Anticancer agent containing NKT cell ligand-containing liposome composition

Abstract

(1) The purpose of the present invention is to provide an antitumor agent in which an effect of an immune checkpoint inhibitor is enhanced. (2) The purpose of the present invention is also to provide a drug that could exhibit an effect even under conditions in which no antigen is present.　(1) Provided is an antitumor agent obtained by combining an immune checkpoint inhibitor with an NKT cell ligand-containing liposome composition. (2) Also provided is a liposome composition containing an NKT cell ligand and an antigen.

Description

Anticancer drug comprising NKT cell ligand-containing liposome composition

[0001] The present invention relates to: (1) a pharmaceutical obtained by combining a liposome composition containing an NKT cell ligand (a-GalCer, RK-163, RCAI-61, RCAI-137, etc.) that has anti-tumor activity through the artificial activation of NKT cells in cancer-bearing living organisms with an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody, etc.) (Invention-1); and (2) a liposome composition containing an NKT cell ligand and an antigen that can prevent the onset of cancer or infectious diseases by inducing long-term immune memory (Invention-2).

[0002] Natural killer T (NKT) cells were discovered as immune cells belonging to a novel lymphocyte lineage that exhibit characteristics distinct from other lymphocyte lineages (T, B, and NK cells) (Non-Patent Document 1). NKT cells express only one antigen receptor (Va24Ja18 in humans and Va24Ja18 in mice) that is specific to NKT cells. Therefore, these cells are novel cells that are fundamentally different from other immune cell groups. Their role is to induce the activation of immune cell groups in the innate and adaptive immune systems (Non-Patent Document 2), enhance antitumor effects (Non-Patent Documents 3, 4, and 5), and are essential for inducing long-term immunological memory (Non-Patent Document 6).

[0003] The most important characteristic of NKT cells is that the only antigen receptor a-chain expressed on NKT cells (Va24Ja18 in humans, Va14Ja18 in mice) recognizes and activates glycolipid antigens bound to CD1d molecules, which are similar to major histocompatibility complex (MHC) class I molecules, of which there is only one species (Non-Patent Documents 7 and 8). In other words, both the NKT cell antigen receptor and the CD1d molecule are unique within a species, and their sequences are extremely similar across species. In terms of function, mouse NKT cell antigen receptors can recognize NKT cell ligands bound to human CD1d. Conversely, human NKT cell receptors can also recognize NKT cell ligands bound to mouse CD1d. Since human NKT cell receptors and CD1d molecules are common to all humans, NKT cell ligand-containing liposome compositions can be used as drugs for all humans and can also be applied to many mammals, including dogs and cats.

[0004] It has been reported that α-galactosylceramide (α-GalCer) artificially activates NKT cells strongly (Non-Patent Document 9). Compounds (RK-163, RCAI-61, RCAI-137, etc.) with activity several tens of times more potent than α-galactosylceramide have also been discovered (Patent Documents 1 to 5, Non-Patent Document 10). The present inventors, together with other inventors, have also developed novel NKT cell ligand-containing liposome compositions containing these compounds (International Application No. PCT / JP2023 / 005048 (WO2023 / 153527): Patent Document 6).

[0005] When administered in vivo, the novel NKT cell ligand-containing liposome composition is taken up by antigen-presenting cells (APCs), binds to CD1d molecules, which are similar to major histocompatibility complex (MHC) class I molecules, and is presented on the antigen-presenting cell membrane. NKT cells are artificially activated by recognizing the complex of the CD1d molecule and the novel NKT cell ligand presented on the antigen-presenting cells using the Va24Ja18 antigen receptor α chain, which is unique to human NKT cells in humans. This induces the activation and rapid proliferation of cancer immune cells, enhances anti-tumor effects, and induces long-term immune memory against cancer, thereby enabling the suppression of cancer progression, recurrence, and metastasis.

[0006] NKT cells are found in extremely low numbers in peripheral blood, accounting for only 0.1-0.01% of all white blood cells. However, NKT cells normally congregate in the liver, bone marrow, and lungs. However, once a lesion develops, they arrive at the lesion site ahead of other immune cells. This is because, even when NKT cells are not activated, they express the CXCL16 chemokine receptor CXCR6 more strongly than any other cell type. Therefore, when CXCL16, which attracts NKT cells from the lesion site, is released into the blood, they rush to the scene immediately (Non-Patent Document 11). As a result, NKT cells resident in the liver, bone marrow, and lungs migrate toward areas with high CXCL16 concentrations and arrive at the lesion site first. However, at that point, the NKT cells are not activated. When the lesion is infected with a pathogen such as bacteria, NKT cells that arrive at the lesion are immediately activated by adjuvant substances contained in the pathogen components. However, in the case of cancer, because cancer cells are themselves mutated cells, there are no adjuvant substances to activate NKT cells, and NKT cells cannot be activated. As a result, they cannot induce anti-tumor enhancing effects or long-term immune memory against cancer, making them powerless against cancer cells. However, by artificially activating NKT cells, it becomes possible to induce anti-tumor enhancing effects and long-term immune memory against cancer, making it possible to prevent and treat cancer onset. In fact, artificial activation of NKT cells has shown extremely high efficacy in clinical trials of advanced lung cancer cases (Non-Patent Document 12).

