patient with a compound at a toxic concentration that modifies sphingolipid metabolism to induce immunogenic cell death in the collected cancer cells.

[0138] Description 2: An ex vivo method of producing immunologically primed cancer cells collected from a patient, the method comprising treating the cancer cells collected from the patient with a compound at a toxic concentration that modifies sphingolipid metabolism to induce immunogenic cell death in the collected cancer cells, thereby producing immunologically primed cancer cells.

[0139] Description 3: Ex vivo use of a compound that modifies sphingolipid metabolism to induce immunogenic cell death in cancer cells collected from a patient.

[0140] Description 4a: The method according to Description 1 or Description 2 or the use according to Description 3, wherein the compound that modifies sphingolipid metabolism is an inhibitor of sphingosine kinase.

[0141] Description 4b: The method according to Description 1 or Description 2, or the use according to Description 3, wherein the toxic concentration of the compound that modifies sphingolipid metabolism is from about 20 μM to about 60 μM.

[0142] Description 5: The method or use according to Description 4a or 4b, wherein the inhibitor of sphingosine kinase is a selective inhibitor of sphingosine kinase-2 (SK2).

[0143] Description 6: The method or use according to Description 5, wherein the selective inhibitor of SK2 is 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide or a pharmaceutically acceptable salt thereof.

[0144] Description 7: The method or use according to any one of Descriptions 1, 2, or 4a to 6, wherein the collected cancer cells are treated for at least 24 hours, or the method or use according to any one of Descriptions 3 to 6.

[0145] Description 8: The method or use according to any one of Descriptions 1, 2, or 4a to 7, wherein the cancer cells are immune cells.

[0146] Description 9: The method or use according to Description 8, wherein the immune cells include T cells, natural killer (NK) cells, or dendritic cells.

[0147] Description 10: The method or use according to any one of Descriptions 1, 2, or 4a to 7, wherein the cancer cells are blood cancer cells.

[0148] Description 11: The method or use according to Description 10, wherein the blood cancer cells are leukemia cells.

[0149] Description 12: The method or use according to any one of Descriptions 1, 2, or 4a to 7, wherein the cancer cells are solid tumor cells.

[0150] Description 13: The method according to any one of Descriptions 1, 2, or 4a to 7, or the use according to any one of Descriptions 3 to 7, wherein the cancer cells are circulating tumor cells.

[0151] Description 14: The method according to any one of Descriptions 1, 2, or 4a to 13, or the use according to any one of Descriptions 3 to 13, further comprising transporting at least a portion of the immunologically primed cancer cells to a patient care location.

[0152] Description 15: The method or use according to Description 14, wherein a portion of the transported primed cancer cells comprises stained cancer cells.

[0153] Description 16: The method or use according to Description 14 or 15, wherein the patient care location is a hospital.

[0154] Description 17: The method or use according to Description 14 or 15, wherein the patient care location is a cancer center.

[0155] Description 18: Immunologically primed cancer cells produced by the method according to any one of Descriptions 2 or 4 to 13 for use in medicine.

[0156] Description 19: Immunologically primed cancer cells produced by the method according to any one of Descriptions 2 or 4 to 13 for promoting an immune response against cancer cells in a patient.

[0157] Description 20: Immunologically primed cancer cells produced by the method according to any one of Descriptions 2 or 4 to 13 for use in delaying or halting the growth of cancer in a patient.

[0158] Description 21: Immunologically primed cancer cells produced by the method according to any one of Descriptions 2 or 4 to 13 for use in halting the metastasis of cancer in a patient.

[0159] An immunologically primed cancer cell produced by the method according to any one of descriptions 2 or 4 to 13 for use in making a patient's immune system more efficient when killing cancer cells.

[0160] Description 23: An immunologically primed cancer cell produced by the method according to any one of descriptions 2 or 4 to 13 for use in a method of treating cancer.

[0161] Description 24: An immunologically primed cancer cell for use according to description 20, wherein the method further comprises administering an effective amount of at least one checkpoint inhibitor.

