Combined use of ubenimex and immune checkpoint inhibitors

Abstract

The purpose of the present invention is to provide a more effective treatment method for cancer treatment that utilizes the tumor immune system. A further purpose of the present invention is to provide, as a more effective tumor immunotherapy, a drug that enhances antitumor, pro-inflammatory and anti-inflammatory cytokine production. A further purpose of the present invention is to provide, as a more effective tumor immunotherapy, a drug that increases tumor-infiltrating lymphocytes.　This anti-tumor agent has ubenimex as the active component, and is dosed in combination with anti-PD-1 antibodies, an anti-PD-L1 antibodies and / or an anti-CTLA-4 antibodies, which are immune checkpoint inhibitors (ICIs). This ubenimex and ICI combination therapy brings about improvement of the tumor immune system when compared with the use of either of these alone, and brings about many benefits in the treatment of malignant tumors.

Description

Ubenimex in combination with immune checkpoint inhibitors

[0001] The present invention relates to a pharmaceutical for treating malignant tumors, which comprises administering ubenimex in combination with immune checkpoint inhibitors (ICIs) such as an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody.

[0002] In recent years, the relationship between cancer progression and inflammatory cells and immune cells has been elucidated. Based on this knowledge, therapeutic agents based on the tumor immune mechanism have been developed. Ubenimex ((2S)-2-[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoylamino]-4-methylpentanoic acid / Bestatin (registered trademark)) is a drug that exerts antitumor effects by nonspecifically activating macrophages, T cells, NK cells, etc., and / or by binding to the surface of immunocompetent cells via aminopeptidases, thereby activating antitumor immunity. It has been used clinically to "extend survival time in adult acute nonlymphocytic leukemia when used in combination with maintenance and intensification chemotherapy agents after induction of complete remission" (Non-Patent Document 1).

[0003] On the other hand, it has been discovered that cancer cells contain various immune checkpoint molecules that inhibit the immune response against cancer. Research is actively being conducted into therapeutic agents that can reverse this immune suppression mechanism and achieve antitumor effects. Known immune checkpoint molecules include programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1), and cytotoxic T-lymphocyte antigen 4 (CTLA-4), and ICIs targeting these molecules have been developed. PD-1 is expressed on activated T cells, and when it binds to PD-L1 or PD-L2, T cell activity is suppressed, causing dysfunction and suppressing the antitumor immune response. PD-L1 expressed on cancer cells, etc., binds to PD-1 on activated T cells to control T cell activity and suppress the antitumor immune response. CTLA-4 is expressed on regulatory T cells (Tregs) and activated T cells, etc., and inhibits the binding of CD80 / CD86 on antigen-presenting cells to CD28 on T cells, thereby suppressing T cell activity and suppressing the antitumor immune response. Thus, the primary function of immune checkpoint molecules is to suppress T cell activity, and although the targets of ICIs are different, namely PD-1, PD-L1, and CTLA-4, the essence of their action is to relieve the suppression of T cell activity. Thus, the PD-1 / PD-L1 and CTLA-4 signaling pathways are important regulators of tumor immune responses, and anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies have been developed as PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors that block these pathways, achieving remarkable anti-tumor effects in multiple malignant tumors. Anti-PD-1 antibodies include nivolumab (Opdivo®) and pembrolizumab (Keytruda®), anti-PD-L1 antibodies include atezolizumab (Tecentriq®), durvalumab (Imfinzi®), and avelumab (Bavencio®), and anti-CTLA-4 antibodies include ipilimumab (Yervoy®).

[0004] Because the effectiveness of ICI in treating malignant tumors is limited, in order to seek more effective treatment, combination therapies combining multiple antitumor agents have been attempted, and combination therapies including combinations with various antitumor agents such as cytotoxic drugs and molecular targeted drugs, as well as their administration doses and administration schedules, are being considered. Combinations with drugs involved in tumor immunity are also being considered. For example, Patent Document 1 describes a combination therapy of an anti-PD-1 antibody and an anti-CTLA-4 antibody. Regarding combinations with small molecule drugs involved in tumor immunity, Patent Documents 2 and 3 describe the combination of ICI with Smac (pro-apoptotic protein)-like compounds (Smac mimetic compounds (SMC)), which are apoptosis regulators, and Patent Document 4 describes the combination of an inhibitor of apoptosis protein (IAP) antagonist and an anti-PD-1 molecule.

[0005] Pharmaceutical interview form: "Antineoplastic agent Bestatin Capsules 10 mg, Bestatin Capsules 30 mg" (Nippon Kayaku Co., Ltd., October 2020 (revised 7th edition))

[0006] Japanese Patent Application Laid-Open No. 2012-158605 Special Publication No. 2019-506438 Special Publication No. 2020-515600 Special Publication No. 2021-506781

[0007] An objective of the present invention is to provide a more effective cancer treatment method utilizing the tumor immune mechanism. Another objective of the present invention is to provide a drug that enhances the production of antitumor cytokines as a more effective tumor immunotherapy. Alternatively, an objective of the present invention is to provide a drug that increases tumor-infiltrating lymphocytes as a more effective tumor immunotherapy.

[0008] The present inventors have found that combined therapy of ubenimex and ICI improves tumor immune function and provides many benefits in the treatment of malignant tumors, compared to the use of either agent alone. The present application provides the following inventions: [1] An antitumor agent containing ubenimex as an active ingredient, for administration in combination with immune checkpoint inhibitors such as anti-PD-1 antibody, anti-PD-L1 antibody, and / or anti-CTLA-4 antibody. [2] The antitumor agent according to [1], wherein the dose of ubenimex is such that the area under the blood concentration-time curve (AUC) after a single administration is 3.00 to 50.00 μg hr / mL. [3] The antitumor agent according to [1] or [2], wherein ubenimex is administered daily.