[0007] WO2008 / 102888WO2009 / 119692WO2010 / 030012WO2011 / 096536WO2013 / 162016WO2023 / 153527

[0008] Proc. Natl. Acad. Sci. USA 1990, 87:5248-5252Nature Immunol 2003, 4: 1164-1165Cancer Sci 2008; 99: 638-645Science, 1997, 278, 1623-1626J Immunol 1999; 163: 2387-2391Front. Immunol., 2017, 25 SeptemberJ. Immunol. 1998, 161, 3271-3281Cancer Res 1999; 59: 5102-5105Science, 1997, 278, 1626-1629Bioorganic & Medicinal Chemistry, 2013, 21: 3066-3079J Immunol., 2005, 175: 2051-2555J Immunol 2009, 182: 2492-2501Immunology 2012, 138:105-115J.Exp.Med., 2003, 198:267-279British J Cancer 2011, 105, 93-103Oncotarget 2018; 9:6201-6212Cytometry Research, 27(2): 39-44,2017

[0009] Immune checkpoint inhibitors are becoming the standard treatment for some intractable malignant tumors, but the response rate is approximately 10-30%, depending on the cancer type, and some patients experience no response at all. This is because tumor tissue is immunocompromised, and only a small subset of immune cells stimulated by cancer antigens are activated, ultimately expressing PD-1. Combination studies with multiple immune checkpoint inhibitors and other anticancer drugs, including chemotherapeutic agents, have been actively conducted. However, these studies have not necessarily been combined with therapies that theoretically induce the expression of PD-1 or its ligand, PD-L1, and therefore have not demonstrated the expected combined effects (Non-Patent Document 17). Furthermore, no drugs have been developed that induce PD-1 expression in immune cells within tumor tissue, leading to the accumulation of PD-1-positive immune cells within the tumor, or that enhance the expression of immune checkpoint ligand molecules, such as PD-L1, on tumor cells, thereby enhancing the efficacy of anti-PD-1 antibodies. Therefore, the development of drugs that enhance the efficacy of immune checkpoint inhibitors is desirable (Objective 1).

[0010] Furthermore, previous NKT cell ligand-containing liposome compositions were developed as compositions that artificially activate NKT cells in the body of cancer-bearing patients, thereby further activating cancer-specific immune cell populations present in the patient's body through their adjuvant action, enhancing anti-tumor function, and inducing long-term immune memory to control cancer. These compositions were used under conditions where cancer antigens were already present in the cancer-bearing patient. Therefore, there is a need to develop drugs that can be effective even when cancer antigens are not present (second challenge).

[0011] As a result of intensive research aimed at solving the above-mentioned problem (Problem 1), the present inventors discovered that administration of NKT cell ligand-containing liposomes artificially activates NKT cells, and the adjuvant effect mediated by IFNγ produced by the artificially activated NKT cells potently activates and proliferates insufficiently activated immune cell populations in tumor tissue, resulting in enhanced PD-1 expression (see Test Example 1, described below). Furthermore, they found that administration of NKT cell ligand-containing liposomes enhances the expression of PD-L1 ligand molecules in tumor cells (see Test Examples 2 to 4, described below). Indeed, when an NKT cell ligand-containing liposome composition and an anti-PD-1 antibody were administered in combination to a tumor-bearing animal model, a synergistic antitumor effect was exerted, enabling almost complete suppression of tumor growth compared to administration of either agent alone (see Test Example 5, described below). Based on these findings, further research led to the completion of the present invention (Invention 1).

[0012] Furthermore, as a result of extensive research aimed at achieving the above-mentioned object (the second object), the present inventors have found that administration of a liposome composition containing an NKT cell ligand and an antigen (e.g., a cancer antigen) can exert excellent effects (e.g., antitumor effects) even in conditions in which the antigen (e.g., a cancer antigen) is not yet present. That is, for example, when the antigen is a cancer antigen, administration of a liposome composition containing an NKT cell ligand and a cancer antigen results in the liposome composition being taken up by antigen-presenting cells, and the cancer antigen is then expressed on the patient's individual major histocompatibility complex (HLA class I and HLA class II) molecules, thereby activating cancer antigen-specific CD4 T / CD8 T cell populations. At the same time, the NKT cell ligand contained in the liposome composition is also expressed together with CD1d molecules on antigen-presenting cells, thereby artificially activating NKT cells in vivo. The cancer antigen-specific T cell population activated by the administered cancer antigen is further activated and enhanced by the adjuvant effect of the artificially activated NKT cells, which then induces long-term cancer immune memory, and this long-term immune cell population can suppress the onset, recurrence, and metastasis of cancer. Based on these new findings, further investigations led to the completion of the present invention (Invention 2).

[0013] That is, the present invention is as follows.

[0014] [1] An antitumor agent comprising a combination of an immune checkpoint inhibitor and a liposome composition containing an NKT cell ligand. [2] The antitumor agent according to the above-mentioned [1], wherein the NKT cell ligand is a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof. Formula (I):

[0015]

[0016] [wherein X represents an alkylene group or —NH—; R １ and R ２ are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group, or an aryl group which may have a substituent, R １ and R ２ may form a 5- or 6-membered ring together with the adjacent nitrogen atom; R ３ represents a hydrocarbon group having 1 to 20 carbon atoms; R ４ represents a hydrocarbon group having 1 to 30 carbon atoms.] Formula (II):

[0017]

[0018] [In the formula, R ５ represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom; R ６ and R ７ each independently represents a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents —CH ２ [3] The antitumor agent according to the above-mentioned [2], wherein the glycolipid is a compound represented by formula (I) or a salt thereof. [4] The glycolipid is a compound represented by the following formula:

[0019]

[0020] or a salt thereof. [5] An agent for enhancing the expression of immune checkpoint molecules by activating cancer immune cell groups, comprising an NKT cell ligand-containing liposome composition. [6] An agent for enhancing the expression of immune checkpoint ligand molecules on cancer cells by artificial activation of NKT cells, comprising an NKT cell ligand-containing liposome composition. [7] An NKT cell ligand-containing liposome composition for enhancing the therapeutic effect of an immune checkpoint inhibitor. [8] An immune checkpoint inhibitor for enhancing the therapeutic effect of an NKT ligand-containing liposome composition.

[0021] [9] A method for treating malignant tumors, comprising administering to a subject an immune checkpoint inhibitor and an NKT cell ligand-containing liposome composition.