[0162] A method of treating cancer by enhancing or inducing immunogenic cell death in a cell in a subject in need of treatment for cancer, the method comprising administering to the subject an effective amount of a compound that modifies sphingolipid metabolism and an inhibitor of a checkpoint pathway. In an embodiment, the compound that modifies sphingolipid metabolism is an inhibitor of sphingosine kinase. In an embodiment, the compound that is an inhibitor of sphingosine kinase is a selective inhibitor of sphingosine kinase-2 (SK2). In an embodiment, the selective inhibitor of SK2 is 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide or a pharmaceutically acceptable salt thereof. In an embodiment, the immunogenic cell death comprises increased expression of calreticulin on the surface of the cancer cell. In an embodiment, the cancer cell is a melanoma cell. In an embodiment, the cancer cell is a lung cancer cell. In an embodiment, the inhibitor of the checkpoint pathway is an anti-PD-L1 antibody, an anti-PD-1 antibody, or a combination thereof. In an embodiment, the checkpoint inhibitor pathway is an anti-CTLA4 antibody. In an embodiment, The anti-PD-L1 antibody or anti-PD-1 antibody is a monoclonal antibody. In an embodiment, the anti-CTLA4 antibody is a monoclonal antibody. In an embodiment, the monoclonal antibody is a human antibody or a humanized antibody. In an embodiment, the administration is performed multiple times. In an embodiment, a second cancer therapy is further administered to the subject. In an embodiment, the second cancer therapy includes surgery, radiation therapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, or gene therapy.

[0163] A kit comprising 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide compound or a pharmaceutically acceptable salt thereof, at least one checkpoint inhibitor, and instructions for use. In an embodiment, the at least one checkpoint inhibitor is a CTLA-4 receptor inhibitor, a PD-1 receptor inhibitor, a PD-L1 ligand inhibitor, a PD-L2 ligand inhibitor, a LAG-3 receptor inhibitor, a TIM-3 receptor inhibitor, a BTLA receptor inhibitor, a KIR receptor inhibitor, or any combination of the foregoing checkpoint inhibitors. In an embodiment, the checkpoint inhibitor is an antibody or an antibody fragment. In an embodiment, the at least one checkpoint inhibitor is an anti-CTLA-4 receptor antibody, an anti-PD-1 receptor antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, or any combination of the foregoing antibodies. In an embodiment, the at least one checkpoint inhibitor is in the form of a lyophilized solid. In an embodiment, the kit further comprises an aqueous reconstitution solvent. In an embodiment, the at least one checkpoint inhibitor is incorporated into a first pharmaceutically acceptable formulation, and the 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide compound or a pharmaceutically acceptable salt thereof is incorporated into a second pharmaceutically acceptable formulation.