[0009] [4] An antitumor agent containing, as an active ingredient, an ICI selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-CTLA-4 antibody, for administration in combination with ubenimex. [5] The antitumor agent according to [4], wherein the dose of ubenimex is such that the AUC after a single administration is 3.00 to 50.00 μg hr / mL.

[0010] The combination of ubenimex and ICI enhances the production of antitumor cytokines that contribute to the activation mechanism of tumor immunity. Thus, the present invention provides use as an antitumor cytokine-producing agent. [6] A pharmaceutical comprising ubenimex as an active ingredient for the production of antitumor cytokines by administration in combination with immune checkpoint inhibitors such as anti-PD-1 antibody, anti-PD-L1 antibody, and / or anti-CTLA-4 antibody. [7] The pharmaceutical according to [6], wherein the dose of ubenimex is such that the AUC after a single administration is 3.00 to 50.00 μg hr / mL.

[0011] The combination of ubenimex and ICI has the effect of increasing cytotoxic T cells (CTLs), a key factor in tumor immunity, in tumors, contributing to effective tumor immunotherapy. Therefore, the present invention provides use as a tumor-infiltrating lymphocyte activator. [8] A pharmaceutical containing ubenimex as an active ingredient for increasing tumor-infiltrating lymphocytes by administering it in combination with immune checkpoint inhibitors, such as anti-PD-1 antibody, anti-PD-L1 antibody, and / or anti-CTLA-4 antibody. [9] The pharmaceutical according to [8], wherein the dose of ubenimex is such that the AUC after a single administration is 3.00 to 50.00 μg hr / mL.

[0012] The combination of ubenimex and ICI provides a pharmaceutical application applicable to the treatment of a wide range of malignant tumors.

[10] A pharmaceutical containing ubenimex as an active ingredient, which is used in the treatment of malignant tumors selected from the group consisting of small cell lung cancer, non-small cell lung cancer, esophageal cancer, head and neck cancer, brain tumor, colorectal cancer, colon adenocarcinoma, gastric cancer, breast cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, kidney cancer, prostate cancer, bladder cancer, ovarian cancer, cervical cancer, thyroid cancer, and melanoma, by administering it in combination with immune checkpoint inhibitors such as anti-PD-1 antibody, anti-PD-L1 antibody, and / or anti-CTLA-4 antibody.

[11] The pharmaceutical according to

[10] , wherein the dose of ubenimex is such that the area under the blood concentration-time curve (AUC) after a single administration is 3.00 to 50.00 μg hr / mL.

[12] The medicine according to

[10] or

[11] , wherein ubenimex is administered daily.

[0013] The combination of ubenimex and ICI enhances the antitumor effect compared to the use of either drug alone, and also increases antitumor cytokines and tumor-infiltrating lymphocytes, providing a more effective tumor immunotherapy.

[0014] 1 shows the time course of the average tumor volume for each group in a test of the antitumor effect of administration of ubenimex and anti-mouse PD-1 antibody (J43) on mouse melanoma cells B16-F0. This figure shows the individual values ​​of the relative tumor volume ratio (T / C) for each group to the average tumor volume for the control group 15 days after transplantation in a test of the antitumor effect of administration of ubenimex and anti-mouse PD-1 antibody (J43) on mouse melanoma cells B16-F0. This figure shows plots of individual and average values ​​for plasma IFN-γ for each group after administration of ubenimex and anti-mouse PD-1 antibody (J43). Error bars indicate standard deviation. (*: P<0.05) This figure shows plots of individual and average values ​​for plasma TNF-α for each group after administration of ubenimex and anti-mouse PD-1 antibody (J43). Error bars indicate standard deviation. (*: P<0.05) Figure 1 shows individual plots and average values ​​of the number of CD8-positive T cells per 10,000 viable cells in a single-cell suspension dispersed from tumor fragments following administration of ubenimex and anti-mouse PD-1 antibody (J43). Error bars indicate standard deviation. Figure 1 shows the correlation between the number of CD8-positive T cells and relative tumor volume following administration of ubenimex and anti-mouse PD-1 antibody (J43). The horizontal axis shows the number of CD8-positive T cells per 10,000 viable cells, and the vertical axis shows the relative tumor volume ratio (TGI) for each individual relative to the mean tumor volume in the control group. The area enclosed by a dotted line indicates an individual with a high number of CD8-positive T cells in the group showing significant tumor growth inhibition. Figure 1 shows individual plots and average values ​​of the number of regulatory T lymphocytes (Treg) in peripheral blood following administration of ubenimex and anti-mouse PD-1 antibody (J43). Error bars indicate standard deviation. This figure shows the correlation between the number of Tregs in peripheral blood and relative tumor volume following administration of ubenimex and an anti-mouse PD-1 antibody (J43). The horizontal axis shows the number of Tregs in peripheral blood, and the vertical axis shows the relative tumor volume ratio (TGI) for each individual relative to the mean tumor volume in the control group. The area enclosed by the dotted line indicates individuals with low Treg counts in a group showing significant tumor growth inhibition. This figure shows the time course of the mean tumor volume for each group in a test of the antitumor effect of administration of ubenimex and an anti-mouse PD-1 antibody (J43) on mouse colon adenocarcinoma cell line MC38.(*: P<0.05, **: P<0.01) This figure shows the change over time in the mean plasma IFN-γ levels in each group following administration of ubenimex and anti-mouse PD-1 antibody (J43). (**: P<0.01) This figure shows the change over time in the mean plasma TNF-α levels in each group following administration of ubenimex and anti-mouse PD-1 antibody (J43). (*: P<0.05, **: P<0.01) This figure shows the change over time in the mean plasma IL-6 levels in each group following administration of ubenimex and anti-mouse PD-1 antibody (J43). (*: P<0.05) This figure shows the change over time in the mean plasma KC / GRO (IL-8) levels in each group following administration of ubenimex and anti-mouse PD-1 antibody (J43). This figure shows the change over time in the mean plasma IL-10 levels in each group following administration of ubenimex and anti-mouse PD-1 antibody (J43). (**: P<0.01).