[10] The method for treatment according to [9] above, wherein the NKT cell ligand is a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and a salt thereof. Formula (I):

[0022]

[0023] [wherein X represents an alkylene group or —NH—; R １ and R ２ are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group, or an aryl group which may have a substituent, R １ and R ２ may form a 5- or 6-membered ring together with the adjacent nitrogen atom; R ３ represents a hydrocarbon group having 1 to 20 carbon atoms; R ４ represents a hydrocarbon group having 1 to 30 carbon atoms.] Formula (II):

[0024]

[0025] [In the formula, R ５ represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom; R ６ and R ７ each independently represents a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents —CH ２

[11] The method of treatment according to the above-mentioned

[10] , wherein the glycolipid is a compound represented by formula (I) or a salt thereof.

[12] The glycolipid is a compound represented by the following formula:

[0026]

[0027] or a salt thereof.

[13] A method for enhancing the expression of immune checkpoint molecules by activating a cancer immune cell population in a subject, comprising administering to the subject an NKT cell ligand-containing liposome composition.

[14] A method for enhancing the expression of immune checkpoint ligand molecules on cancer cells in a subject by artificially activating NKT cells, comprising administering to the subject an NKT cell ligand-containing liposome composition.

[0028]

[15] A pharmaceutical composition for use in the treatment of malignant tumors, comprising a combination of an immune checkpoint inhibitor and a liposome composition containing an NKT cell ligand.

[16] The pharmaceutical composition for use according to the above-mentioned

[15] , wherein the NKT cell ligand is a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof. Formula (I):

[0029]

[0030] [wherein X represents an alkylene group or —NH—; R １ and R ２ are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group, or an aryl group which may have a substituent, R １ and R ２ may form a 5- or 6-membered ring together with the adjacent nitrogen atom; R ３ represents a hydrocarbon group having 1 to 20 carbon atoms; R ４ represents a hydrocarbon group having 1 to 30 carbon atoms.] Formula (II):

[0031]

[0032] [In the formula, R ５ represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom; R ６ and R７ each independently represents a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents —CH ２

[17] The pharmaceutical composition for use according to the above-mentioned

[16] , wherein the glycolipid is a compound represented by formula (I) or a salt thereof.

[18] The pharmaceutical composition for use according to the above-mentioned

[16] , wherein the glycolipid is a compound represented by the following formula:

[0033]

[0034] or a salt thereof.

[19] A liposome composition containing an NKT cell ligand for use in enhancing the expression of immune checkpoint molecules by activating cancer immune cells.

[20] A liposome composition containing an NKT cell ligand for use in enhancing the expression of immune checkpoint ligand molecules on cancer cells by artificial activation of NKT cells.

[21] A liposome composition containing an NKT cell ligand for use in improving the therapeutic effect of an immune checkpoint inhibitor.

[22] An immune checkpoint inhibitor for use in improving the therapeutic effect of an NKT ligand-containing liposome composition.

[23] A liposome composition containing an immune checkpoint inhibitor and an NKT cell ligand for use as a pharmaceutical.

[0035]

[24] Use of a combination of an immune checkpoint inhibitor and a liposome composition containing an NKT cell ligand in the manufacture of an antitumor agent.

[25] The use according to

[24] above, wherein the NKT cell ligand is a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and a salt thereof. Formula (I):

[0036]

[0037] [wherein X represents an alkylene group or —NH—; R １ and R ２ are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group, or an aryl group which may have a substituent, R １ and R ２ may form a 5- or 6-membered ring together with the adjacent nitrogen atom; R ３represents a hydrocarbon group having 1 to 20 carbon atoms; R ４ represents a hydrocarbon group having 1 to 30 carbon atoms.] Formula (II):

[0038]

[0039] [In the formula, R ５ represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom; R ６ and R ７ each independently represents a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents —CH ２

[26] The use according to the above-mentioned

[25] , wherein the glycolipid is a compound represented by formula (I) or a salt thereof.

[27] The use according to the above-mentioned

[25] , wherein the glycolipid is a compound represented by the following formula:

[0040]

[0041] or a salt thereof.

[28] Use of an NKT cell ligand-containing liposome composition in the manufacture of an agent for enhancing the expression of immune checkpoint molecules by activating a cancer immune cell population.

[29] Use of an NKT cell ligand-containing liposome composition in the manufacture of an agent for enhancing the expression of immune checkpoint ligand molecules on cancer cells by artificial activation of NKT cells.

[30] Use of an NKT cell ligand-containing liposome composition in the manufacture of an agent for improving the therapeutic effect of an immune checkpoint inhibitor.

[31] Use of an immune checkpoint inhibitor in the manufacture of an agent for improving the therapeutic effect of an NKT ligand-containing liposome composition.

[32] A pharmaceutical composition comprising a combination of an immune checkpoint inhibitor and an NKT cell ligand-containing liposome composition.

[0042]

[33] A liposome composition containing an NKT cell ligand and an antigen.

[34] The composition according to

[33] above, wherein the NKT cell ligand is a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and a salt thereof. Formula (I):

[0043]

[0044] [wherein X represents an alkylene group or —NH—; R １ and R ２ are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group, or an aryl group which may have a substituent, R １ and R ２ may form a 5- or 6-membered ring together with the adjacent nitrogen atom; R ３ represents a hydrocarbon group having 1 to 20 carbon atoms; R ４ represents a hydrocarbon group having 1 to 30 carbon atoms.] Formula (II):

[0045]

[0046] [In the formula, R ５ represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom; R ６ and R ７ each independently represents a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents —CH ２

[35] The composition according to the above-mentioned

[34] , wherein the glycolipid is a compound represented by formula (I) or a salt thereof.

[36] The composition according to the above-mentioned

[34] , wherein the glycolipid is a compound represented by the following formula:

[0047]

[0048] or a salt thereof.

[37] The composition according to any of

[33] to

[36] above, for inducing immunological memory.