[0164] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. The present invention includes the following aspects. [Item 1] A method comprising preparing immunologically primed cancer cells by treating cancer cells collected from a patient in need of treatment with a compound at a toxic concentration that modifies sphingolipid metabolism ex vivo, wherein the toxic concentration is sufficient to induce immunogenic cell death of the cancer cells. [Item 2] The method according to Item 1, wherein the compound that modifies sphingolipid metabolism is an inhibitor of sphingosine kinase. [Item 3] The method according to Item 2, wherein the inhibitor of sphingosine kinase is a selective inhibitor of sphingosine kinase-2 (SK2). [Item 4] The method according to Item 3, wherein the selective inhibitor of SK2 is 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide or a pharmaceutically acceptable salt thereof. [Item 5] The method according to Item 1, wherein the cancer cells are treated with the compound for at least 24 hours. [Item 6] The method according to Item 3 or 4, wherein the toxic concentration of the selective inhibitor of SK2 is from about 20 μM to about 60 μM. [Item 7] The method according to Item 1, wherein the immunologically primed cancer cells overexpress calreticulin on their surfaces. [Item 8] The method according to Item 1, wherein the cancer cells are immune cells. [Item 9] The method according to Item 1, wherein the cancer cells are blood cancer cells. [Item 10] The method according to Item 1, wherein the cancer cells are solid tumor cells. [Item 11] The method according to Item 1, wherein the cancer cells are circulating tumor cells. [Item 12] The method according to Item 1, further comprising collecting at least a portion of the immunologically primed cancer cells and suspending the cells in phosphate buffered saline. [Item 13] The method according to Item 12, further comprising transporting at least a portion of the immunologically primed cancer cells to the patient's care location. [Item 14] The method according to Item 13, wherein the patient's care location is a hospital. [Item 15] The method according to Item 13, wherein the patient's care location is a cancer center. [Item 16] The method according to Item 13, further comprising administering at least a portion of the transported immunologically primed cancer cells to the patient to induce an immune response. [Item 17] The method according to item 16, wherein the immune response delays or halts cancer growth in the patient. [Item 18] The method according to item 16, wherein the immune response halts cancer metastasis in the patient. [Item 19] The method according to item 16, wherein the immune response makes the patient's immune system more efficient in killing cancer cells. [Item 20] The method according to item 16, further comprising administering an effective amount of at least one checkpoint inhibitor. [Item 21] A method for treating cancer in a patient in need of cancer treatment by enhancing or inducing immunogenic cell death in cancer cells, administering to the patient an effective amount of a compound that modifies sphingolipid metabolism, administering to the patient an effective amount of an inhibitor of the checkpoint pathway, comprising a method. [Item 22] The method according to item 21, wherein the compound that modifies sphingolipid metabolism is an inhibitor of sphingosine kinase. [Item 23] The method according to item 22, wherein the inhibitor of sphingosine kinase is a selective inhibitor of sphingosine kinase-2 (SK2). [Item 24] The method according to item 23, wherein the selective inhibitor of SK2 is 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide or a pharmaceutically acceptable salt thereof. [Item 25] The method according to item 21, wherein the immunogenic cell death comprises increased expression of calreticulin on the surface of the cancer cells. [Item 26] The method according to item 21, wherein the cancer cells are cells derived from a cancer that is commonly treated with a checkpoint inhibitor. [Item 27] The method according to item 26, wherein the cancer is selected from the group consisting of melanoma, Merkel cell carcinoma, squamous cell carcinoma, esophageal squamous cell carcinoma, lung, small cell lung, non-small cell lung kidney, Hodgkin lymphoma, head and neck, mediastinal primary B-cell large cell lymphoma, kidney, bladder, urinary tract, liver, colorectal, cervical, uterine, and gastric cancer. [Item 28] The method according to item 21, wherein the inhibitor of the checkpoint pathway is an anti-PD-L1 antibody, an anti-PD-1 antibody, or a combination thereof. [Item 29] The method according to item 28, wherein the anti-PD-L1 antibody and the anti-PD-1 antibody are monoclonal antibodies. [Item 30] The method according to item 29, wherein the monoclonal antibody is a human antibody or a humanized antibody. [Item 31] The method according to item 21, wherein the inhibitor of the checkpoint pathway is an anti-CTLA4 antibody. [Item 32] The method according to item 31, wherein the anti-CTLA4 antibody is a monoclonal antibody. [Item 33] The method according to item 32, wherein the monoclonal antibody is a human antibody or a humanized antibody. [Item 34] The method according to item 21, wherein the administration is performed multiple times over a sufficient period of time. [Item 35] A kit comprising 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide or a pharmaceutically acceptable salt thereof, at least one checkpoint inhibitor, and instructions for use. [Item 36] The kit according to item 35, wherein the at least one checkpoint inhibitor is a CTLA-4 receptor inhibitor, a PD-1 receptor inhibitor, a PD-L1 ligand inhibitor, a PD-L2 ligand inhibitor, a LAG-3 receptor inhibitor, a TIM-3 receptor inhibitor, a BTLA receptor inhibitor, a KIR receptor inhibitor, or any combination of the checkpoint inhibitors. [Item 37] The kit according to item 35, wherein the at least one checkpoint inhibitor is an antibody or an antibody fragment. [Item 38] The kit according to item 35, wherein the at least one checkpoint inhibitor is an anti-CTLA-4 receptor antibody, an anti-PD-1 receptor antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, or any combination of the antibodies. [Item 39] The kit according to item 35, wherein the at least one checkpoint inhibitor is in the form of a lyophilized solid. [Item 40] The kit according to item 35, further comprising an aqueous reconstitution solvent. [Item 41] The kit according to item 35, wherein the at least one checkpoint inhibitor is incorporated into a first pharmaceutically acceptable formulation, and the 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide is incorporated into a second pharmaceutically acceptable formulation.