[0015] The gist of the present invention is an antitumor agent that combines ubenimex and ICI. Details are described below. Ubenimex is (2S)-2-[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoylamino]-4-methylpentanoic acid, a dipeptide compound with aminopeptidase inhibitory activity. In the present invention, ubenimex may be used in the form of a free salt or a pharmaceutically acceptable salt such as hydrochloride or phosphate. Ubenimex is available in 10 mg and 30 mg formulations under the clinical application of "Bestatin (registered trademark) Capsules," and these may also be used.

[0016] The dosage of ubenimex may be a pharmaceutically effective amount. Preferably, a single dose is administered at a dose that results in an AUC of greater than 3.00 μg·hr / mL but less than 50.00 μg·hr / mL. Also, a maximum blood concentration (Cmax) of greater than 2.0 μg / mL is preferably administered. A more preferred dosage is one that results in an AUC of greater than 5.00 μg·hr / mL but less than 50.00 μg·hr / mL, with a dosage of greater than 10.00 μg·hr / mL but less than 50.00 μg·hr / mL being particularly preferred. Ubenimex may be administered orally or parenterally, such as intravenously. Oral administration is preferred. Ubenimex is preferably administered daily, and preferably orally daily throughout the treatment cycle.

[0017] Anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies are therapeutic agents that bind to PD-1, PD-L1, or CTLA-4, which are immune checkpoint molecules that regulate tumor immunity, and block the PD-1 / PD-L1 or CTLA-4 signaling pathway, thereby relieving the inhibitory mechanism against activated T cells and achieving an anti-tumor effect. The anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies of the present invention can be used without particular limitation, as long as they bind to PD-1, PD-L1, or CTLA-4 and have the ability to relieve tumor immunity suppression function. Many anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies are commercially available or under development, and these may also be used. Examples of anti-PD-1 antibodies include nivolumab (Opdivo (registered trademark)) and pembrolizumab (Keytruda (registered trademark)). Pidilizumab (CT-011), PDR-001, JS001, STI-A1110, AMP-224, AMP-514 (MEDI0680), etc. may also be used. Anti-PD-L1 antibodies include atezolizumab (Tecentriq (registered trademark)), durvalumab (Imfinzi (registered trademark)), and avelumab (Bavencio (registered trademark)). BMS-936559 (MDX1105), LY3300054, etc. may also be used. Anti-CTLA-4 antibodies include ipilimumab (Yervoy (registered trademark)). Tremelimumab (CP-675206), etc. may also be used. In the present invention, the ICI used in combination with ubenimex is preferably an anti-PD-1 antibody and / or anti-PD-L1. A combination of ubenimex and an anti-PD-1 antibody is more preferred.

[0018] The anti-PD-1 antibody, anti-PD-L1 antibody, or anti-CTLA-4 antibody may be used in a pharmaceutically effective amount. For example, when using the anti-PD-1 antibody nivolumab (Opdivo (registered trademark)), it is usually administered by intravenous infusion at a dose of 240 mg every two weeks. When using pembrolizumab (Keytruda (registered trademark)), it is usually administered by intravenous infusion at a dose of 200 mg every three weeks. When using the anti-PD-L1 antibody atezolizumab (Tecentriq (registered trademark)), it is usually administered by intravenous infusion at a dose of 840 mg every two weeks, or at a dose of 1200 mg every three weeks. When using durvalumab (Imfinzi®), it is usually administered by intravenous infusion at a dose of 10 mg / kg every two weeks, or 1500 mg every three weeks. When using avelumab (Bavencio®), it is usually administered by intravenous infusion at a dose of 10 mg / kg every two weeks. When using ipilimumab (Yervoy®), an anti-CTLA-4 antibody, it is usually administered by intravenous infusion at a dose of 3 mg / kg every three weeks for four doses.

[0019] The present invention relates to a combination therapy using ubenimex and an ICI in combination. The combination therapy of the present invention refers to the simultaneous or sequential administration of ubenimex and an ICI, and includes a series of treatment methods including the administration of ubenimex and an ICI in a treatment plan including the administration of multiple drugs. Simultaneous administration includes the administration of both drugs at substantially the same time. Sequential administration includes the administration of one drug for one or several days, followed by an optional drug holiday, followed by the administration of the other drug. In the present invention, a combination therapy in which ubenimex is administered daily and an ICI is administered every 1 to 3 weeks during that administration period is preferred.

[0020] The combination therapy of ubenimex and ICI of the present invention is provided for use in treating malignant tumors, including small cell lung cancer, non-small cell lung cancer, esophageal cancer, head and neck cancer, brain tumor, colorectal cancer, colon adenocarcinoma, gastric cancer, breast cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, kidney cancer, prostate cancer, bladder cancer, ovarian cancer, cervical cancer, thyroid cancer, and melanoma. For example, it is preferably used to treat non-small cell lung cancer.

[0021] The combined therapy of ubenimex and ICI produces a tumor growth inhibitory effect superior to the antitumor effect achieved by administration of either drug alone. That is, the present invention provides a new use of ubenimex in combination with ICI, or encompasses a new use of ICI in combination with ubenimex.