[38] The composition according to any of

[33] to

[36] above, wherein the antigen is a cancer antigen.

[39] The composition according to

[38] above, for prophylactically inducing the activation and proliferation of immune cells that recognize a cancer antigen, even in the absence of cancer.

[40] The composition according to

[38] above, for prophylactically inducing immunological memory against cancer, even in the absence of cancer.

[41] The composition according to

[38] above, for enhancing the inhibitory effect on cancer onset, recurrence, and metastasis.

[0049]

[42] A method for inducing immunological memory, comprising administering to a subject the composition described in any of

[33] to

[36] above.

[43] A method for prophylactically inducing activation and proliferation of immune cells that recognize cancer antigens, even in the absence of cancer in the subject, comprising administering to a subject the composition described in

[38] above.

[44] A method for prophylactically inducing immunological memory against cancer, even in the absence of cancer in the subject, comprising administering to a subject the composition described in

[38] above.

[45] A method for suppressing the onset, recurrence, and metastasis of cancer, comprising administering to a subject the composition described in

[38] above.

[0050]

[46] The composition according to any of

[33] to

[36] above, for use in inducing immunological memory.

[47] The composition according to

[38] above, for use in prophylactically inducing activation and proliferation of immune cell populations that recognize cancer antigens, even in the absence of cancer.

[48] The composition according to

[38] above, for use in prophylactically inducing immunological memory against cancer, even in the absence of cancer.

[49] The composition according to

[38] above, for use in improving the inhibitory effect on cancer onset, recurrence, and metastasis.

[50] The composition according to any of

[33] to

[36] above, for use as a pharmaceutical.

[0051]

[51] Use of the composition according to any one of

[33] to

[36] above for producing an agent for inducing immunological memory.

[52] Use of the composition according to

[38] above for producing an agent for prophylactically inducing activation and proliferation of immune cell populations that recognize cancer antigens, even in the absence of cancer.

[53] Use of the composition according to

[38] above for producing an agent for prophylactically inducing immunological memory against cancer, even in the absence of cancer.

[54] Use of the composition according to

[38] above for producing an agent for suppressing the onset, recurrence, and metastasis of cancer.

[0052] According to the present invention (Invention-1), the combined use of an NKT cell ligand-containing liposome composition and an immune checkpoint inhibitor can provide a useful therapeutic agent for treating cancer, as well as cancer metastasis and recurrence.

[0053] NKT cell ligand-containing liposomes activate cancer immune cells that accumulate in tumor tissue in cancer-bearing subjects, resulting in the strong expression of immune checkpoint molecules such as PD-1 and increased expression of immune checkpoint ligand molecules (such as PD-L1) on tumor cells. Based on these studies, administration of novel NKT cell ligand-containing liposomes and an immune checkpoint inhibitor (Invention 1) artificially activates NKT cells in the body, activating the immune system against cancer and increasing the expression of immune checkpoint ligand molecules (such as PD-L1) on tumor cells. As a result, the inhibitory effect of the immune checkpoint inhibitor is enhanced, inducing a strong antitumor effect and enabling powerful cancer treatment. Furthermore, this combination treatment can achieve stronger antitumor effects even against cancer types that have low expression of immune checkpoint ligand molecules, have limited therapeutic effect when used alone, and have previously shown limited antitumor effects.

[0054] PD-1 is a receptor molecule belonging to the CD28 family (a group of molecules that positively and negatively regulate T cell activation) that is expressed on activated lymphocytes (such as CD4 T cells and CD8 T cells of the adaptive immune system and NK cells of the innate immune system). PD-1 binds to PD-1 ligands (PD-L1 and PD-L2) expressed on cancer cells, transmitting inhibitory signals to activated lymphocytes (CD4 T, CD8 T, NK), negatively regulating the activation state of lymphocytes and suppressing the function of activated lymphocytes, thereby helping cancer cells survive. Specifically, the PD-1 / PD-L1 pathway (immune checkpoint pathway) was originally a so-called fail-safe mechanism for avoiding attacks on autologous cells by the immune system, but because cancer cells are autologous, it is thought to be one of the mechanisms for avoiding attacks by the immune system, etc., and various immune checkpoint inhibitors, including anti-PD-1 antibodies or anti-PD-L1 ligand antibodies, are being used to maintain and enhance the function of activated lymphocytes against tumors over the long term, and are being clinically developed as drugs that enhance attacks on cancer cells. Therefore, by using an NKT cell ligand-containing liposome composition in combination, NKT cells are artificially activated, which in turn activates and induces the proliferation of cancer-attacking lymphocytes and strongly expresses the PD-1 ligand PD-L1 on tumor cells, thereby enhancing the effects of immune checkpoint inhibitors and expected to have a stronger anti-cancer effect. It is known that tumor-attacking lymphocytes (TILs) accumulate within tumors, and that the amount of TIL accumulation correlates with the patient's prognosis (Non-Patent Document 15). Data has also been reported showing that the greater the amount of PD-1-positive lymphocytes infiltrating within the TILs accumulated within the tumor, the better the prognosis (Non-Patent Document 16). Among immune checkpoint inhibitors, anti-PD-1 antibodies were the first to be approved for manufacture and sale. Currently, anti-PD-1 antibodies have been shown to have tumor shrinkage effects and prolonged survival times superior to chemotherapy in various cancer types, including melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, and head and neck cancer, and are becoming the standard treatment for advanced cancers. However, it has also been reported that these antibodies are ineffective in some cancer types (Non-Patent Document 16).

[0055] Therefore, the combined use of NKT cell ligand-containing liposomes and anti-PD-1 antibodies (Invention 1) is expected to have a stronger anti-cancer effect than when either is used alone. Furthermore, even for some cancer types for which treatment with anti-PD-1 antibodies alone is ineffective, it is possible to induce enhanced expression of PD-L1 on cancer cells by artificially activating NKT cells, thereby enabling the expectation of a stronger anti-tumor effect of anti-PD-1 antibodies (Invention 1).