Claims

1. A pharmaceutical composition for treating cancer, comprising 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide or a pharmaceutically acceptable salt thereof and an inhibitor of the checkpoint pathway, wherein the inhibitor of the checkpoint pathway is an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CTLA4 antibody or a combination thereof.

2. The pharmaceutical composition according to claim 1, wherein the anti-PD-L1 antibody and the anti-PD-1 antibody are monoclonal antibodies.

3. The pharmaceutical composition according to claim 2, wherein the monoclonal antibody is a human antibody or a humanized antibody.

4. The pharmaceutical composition according to claim 1, wherein the anti-CTLA4 antibody is a monoclonal antibody.

5. The pharmaceutical composition according to claim 4, wherein the monoclonal antibody is a human antibody or a humanized antibody.

6. A kit for treating cancer, comprising 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide or a pharmaceutically acceptable salt thereof, an inhibitor of the checkpoint pathway, and instructions for use, wherein the inhibitor of the checkpoint pathway is one of an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CTLA4 antibody or a combination thereof.

7. The kit according to claim 6, wherein the inhibitor of the checkpoint pathway is an anti-PD-L1 antibody, an anti-PD-1 antibody, or a combination thereof.

8. The kit according to claim 7, wherein the anti-PD-L1 antibody and the anti-PD-1 antibody are monoclonal antibodies.

9. The kit according to claim 8, wherein the monoclonal antibody is a human antibody or a humanized antibody.

10. The kit according to claim 6, wherein the inhibitor of the checkpoint pathway is an anti-CTLA4 antibody.

11. The kit according to claim 10, wherein the anti-CTLA4 antibody is a monoclonal antibody.

12. The kit according to claim 11, wherein the monoclonal antibody is a human antibody or a humanized antibody.

13. A medicament for treating cancer, comprising 3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide or a pharmaceutically acceptable salt thereof, and an inhibitor of the checkpoint pathway, wherein the inhibitor of the checkpoint pathway is one of an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CTLA4 antibody, or a combination thereof.

14. The medicament according to claim 13, wherein the cancer is selected from the group consisting of melanoma, Merkel cell carcinoma, squamous cell carcinoma, esophageal squamous cell carcinoma, lung, small cell lung, non-small cell lung, kidney, Hodgkin lymphoma, head and neck, mediastinal primary B-cell large cell lymphoma, kidney, bladder, urinary tract, liver, colorectal, cervical, uterine, and gastric cancer.

15. The medicament according to claim 13, wherein the inhibitor of the checkpoint pathway is an anti-PD-L1 antibody, an anti-PD-1 antibody, or a combination thereof.

16. The medicament according to claim 15, wherein the anti-PD-L1 antibody and the anti-PD-1 antibody are monoclonal antibodies.

17. The medicament according to claim 16, wherein the monoclonal antibody is a human antibody or a humanized antibody.

18. The medicament according to claim 13, wherein the inhibitor of the checkpoint pathway is an anti-CTLA4 antibody.

19. The medicament according to claim 18, wherein the anti-CTLA4 antibody is a monoclonal antibody.

20. The pharmaceutical according to claim 19, wherein the monoclonal antibody is a human antibody or a humanized antibody.

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