[0022] Ubenimex and ICI are drugs that suppress tumor growth via antitumor immunity. Ubenimex activates macrophages, NK cells, T cells, bone marrow cells, etc., and exerts its antitumor effect by directly activating these cells and by sequentially activating the immune system network through various cytokines, such as interleukin-1 and -2, produced by these cells. ICI inhibits the binding of PD-1 / PD-L1 and / or CTLA-4, thereby releasing immunosuppressive signals and achieving antitumor effects by activating cancer antigen-specific T cells and cytotoxicity. The present invention has discovered that combined administration of ubenimex and ICI enhances antitumor immunity compared to the use of either drug alone, and also provides a new use of these drugs as antitumor immunity enhancers.

[0023] In another embodiment of the present invention, the combined administration of ubenimex and ICI has the effect of enhancing the production of antitumor cytokines such as interferon (IFN)-γ and TNF-α, inflammatory cytokines such as IL-6 and IL-8, and anti-inflammatory cytokines such as IL-10. As described above, ubenimex is known to activate immune cells, resulting in the production of cytokines such as IL-1 and IL-2. However, administration of ubenimex in combination with ICI clearly enhances the production of antitumor and inflammatory cytokines compared to administration of ubenimex alone. Therefore, the present invention provides a use of ubenimex as an active ingredient in combination with ICI as an agent for enhancing the production of antitumor and inflammatory cytokines. Ubenimex may be administered in a pharmaceutically effective dose to induce the production of antitumor and inflammatory cytokines. Preferably, a single dose is administered such that the AUC is greater than 3.00 μg hr / mL and less than 50.00 μg hr / mL. Preferably, the dose is applied at a level that results in a Cmax of more than 2.0 μg / mL, more preferably at a level that results in an AUC in the range of more than 5.00 μg hr / mL but less than 50.00 μg hr / mL, and even more preferably at a level that results in an AUC in the range of more than 10.00 μg hr / mL but less than 50.00 μg hr / mL.

[0024] In another aspect of the present invention, the combined administration of ubenimex and ICI has the effect of enhancing the induction of tumor-infiltrating lymphocytes. As described above, ubenimex and ICI have the effect of inducing and activating CTLs. However, administration of ubenimex in combination with ICI results in the induction of more tumor-infiltrating lymphocytes than administration of ubenimex alone. Therefore, the present invention provides a use of ubenimex as an active ingredient in a tumor-infiltrating lymphocyte induction enhancer administered in combination with ICI. To induce tumor-infiltrating lymphocytes, ubenimex may be administered at a pharmaceutically effective dose. Preferably, a single dose is administered at a dose such that the AUC is greater than 3.00 μg hr / mL and less than 50.00 μg hr / mL. Furthermore, a dose such that the Cmax is greater than 2.0 μg / mL is preferably administered. A more preferred dose is one that results in an AUC in the range of greater than 5.00 μg·hr / mL and less than 50.00 μg·hr / mL, and a particularly preferred dose is one that results in an AUC in the range of greater than 10.00 μg·hr / mL and less than 50.00 μg·hr / mL.

[0025] In another embodiment of the present invention, the combined administration of ubenimex and ICI has the effect of suppressing the induction of Tregs, which are suppressive factors in anti-tumor immunity. Tumor growth suppression via anti-tumor immunity is effective when it functions in conjunction with the induction and activation of CTLs that directly attack cancer cells, as well as the release of tumor immune suppression mechanisms. As described above, the combined administration of ubenimex and ICI according to the present invention suppresses the induction of Tregs while inducing tumor-infiltrating lymphocytes to cancer cells, thereby providing efficient anti-tumor immunotherapy. To suppress the induction of Tregs, ubenimex may be administered at a pharmaceutically effective dose. Preferably, a single dose is administered at a dose that results in an AUC of greater than 3.00 μg hr / mL and less than 50.00 μg hr / mL. Preferably, a dose that results in a Cmax of greater than 2.0 μg / mL is administered. A more preferred dose is one that results in an AUC in the range of greater than 5.00 μg·hr / mL and less than 50.00 μg·hr / mL, and a particularly preferred dose is one that results in an AUC in the range of greater than 10.00 μg·hr / mL and less than 50.00 μg·hr / mL.

[0026] The combination therapy of ubenimex and ICI of the present invention may further include other antitumor agents. The other antitumor agents are not particularly limited, and any approved pharmaceuticals can be used. For example, alkylating agents such as cyclophosphamide, ifosfamide, and temozolomide; anthracycline antitumor agents such as doxorubicin, epirubicin, pirarubicin, amrubicin, and doxorubicin-encapsulated liposomal formulation (Doxil®); antitumor platinum complexes such as cisplatin, carboplatin, and oxaliplatin; etoposides such as etoposide, etoposide phosphate, and teniposide; camptothecins such as irinotecan and nogitecan; and taxanes such as paclitaxel, docetaxel, cabazitaxel, and albumin-bound paclitaxel (Abraxane®). Vinca alkaloids such as vincristine, vinblastine, vindesine, and vinorelbine. Tubulin polymerization inhibitors such as eribulin. Nucleic acid antimetabolites such as 5-fluorouracil, tegafur, tegafur / uracil combination (UFT), tegafur / gimeracil / oteracil potassium combination (S-1), trifluridine / tipiracil combination (Lonsurf (registered trademark)), furtulon, capecitabine, gemcitabine, cytosine arabinoside, and azacitidine. Folate antimetabolites such as methotrexate and pemetrexed. Steroids such as prednisolone and dexamethasone. Also included are tyrosine kinase inhibitors such as erlotinib, lapatinib, gefitinib, afatinib, osimertinib, axitinib, sunitinib, sorafenib, regorafenib, lenvatinib, alectinib, crizotinib, and pazopanib. Proteasome inhibitors such as bortezomib, carfilzomib, and ixazomib. Cyclin-dependent kinase inhibitors such as palbociclib. mTOR inhibitors such as everolimus and temsirolimus. Anti-growth factor (receptor) antibodies such as trastuzumab, cetuximab, necitumumab, bevacizumab, panitumumab, and ramucirumab. Other known antitumor agents may also be used. It is preferable to use other antitumor agents in combination in a pharmaceutically effective amount using a known method of administration.