[0056] Furthermore, the following effects can be expected when an NKT cell ligand-containing liposome composition is administered to a living body. (1) The adjuvant effect of Th1 cytokines, such as interferon-γ, rapidly and massively produced by artificially activated NKT cells artificially activated by an NKT cell ligand, strongly induces the expression of checkpoint ligand molecules (e.g., PD-L1) on the cancer cell membrane to protect the cancer cell from attack by immune cells. (2) At the same time, it induces the activation and rapid proliferation of effector immune cells, such as NK cells, CD4 T cells, CD8 T cells, and γδ T cells (Non-Patent Document 2), enhancing antitumor activity and resulting in strong expression of PD-1 by immune cells. (3) As a result, it activates immune cells against cancer, enhancing antitumor effects and inducing long-term cancer immune memory lasting up to one year, thereby enabling sustained attack and suppressing cancer progression, recurrence, and metastasis (Non-Patent Document 6). (4) In addition to activating other immune cell groups, NKT cells themselves express various cytotoxic factors, such as perforin, and can therefore directly cytotoxicate target cells (Non-Patent Document 8). (5) Their cytotoxic activity directly kills immunosuppressive macrophages (MDSCs) and tumor-associated macrophages (TAMs), which cancer cells induce to protect themselves from immune attack, thereby improving the immune environment within cancer tissue (Non-Patent Document 13). (6) NKT cells are the only cells in the immune system that can interact with immature dendritic cells, which lack antigen-presenting capacity, via NKT cell ligands. They are also the only cells that can induce differentiation of immature dendritic cells into mature dendritic cells and improve the immune environment within cancer tissue (Non-Patent Document 14).

[0057] According to the present invention (Invention-2), a liposome composition containing an NKT cell ligand capable of artificially activating NKT cells and a cancer antigen can serve as an antitumor vaccine agent useful for preventing the onset, recurrence, and metastasis of cancer. The WT-1 antigen peptide, which is expressed in most carcinomas, is a promising cancer antigen peptide, but other peptides with sequences characteristic of carcinomas can also be used. Furthermore, by using a pathogen antigen instead of a cancer antigen, the composition can also serve as an agent (vaccine) for preventing infectious diseases.

[0058] The NKT cell ligand-containing liposome composition is a formulation developed assuming the presence of cancer cells and cancer antigens in the body, i.e., a cancer-bearing state, but the NKT cell ligand- and cancer antigen-containing liposome composition (Invention-2), which was developed to be effective in the normal state, i.e., a state in which cancer antigens are absent, is effective as a measure against cancer onset, recurrence, and metastasis. The state in which cancer antigens are absent in the body includes, for example, a normal state, a tumor removed by surgery but with the possibility of residual tumor or micrometastasis, a genetic risk of developing familial cancer, and an elevated tumor marker but no cancer lesions can be found. In these cases, the NKT cell ligand- and cancer antigen-containing liposome composition (Invention-2) is effective when there are no cancer antigens or extremely few cancer antigens and there is concern about rapid cancer growth.

[0059] The liposome composition containing an NKT cell ligand and an antigen (e.g., a cancer antigen) (Invention-2) is a liposome composition that simultaneously carries an NKT cell ligand and an antigen (e.g., a cancer antigen), in which the NKT cell ligand is present on the surface of the NKT cell-containing liposome and the antigen (e.g., a cancer antigen) is encapsulated inside the liposome. That is, it can be said that the cancer antigen is encapsulated in the novel NKT cell ligand-containing liposome composition (International Application No. PCT / JP2023 / 005048 (WO2023 / 153527)). Therefore, the liposome composition containing an NKT cell ligand and an antigen (Invention-2) is most effective when the antigen is a cancer antigen, for example, in cases where (1) suppression of cancer onset is expected before the onset of familial cancer, (2) suppression of cancer recurrence when cancer is removed by surgery or the like and the cancer antigen has disappeared from the body, or (3) prophylactic treatment when tumor marker elevation is observed but cancer lesions cannot be found. In other words, in these near-normal patient states, although T cell populations that recognize cancer antigens are present in the body, the clone size of each is small and anti-cancer effects cannot be expected, and even during the cancer-bearing state, long-term immune memory against cancer has not been established, so the number of immune cells is not sufficient to suppress the onset of cancer. However, even in such a state, if cancer-specific immune cell populations are increased in advance and long-term cancer immune memory is induced, the immune system can respond to cancer onset and recurrence.

[0060] FIG. 1 shows the results of Test Example 1. In FIG. 1, RK-163-LIPO indicates the group administered with the RK-163-containing liposome composition, RCAI-61-LIPO indicates the group administered with the RCAI-61-containing liposome composition, RCAI-137-LIPO indicates the group administered with the RCAI-137-containing liposome composition, and PBS indicates the group administered with PBS (control). FIG. 1(A) shows the results of PD-1 expression due to activation of CD8 T cells, FIG. 1(B) shows the results of PD-1 expression due to activation of CD4 T cells, and FIG. 1(C) shows the results of PD-1 expression due to activation of NK cells. FIG. 2 shows the results of Test Example 2. In Figure 2, RK-163-LIPO indicates the group administered with the RK-163-containing liposome composition, RCAI-61-LIPO indicates the group administered with the RCAI-61-containing liposome composition, RCAI-137-LIPO indicates the group administered with the RCAI-137-containing liposome composition, and PBS indicates the group administered with PBS (control). Figure 3 shows the results of Test Example 3. In Figure 3, RK-163-LIPO indicates the group in which the RK-163-containing liposome composition was used in the mixed culture, RCAI-61-LIPO indicates the group in which the RCAI-61-containing liposome composition was used in the mixed culture, RCAI-137-LIPO indicates the group in which the RCAI-137-containing liposome composition was used in the mixed culture, and PBS indicates the group in which PBS was used (control). Figure 4 shows the results of Test Example 4. Figure 5-1 shows the results of Test Example 5 (using an RK-163-containing liposome composition as the NKT cell ligand-containing liposome composition). In Figure 5-1, PBS+anti-PD-1 indicates a group administered with an anti-PD-1 antibody alone, RK-163-LIPO indicates a group administered with an RK-163-containing liposome composition alone, and RK-163-LIPO+anti-PD-1 indicates a group administered with a combination of an RK-163-containing liposome composition and an anti-PD-1 antibody. Figure 5-2 shows the results of Test Example 5 (using an RCAI-61-containing liposome composition as the NKT cell ligand-containing liposome composition). In Figure 5-2, PBS + anti-PD-1 indicates a group administered with an anti-PD-1 antibody alone, RCAI-61-LIPO indicates a group administered with an RCAI-61-containing liposome composition alone, and RCAI-61-LIPO + anti-PD-1 indicates a group administered with a combination of an RCAI-61-containing liposome composition and an anti-PD-1 antibody.Figure 5-3 shows the results of Test Example 5 (using an RCAI-137-containing liposome composition as the NKT cell ligand-containing liposome composition). In Figure 5-3, PBS+anti-PD-1 indicates a group administered with an anti-PD-1 antibody alone, RCAI-137-LIPO indicates a group administered with an RCAI-137-containing liposome composition alone, and RCAI-137-LIPO+anti-PD-1 indicates a group administered with a combination of an RCAI-137-containing liposome composition and an anti-PD-1 antibody. Figure 6 shows the results of Test Example 6. In Figure 6, "0.1 ng" represents the group administered with a liposome composition (RK-LIPO-OVA) containing an NKT cell ligand (RK-163) and a cancer-mimetic antigen (OVA) at 0.1 ng / 25 g body weight, calculated as the amount of RK-163; "1 ng" represents the group administered with RK-LIPO-OVA at 1 ng / 25 g body weight, calculated as the amount of RK-163; "10 ng" represents the group administered with RK-LIPO-OVA at 10 ng / 25 g body weight, calculated as the amount of RK-163; and PBS represents the PBS-administered group (control). The middle graph in Figure 6 is a bar graph showing the tumor size plot 17 days after tumor inoculation shown in the left graph. The RK-LIPO-OVA dose-dependent tumor size (calculated as the amount of RK-163) shown in the middle graph was converted into a curve graph (right graph) to calculate the ED50. FIG. 7 shows the results of Test Example 7. In FIG. 7, RK-LIPO-OVA indicates the group administered with a liposome composition containing an NKT cell ligand (RK-163) and a pseudo cancer antigen (OVA), and PBS indicates the group administered with PBS (control). FIG. 8 shows the results of Test Example 8. In FIG. 8, RK-LIPO-OVA indicates the group administered with a liposome composition containing an NKT cell ligand (RK-163) and a pseudo cancer antigen (OVA), and PBS indicates the group administered with PBS (control). FIG. 9 shows the results of Test Example 9. FIG. 10 shows the results of Test Example 10. In FIG. 10, RK-LIPO-OVA indicates the group administered with a liposome composition containing an NKT cell ligand (RK-163) and a pseudo cancer antigen (OVA), and PBS indicates the group administered with PBS (control).

[0061] 1. Invention-1 The present invention (Invention-1) is described in detail below. The present invention relates to an antitumor agent comprising a combination of an immune checkpoint inhibitor and an NKT cell ligand-containing liposome composition (hereinafter also referred to as the combination agent of the present invention).

[0062] [1. NKT cell ligand] The NKT cell ligand-containing liposome composition of the present invention contains an NKT cell ligand. In the present invention, the NKT cell ligand is preferably a glycolipid selected from the group consisting of a compound represented by the following formula (I), a compound represented by formula (II), and a salt thereof. An NKT cell ligand refers to a compound that, when presented on a CD1d molecule, is specifically recognized by an NKT cell-specific antigen-recognition receptor (NKT cell receptor) on an NKT cell, and can specifically activate the NKT cell. In this specification, the compound represented by formula (I), the compound represented by formula (II), and salts thereof are also collectively referred to as the "NKT cell ligand of the present invention."

[0063] First, the compound represented by formula (I) (also referred to as compound (I) in this specification) and its salts will be described. Formula (I):

[0064]

[0065] [wherein X represents an alkylene group or —NH—; R １ and R ２ are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group, or an aryl group which may have a substituent, R １ and R ２ may form a 5- or 6-membered ring together with the adjacent nitrogen atom; R ３ represents a hydrocarbon group having 1 to 20 carbon atoms; R ４ represents a hydrocarbon group having 1 to 30 carbon atoms.]

[0066] X represents an alkylene group or -NH-. The "alkylene group" is, for example, a linear or branched alkylene group having 1 to 8 carbon atoms, and specific examples thereof include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, propylene, ethylethylene, dimethylmethylene, and dimethyltrimethylene.

[0067] R １ and R ２are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group, or an aryl group which may have a substituent, R １ and R ２ may be combined with the adjacent nitrogen atom to form a 5- or 6-membered ring.

[0068] The "alkyl group" is, for example, C １～２４ , more preferably C １～１６ , more preferably C １～１０ , particularly preferably C １～６ Specific examples of R include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl. １ and R ２ The alkyl group of C is preferably １～６ It is an alkyl group (e.g., methyl, ethyl).

[0069] The "alkoxy group" is, for example, C １～２４ , more preferably C １～１６ , more preferably C １～１０ , particularly preferably C １～６ Specific examples of the alkoxy group include methoxy, ethoxy, propoxy, isopropoxy, butoxy, and tert-butoxy. １ and R ２ The alkoxy group of the formula (I) is preferably C １～６ It is an alkoxy group (e.g., methoxy).