[0027] When the present invention is applied as a therapeutic agent for non-small cell lung cancer, it is preferably applied in combination with known therapeutic methods. For example, paclitaxel, albumin-suspended paclitaxel (Abraxane (registered trademark)), docetaxel, tegafur-gimeracil-oteracil potassium combination (S-1), gemcitabine, pemetrexed, vinorelbine, gefitinib, erlotinib, afatinib, osimertinib, necitumumab, etc. are preferably used in combination as concomitant drugs. It is also preferable to combine these antitumor agents with chemotherapy that further combines an antitumor platinum complex of cisplatin or carboplatin. More preferred are combinations of paclitaxel and an antitumor platinum complex, combinations of albumin-suspended paclitaxel (Abraxane (registered trademark)) and an antitumor platinum complex, combinations of pemetrexed and an antitumor platinum complex, and combinations of tegafur-gimeracil-oteracil potassium (S-1) and an antitumor platinum complex. These other antitumor agents used in combination are preferably administered at dosages and methods that have been confirmed to be effective in treating non-small cell lung cancer.

[0028] The present invention will be described in more detail below with reference to the following examples. Materials used in the examples were obtained as follows and subjected to the respective tests. (1) Tumor cells Mouse melanoma B16-F0 was obtained from the ATCC (The American Type Culture Collection). These were cultured in DMEM (Thermo Fisher Scientific Inc.) containing 10% fetal bovine serum (Corning International, Inc.) and 100 μg / mL kanamycin sulfate under CO . ２The mouse colon adenocarcinoma cell line MC38 was obtained from GenOway S.A. and cultured in an incubator (37°C, 5% carbon dioxide). This was then transferred to DMEM (Thermo Fisher Scientific Inc.) containing 10% fetal bovine serum (Corning International, Inc.), 2 mM L-glutamine (Thermo Fisher Scientific Inc.), 10 mM HEPES (Thermo Fisher Scientific Inc.), 1 mM sodium pyruvate (Fujifilm Wako Pure Chemical Corporation), 0.1 mM MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific Inc.), and penicillin / streptomycin (Fujifilm Wako Pure Chemical Corporation). Scientific Inc.) ２ The cells were cultured in an incubator (37°C, 5% carbon dioxide) and used. (2) Test Substance Ubenimex was obtained from Nippon Kayaku Co., Ltd. Anti-mouse PD-1 antibody (clone J43) and isotype control antibody IgG (polyclonal Armenian hamster IgG) were obtained from BioXcell. Ubenimex was dissolved in distilled water for injection (Otsuka Pharmaceutical Factory, Inc.) and used. The anti-mouse PD-1 antibody and IgG were diluted with PBS(-) (Thermo Fisher Scientific Inc.).

[0029] Example 1 The antitumor effect of combined administration of ubenimex and anti-mouse PD-1 antibody was evaluated using a subcutaneous transplant model of mouse melanoma cells B16-F0. At the same time, changes in cytokines and immune system markers were also monitored. Female C57BL / 6N mice (Charles River Biosciences Japan) were subcutaneously transplanted with 2 x 10 ５A subcutaneous transplantation model was prepared by transplanting B16-F0 cells. Ubenimex was orally administered to the B16-F0 subcutaneous transplantation model mice at a dose of 5 or 50 mg / kg once daily from the day after transplantation until 14 days after transplantation, for a total of 14 doses. Furthermore, an anti-mouse PD-1 antibody was intraperitoneally administered at a dose of 200 μg / body on days 4, 7, 11, and 14 after transplantation. As a control, an IgG antibody, which is an isotype control for the anti-mouse PD-1 antibody, was administered under the same conditions as the anti-mouse PD-1 antibody. The composition of each administration group is summarized in Table 1.

[0030] *: 4, 7, 11, 14 days after transplantation

[0031] The major and minor diameters of the tumor were measured using a digital caliper (Mitutoyo Corporation), and the tumor volume (mm ３ ) to "long diameter x short diameter" ２ Using the tumor volume values ​​15 days after implantation, the relative tumor volume (T / C) of each administration group to the control group was calculated using the formula "mean tumor volume of test substance administration group / mean tumor volume of control group × 100."

[0032] All animals were necropsied 15 days after transplantation, the day after the final administration. On the day of necropsy, blood was collected from the jugular vein using a syringe and needle containing EDTA-2K solution, and the total white blood cell count was measured using an XN-2000V automated hematology analyzer (Sysmex Corporation). Under anesthesia, blood was collected from the abdominal aorta using a syringe and needle containing heparin sodium solution, followed by collection of the tumor. Blood and tumor tissue were used for immunophenotyping. Cytokines were measured using ELISA in plasma obtained by centrifugation of the blood. Statistical analysis was performed using the statistical analysis software EXSUS (ver. 10.0, CAC Clore Co., Ltd.) for the control group, each treatment group, the combination group, and each single-drug group. Two-sided statistical analysis was performed with a significance level of P<0.05.