[0070] The "aryl group" in the "aryl group which may have a substituent" is, for example, C ６～１４ , more preferably C ６～１２ R is a monocyclic to tricyclic aryl group, and specific examples thereof include phenyl, naphthyl, anthryl, and phenanthryl. １ and R ２ The aryl group of C is preferably ６～１２An aryl group (e.g., phenyl). Examples of substituents that the "aryl group" may have include halogen atoms (e.g., chlorine atom, fluorine atom, bromine atom, iodine atom); alkyl groups (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl); halogenoalkyl groups (e.g., trifluoromethyl); alkoxy groups (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy); hydroxyl groups; amino groups; alkylamino groups (e.g., methylamino, dimethylamino, ethylamino, diethylamino); cycloalkylamino groups, and the like. The positions and number of substituents are not particularly limited, and the aryl group may have from one to the maximum number of substituents at substitutable positions.

[0071] R １ and R ２ However, the 5- or 6-membered ring that may be formed together with the adjacent nitrogen atom is, for example, a 5- or 6-membered nitrogen-containing saturated heterocycle, and specific examples thereof include pyrrolidine, piperidine, morpholine, thiomorpholine, piperazine, etc. Preferred are pyrrolidine, piperidine, and morpholine.

[0072] R ３ represents a hydrocarbon group having 1 to 20 carbon atoms. １～２０ Alkyl group, C ２～２０ Alkenyl group, C ２～２０ Alkynyl group, C ３～２０ Cycloalkyl group, C ３～２０ Cycloalkenyl group, C ６～２０ The aryl group may be linear, branched or cyclic, may be a saturated or unsaturated hydrocarbon group, and may have an unsaturated bond either in the molecule or at the terminal. ３ As for C １～２０ Alkyl group, C ２～２０ Alkenyl groups, and C ２～２０ Alkynyl groups are preferred, C １２～１４ An alkyl group is more preferred. ３ Specific examples of the group include -(CH ２ ) １３ CH３ etc.

[0073] R ４ represents a hydrocarbon group having 1 to 30 carbon atoms. １～３０ Alkyl group, C ２～３０ Alkenyl group, C ２～３０ Alkynyl group, C ３～３０ Cycloalkyl group, C ３～３０ Cycloalkenyl group, C ６～３０ The concept also includes an aryl group, and may be any of linear, branched, and cyclic, may be a saturated hydrocarbon group, or an unsaturated hydrocarbon group, and may have an unsaturated bond either within the molecule or at the terminal. ４ As for C １～３０ Alkyl group, C ２～３０ Alkenyl groups, and C ２～３０ Alkynyl groups are preferred, C １０～３０ Alkyl groups are more preferred, and C １５～２５ An alkyl group is more preferred. ４ Specific examples of the group include -(CH ２ ) １５ CH ３ , -(CH ２ ) ２３ CH ３ etc.

[0074] R ３ and R ４ The hydrocarbon group represented by may have a substituent. ３ and R ４When the hydrocarbon group represented by the formula (I) has a substituent, examples of the substituent include a halogen atom (preferably a chlorine atom or a fluorine atom); an alkoxy group such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, or tert-butoxy (preferably having 1 to 24 carbon atoms, more preferably having 1 to 16 carbon atoms, even more preferably having 1 to 10 carbon atoms, and particularly preferably having 1 to 4 carbon atoms); an aryloxy group such as phenoxy (preferably having 6 to 14 carbon atoms); a hydroxyl group; an amino group; an alkylamino group such as methylamino, dimethylamino, ethylamino, or diethylamino; a cycloalkylamino group; Examples include electron-donating groups such as alkylcarbonylamino groups such as acetamide; alkylcarbonylamino groups such as acetamide; cycloalkylcarbonylamino groups; and arylcarbonylamino groups such as benzoylamino (preferably, an arylcarbonylamino group where the aryl moiety is an aryl group having 6 to 14 carbon atoms), as well as electron-withdrawing groups such as a carboxyl group; an alkoxycarbonyl group; an acyl group (acyl groups are as described below. Preferably, an alkyl-carbonyl group where the alkyl moiety is a linear or branched alkyl group having 1 to 24 carbon atoms); a carbamoyl group; and trifluoromethyl. The position and number of substituents are not particularly limited, and there may be one to the maximum number of substituents at substitutable positions. When one or more substituents are present, they may be the same or different.

[0075] In this specification, the term "acyl group" refers to, for example, a formyl group; an alkyl-carbonyl group (for example, an alkyl-carbonyl group in which the alkyl moiety is a straight-chain or branched alkyl group having 1 to 24 carbon atoms (preferably 1 to 12 carbon atoms) (e.g., acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl)); a cycloalkyl-carbonyl group (for example, a cycloalkyl-carbonyl group in which the cycloalkyl moiety is a cycloalkyl group having 3 to 10 carbon atoms); an alkenyl-carbonyl group (for example, an alkenyl-carbonyl group in which the alkenyl moiety is a straight-chain or branched alkenyl group having 2 to 12 carbon atoms (e.g., acryloyl, methacryloyl)); an aryl-carbonyl group (for example, an aryl-carbonyl group in which the aryl moiety is an aryl group having 6 to 14 carbon atoms (e.g., benzoyl, naphthoyl)); The aryl group in the aryl-carbonyl group refers to, for example, a monocyclic to tricyclic aromatic hydrocarbon group, and specific examples thereof include phenyl, naphthyl, anthryl, and phenanthryl. Among these, the acyl group is preferably formyl, acetyl, propionyl, butyryl, isobutyryl, benzoyl, naphthoyl, etc., and more preferably acetyl or benzoyl.