[0033] [Example 1.1] Antitumor Effect The progression of tumor growth in each administration group is shown in Figure 1. In terms of the relative mean tumor volume (T / C) compared to the control group (IgG administration) 15 days after implantation, the ubenimex 5 mg / kg and 50 mg / kg administration groups and the anti-PD-1 antibody administration group showed no tumor growth inhibitory effect compared to the control group, whereas the T / C of the group administered a high dose of ubenimex in combination with an anti-PD-1 antibody was 68%, demonstrating a tumor growth inhibitory effect. It was confirmed that the combined use of ubenimex and an anti-PD-1 antibody exerted a tumor growth inhibitory effect that was not observed when either antibody was administered alone. When the antitumor effect of Example 1.1 was analyzed using individual values ​​of relative tumor volume, the number of individuals showing a T / C of less than 100% 15 days after transplantation was less than half of the groups treated with each single agent, whereas in both the group treated with low-dose ubenimex and anti-PD-1 antibody and the group treated with high-dose ubenimex and anti-PD-1 antibody, the number was 5 out of 8, which is the majority (Figure 2).

[0034] [Example 1.2] Antitumor cytokine production The concentrations of IFN-γ and TNF-α as antitumor cytokines in plasma in each administration group were measured using LevisMouse IFN-γ ELISA kit and LevisMouse TNF-α ELISA kit (Fujifilm Wako Shibayagi Co., Ltd.). The results are shown in Figures 3 and 4.

[0035] The combination group of high-dose ubenimex and anti-PD-1 antibody showed a clear enhancement of IFN-γ production compared to the control group, each dose of ubenimex alone, and the anti-PD-1 antibody alone group. The high-dose ubenimex combination group showed a significant increase compared to the control group and the high-dose ubenimex alone group (P value 0.02, Figure 3). Furthermore, the combination group of ubenimex and anti-PD-1 antibody showed an enhancement of TNF-α production compared to the control group, each dose of ubenimex alone, and the anti-PD-1 antibody alone group. The combination group of high-dose ubenimex and anti-PD-1 antibody showed a significant increase compared to the control group and the high-dose ubenimex alone group (P value 0.045, Figure 4). IFN-γ and TNF-α are known as anti-tumor cytokines that contribute to anti-tumor effects by cascadingly activating the immune system network. The combined use of ubenimex and anti-PD-1 antibody showed tumor growth inhibitory effects, suggesting that IFN-γ and TNF-α may contribute to tumor growth inhibition.

[0036] [Example 1.3] Immunophenotyping of tumor-infiltrating lymphocytes After confirming the antitumor effect, tumor tissues collected by autopsy were analyzed for tumor-infiltrating lymphocytes by flow cytometry. Immunophenotyping was performed using anti-mouse CD45 antibody, anti-mouse CD3 antibody, anti-mouse CD4 antibody, anti-mouse CD8 antibody, anti-mouse / human CD11b antibody, anti-mouse CD25 antibody, anti-mouse / human FOXP3 antibody, and anti-mouse CD16 / 32 antibody (BioLegend), Lysing Buffer (BD Biosciences), True-Nuclear Transcription Factor Buffer Set (BioLegend), and Tumor Dissociation Kit, mouse (Milteny Biotec). Tumor fragments were minced in the enzyme solution of the Tumor Dissociation Kit, mechanically dissociated using a gentleMACS Octo Dissociator, and then incubated at 37°C for 40 minutes with gentle shaking. After incubation, mechanical dispersion was repeated. 10% FBS-containing RPMI 1640 was added, and the cells were passed through a 70 μm cell strainer and centrifuged. The cell mass was then resuspended in 10% FBS-containing RPMI 1640, and this was used as a single-cell suspension prepared by dissociating the tumor fragments. A portion of the single-cell suspension was collected, hemolyzed with lysing buffer, and subjected to Fc receptor blocking with anti-mouse CD16 / 32 antibody. The cell surface markers CD45, CD3, CD4, CD8, and CD11b were stained and measured using a flow cytometer LSRII (BD Biosciences). Evaluation was performed using CD8-positive T cells (CD45+, CD3+, CD8+), which are CTLs. The CD8-positive T cell rate was calculated from the total viable cell fraction count and the CD8-positive T cell count in the single-cell suspension measured by flow cytometer. The total number of CD8-positive T cells in the single-cell suspension was calculated using the CD8-positive T cell rate, which was calculated from the total viable cell fraction count and the CD8-positive T cell count. The CD8-positive T cell rate was calculated as the number of CD8-positive T cells per 10,000 viable cells. The results for the number of CD8-positive T cells in each treatment group are shown in Figure 5.

[0037] The number of CD8-positive T cells in the tumor was found to be high in some individuals in the group receiving the combination of high-dose ubenimex and an anti-PD-1 antibody. When examining the correlation between the number of CD8-positive T cells and relative tumor volume, individuals in each combination group showing significant tumor growth inhibition were found to coincide with individuals showing high numbers of CD8-positive T cells. The induction of CD8-positive T cells, which contribute to the tumor growth inhibitory effect, was observed not only with the combination of high-dose ubenimex but also with the combination of low-dose ubenimex (Figure 6). CD8-positive T cells act as CTLs to damage tumor cells. Therefore, the combination of ubenimex and an anti-PD-1 antibody can induce CTLs in tumors and exert a tumor growth inhibitory effect.

[0038] [Example 1.4] Immunophenotyping of Peripheral Blood Lymphocytes After confirming the antitumor effect, blood collected at autopsy was analyzed for peripheral blood lymphocytes by flow cytometry. Peripheral blood was hemolyzed with lysing buffer to prepare peripheral blood mononuclear cells (PBMCs). Fc receptors were blocked with anti-mouse CD16 / 32 antibody, and cell surface markers CD45, CD3, CD4, CD8, and CD25 were stained. PBMCs were fixed and permeabilized, stained for the nuclear factor FOXP3, and measured using a flow cytometer LSRII (BD Biosciences). Evaluation was performed using Tregs identified as CD45+, CD3+, CD4+, CD25+, and FOXP3+ as indicators. The proportion of Tregs present in CD45-positive cells was calculated, and the number of Tregs per μL of peripheral blood was calculated from the total white blood cell count. The results for the number of Tregs in each administration group are shown in Figure 7.

[0039] The number of Tregs in peripheral blood was low in the anti-PD-1 antibody monotherapy group and in the combination therapy with low and high doses of ubenimex (Figure 7). On the other hand, no change was observed in the ubenimex monotherapy group. When examining the correlation between Treg counts and relative tumor volume, individuals in the combination therapy group showing significant tumor growth inhibition were consistent with individuals showing low Treg counts. Not only high-dose ubenimex but also low-dose ubenimex combination therapy demonstrated an inhibitory effect on Treg induction, contributing to the tumor growth inhibitory effect (Figure 8). Tregs have the ability to suppress the activation or damage CTLs, which play a key role in tumor immunity. Therefore, the reduction of Tregs promotes tumor immunity and contributes to tumor growth inhibition.

[0040] Immunophenotyping revealed that the combined administration of ubenimex and anti-PD-1 antibody suppressed the induction of Tregs and induced CTLs in the tumor, suggesting that it is possible to promote tumor immunity locally in the tumor. Based on the above, it was demonstrated that the combined administration of ubenimex and anti-PD-1 antibody can exert anti-tumor effects by activating multiple tumor immune system networks, such as increasing the number of CTLs in the tumor, increasing the production of anti-tumor cytokines, and reversing tumor immune tolerance, based on the lymphocyte activation effect of ubenimex.

[0041] Example 2: Time course of blood concentrations in mice. Fasted 9-week-old female C57BL / 6N mice (Charles River Japan, Inc.) were orally administered a single dose of ubenimex at 5 mg / kg or 50 mg / kg, with nine mice per group. Blood was collected at nine time points: 5, 10, 30 minutes, 1, 3, 6, 8, 12, and 24 hours after administration. From 5 minutes to 6 hours after administration, 0.1 mL of blood was collected from the jugular vein under isoflurane anesthesia. From 8 hours after administration onward, whole blood was collected from the caudal vena cava under isoflurane anesthesia. To ensure a total of three blood samples per time point, blood was collected from each animal at three time points, with sparse blood collection in groups of three. Plasma was prepared from the collected blood, and plasma ubenimex concentrations were measured using a liquid chromatograph mass spectrometer (LC / MS / MS). Pharmacokinetic parameters at each dose are shown in Table 2.

[0042]

[0043] Cmax was proportional to the dose, and MRTinf was constant regardless of the dose. The 50 mg / 5 mg ratio of AUCinf tended to be greater than the dose ratio, but dose proportionality was generally achieved in mice at doses of 5 to 50 mg / kg.

[0044] Example 3 Antitumor Effect The antitumor effect of combined administration of ubenimex and anti-mouse PD-1 antibody was evaluated using a subcutaneously transplanted mouse colon adenocarcinoma cell line MC38 model. Female C57BL / 6N mice (Charles River Biosciences Japan) were subcutaneously injected with 1 x 10 ５ A subcutaneous transplantation model was prepared by transplanting 100 MC38 cells. Ubenimex was orally administered to the MC38 subcutaneous transplantation model mice at a dose of 0.5 or 5 mg / kg once daily from the day after transplantation until 24 days after transplantation, for a total of 24 doses. Furthermore, an anti-mouse PD-1 antibody was intraperitoneally administered at a dose of 50 μg / body on days 7, 11, 14, 18, 21, and 24 after transplantation. As a control, an IgG antibody, which is an isotype control for the anti-mouse PD-1 antibody, was administered under the same conditions as the anti-mouse PD-1 antibody. The composition of each administration group is summarized in Table 3.

[0045] *: 7, 11, 14, 18, 21, 24 days after transplantation

[0046] The major and minor diameters of the tumor were measured using a digital caliper (Mitutoyo Corporation), and the tumor volume (mm ３ ) to "long diameter x short diameter" ２ Using the tumor volume values ​​25 days after implantation, the relative tumor volume (T / C) of each administration group to the control group was calculated using the formula "mean tumor volume of test substance administration group / mean tumor volume of control group × 100."

[0047] Statistical analysis was performed using the statistical analysis software EXSUS (ver. 10.0, EP Clore Co., Ltd.) using a Wilcoxon test on tumor volume values ​​25 days after implantation for the control group, each treatment group, the combination group, and each single-agent group. Two-sided statistical analysis was performed with significance levels of P<0.05 and P<0.01.

[0048] The progression of tumor growth in each administration group is shown in Figure 9. The relative mean tumor volume (T / C) of the ubenimex monotherapy or anti-PD-1 antibody monotherapy groups compared to the control group (IgG administration) on day 25 after transplantation was 62% for the ubenimex 0.5 mg / kg group, 51% for the ubenimex 5 mg / kg group, and 33% for the anti-PD-1 antibody group, demonstrating antitumor efficacy in all groups. The T / C of the combination groups was 37% for the ubenimex 0.5 mg / kg and anti-PD-1 antibody combination group and 19% for the ubenimex 5 mg / kg and anti-PD-1 antibody combination group. A dose-dependent antitumor effect of ubenimex was confirmed. Furthermore, the combination of ubenimex 5 mg / kg and anti-PD-1 antibody showed a stronger effect than the single agents (P value vs. control group: 0.002, P value vs. ubenimex 5 mg / kg group: 0.015, P value vs. ubenimex 0.5 mg / kg and anti-PD-1 antibody combination group: 0.039, Figure 9).

[0049] Example 4 Cytokine Production Using a subcutaneously transplanted mouse colon adenocarcinoma cell line MC38, changes in plasma cytokine levels following combined administration of ubenimex and an anti-mouse PD-1 antibody were evaluated. Female C57BL / 6N mice (Charles River Biosciences Japan) were subcutaneously injected with 1 x 10 ５ A subcutaneous transplantation model was prepared by transplanting 100 MC38 cells. Ubenimex was orally administered to the MC38 subcutaneous transplantation model mice at a dose of 5 mg / kg once daily from the day after transplantation until 24 days after transplantation, for a total of 24 doses. Furthermore, an anti-mouse PD-1 antibody was intraperitoneally administered at a dose of 50 μg / body on days 7, 11, 14, 18, 21, and 24 after transplantation. As a control, an IgG antibody, which is an isotype control for the anti-mouse PD-1 antibody, was administered under the same conditions as the anti-mouse PD-1 antibody. The composition of each administration group is summarized in Table 4.

[0050] *: 7, 11, 14, 18, 21, 24 days after transplantation

[0051] On days 7, 14, 21, or 24 after transplantation, blood was collected from the jugular vein using a syringe and needle containing heparin sodium solution (Mochida Pharmaceutical Co., Ltd.). The collected blood was centrifuged, and cytokines were measured using the resulting plasma by ELISA or electrochemiluminescence. IFN-γ levels were measured using the Levis Mouse IFN-γ ELISA kit (Fujifilm Wako Shibayagi Corporation), and TNF-α, IL-6, KC / GRO (IL-8), and IL-10 levels were measured using the V-PLEX Proinflammatory Panel 1 Mouse Kit (Meso Scale Diagnostics, LLC.). The time courses of these levels are shown in Figures 10 to 14.

[0052] Statistical analysis was performed using the statistical analysis software EXSUS (ver. 10.0, EP Clore Co., Ltd.) using a Wilcoxon test for the control group and each treatment group. Two-sided statistical analysis was performed with significance levels of P<0.05 and P<0.01.

[0053] The ubenimex and anti-PD-1 antibody combination group showed increased cytokine production over time. The combination group showed a significant increase in IFN-γ, TNF-α, IL-6, KC / GRO (IL-8), and IL-10 production compared to the control group, ubenimex alone, and anti-PD-1 antibody alone (Figures 10 to 14). The P values ​​for the combination group compared to the control group at 21 or 24 days after transplantation were 0.003 for IFN-γ, 0.045 for TNF-α, 0.031 for IL-6, and 0.008 for IL-10.

[0054] IFN-γ, TNF-α, IL-6, and IL-8 are known as antitumor or pro-inflammatory cytokines that are thought to contribute to antitumor effects by activating the immune system network in a chain reaction. IL-10 is known as an anti-inflammatory cytokine that regulates inflammatory responses. In Example 3, the group administered the combination of ubenimex and an anti-PD-1 antibody showed a stronger tumor growth inhibitory effect than the groups administered each agent alone. This suggests that the combined administration of ubenimex and an anti-PD-1 antibody increases the production of IFN-γ, TNF-α, IL-6, and KC / GRO (IL-8), contributing to tumor growth inhibition. It is also speculated that IL-10 increases compensatory in response to increased production of the above cytokines, regulating the body's excessive inflammatory response.

Claims

1. An antitumor agent containing ubenimex as an active ingredient, for administration in combination with at least one selected from the group consisting of immune checkpoint inhibitors, anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody.

2. The antitumor agent according to claim 1, wherein the dose of ubenimex is such that the area under the blood concentration-time curve (AUC) upon single administration is 3.00 to 50.00 μg·hr / mL.

3. The antitumor agent according to claim 1 or 2, wherein ubenimex is administered daily.

4. An antitumor agent for administration in combination with ubenimex, the antitumor agent comprising as an active ingredient one selected from the group consisting of immune checkpoint inhibitors anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody.

5. The antitumor agent according to claim 4, wherein the dose of ubenimex is such that the area under the blood concentration-time curve (AUC) upon single administration is 3.00 to 50.00 μg·hr / mL.

6. A medicine containing ubenimex as an active ingredient for enhancing the production of antitumor cytokines by administering it in combination with at least one selected from the group consisting of immune checkpoint inhibitors, anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody.

7. The pharmaceutical composition according to claim 6, wherein the dose of ubenimex is such that the area under the blood concentration-time curve (AUC) upon single administration is 3.00 to 50.00 μg·hr / mL.

8. A medicine containing ubenimex as an active ingredient for increasing tumor-infiltrating lymphocytes by administering it in combination with at least one selected from the group consisting of immune checkpoint inhibitors, anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody.

9. The pharmaceutical composition according to claim 8, wherein the dose of ubenimex is such that the area under the blood concentration-time curve (AUC) upon single administration is 3.00 to 50.00 μg·hr / mL.

10. A medicine containing ubenimex as an active ingredient, which is used to treat a malignant tumor selected from the group consisting of small cell lung cancer, non-small cell lung cancer, esophageal cancer, head and neck cancer, brain tumor, colorectal cancer, colon adenocarcinoma, gastric cancer, breast cancer, hepatocellular carcinoma, pancreatic cancer, biliary tract cancer, kidney cancer, prostate cancer, bladder cancer, ovarian cancer, cervical cancer, thyroid cancer, and melanoma, by administering it in combination with at least one selected from the group consisting of immune checkpoint inhibitors, anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody.

11. The pharmaceutical composition according to claim 10, wherein the dose of ubenimex is such that the area under the blood concentration-time curve (AUC) upon single administration is 3.00 to 50.00 μg·hr / mL.

12. The pharmaceutical composition according to claim 10 or 11, wherein ubenimex is administered daily.