[0076] Examples of the alkyl moiety of the alkylamino group and alkylcarbonylamino group include straight-chain or branched alkyl groups (preferably having 1 to 24 carbon atoms, more preferably having 1 to 16 carbon atoms, even more preferably having 1 to 10 carbon atoms, and particularly preferably having 1 to 4 carbon atoms), such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl. Examples of the cycloalkyl moiety of the cycloalkylamino group and cycloalkylcarbonylamino group include cycloalkyl groups (preferably having 3 to 24 carbon atoms, more preferably having 3 to 16 carbon atoms, even more preferably having 3 to 10 carbon atoms, and particularly preferably having 3 to 6 carbon atoms), such as cyclopentyl and cyclohexyl. Examples of the alkoxy moiety of the alkoxycarbonyl group include the same as those of the alkoxy group.

[0077] The above-mentioned substituents may be further substituted at substitutable positions with at least one of a halogen, an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, a phenyl group, an alkoxy group, a hydroxyl group, an amino group, an alkylamino group, and a cycloalkylamino group. Examples of the halogen, alkoxy group, alkylamino group, and cycloalkylamino group are the same as those described above. Examples of the alkyl group include alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl (preferably having 1 to 24 carbon atoms, more preferably having 1 to 16 carbon atoms, even more preferably having 1 to 10 carbon atoms, and particularly preferably having 1 to 4 carbon atoms). Examples of the cycloalkyl group include cyclopentyl, cyclohexyl, and the like (preferably having 3 to 24 carbon atoms, more preferably having 3 to 16 carbon atoms, even more preferably having 3 to 10 carbon atoms, and particularly preferably having 3 to 6 carbon atoms). Examples of the alkenyl group include vinyl, propenyl, butenyl, and the like (preferably having 2 to 24 carbon atoms, more preferably having 2 to 16 carbon atoms, even more preferably having 2 to 10 carbon atoms, and particularly preferably having 2 to 4 carbon atoms). Examples of the alkynyl group include ethynyl, propargyl, butynyl, pentynyl, and the like (preferably having 2 to 24 carbon atoms, more preferably having 2 to 16 carbon atoms, even more preferably having 2 to 10 carbon atoms, and particularly preferably having 2 to 4 carbon atoms).

[0078] In the present invention, the α-isomer is used among the stereoisomers derived from the cyclic structure of sugar (galactopyranose). When compound (I) has a stereoisomer derived from a structure other than the cyclic structure of sugar (for example, an asymmetric carbon in a portion other than the cyclic structure of sugar), all isomers are encompassed in the present invention, and a mixture of two or more isomers in any ratio (including a racemate) may be used. In particular, compound (I) has optical isomers derived from an asymmetric carbon in a portion other than the cyclic structure of sugar, and in the present invention, it may be a single optically active isomer or a mixture of two or more optically active isomers in any ratio (including a racemate). -NHC(=O)X-R ４ The asymmetric carbon to which is bonded is preferably in the S configuration, and —NHC(═O)X—R ４ The asymmetric carbon atom adjacent to the asymmetric carbon atom to which OH is bonded is preferably in the R configuration. ３ The asymmetric carbon to which is bonded is preferably in the R configuration.

[0079] The salt of compound (I) is preferably a pharmaceutically acceptable salt, and examples thereof include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, etc.; organic acid salts such as succinate, fumarate, acetate, methanesulfonate, toluenesulfonate, etc.; alkali metal salts such as sodium salt, potassium salt, etc.; alkaline earth metal salts such as magnesium salt, calcium salt, etc.; ammonium salts such as ammonium salt, alkylammonium salt, etc.

[0080] Suitable examples of compound (I) or a salt thereof include the following: (a) In formula (I), X is —CH ２ - and R １ and R ２ are the same or different and are a hydrogen atom, C １～６ Alkyl group, hydroxyl group, or C １～６ is an alkoxy group; R ３ But C １～２０ is an alkyl group; R ４ is C １～３０ (b) A compound or a salt thereof, wherein X is —CH ２ - and R １ and R ２ are the same or different and are a hydrogen atom, C１～６ Alkyl group, hydroxyl group, or C １～６ is an alkoxy group; R ３ But C １２～１４ is an alkyl group; R ４ is C １５～２５ (c) A compound or a salt thereof, wherein X is —CH ２ - and R １ and R ２ are the same or different and are a hydrogen atom, C １～６ an alkyl group or a hydroxyl group; R ３ But C １２～１４ is an alkyl group; R ４ is C １５～２５ (d) A compound or a salt thereof, wherein X is —CH ２ - and R １ and R ２ are the same or different and are a hydrogen atom, a methyl group, or a hydroxyl group; R ３ But -(CH ２ ) １３ CH ３ and R ４ Ga-(CH ２ ) ２３ CH ３ A compound or a salt thereof,

[0081] Specific examples of the compound (I) suitable for use in the present invention are shown in Table 1, but the present invention is not limited to these.

[0082]

[0083] Among these, the following compounds are preferred: RCAI-124 (also referred to as RK-163 in this specification).

[0084]

[0085] RCAI-137

[0086]

[0087] The compound represented by formula (I) or a salt thereof can be produced by the methods disclosed in WO2013 / 162016A1, US2015 / 152128A1, etc.

[0088] Next, the compound represented by formula (II) (also referred to as compound (II) in this specification) and its salts will be described. Formula (II):

[0089]

[0090] [In the formula, R ５ represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom; R ６ and R ７ each independently represents a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents —CH ２ -, -CH(OH)- or -CH=CH-.]

[0091] R ５ represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom. ５ The alkyl group having 1 to 7 carbon atoms represented by R represents a substituted or unsubstituted alkyl group, which may form a ring. Examples thereof include methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclopropylmethyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, and cyclohexylmethyl, and methyl and ethyl are preferred. ５ The alkoxy group having 1 to 6 carbon atoms represented by the formula (I) represents an oxygen atom to which a substituted or unsubstituted alkyl group is bonded, and the alkyl portion may form a ring. Examples thereof include methoxy, ethoxy, n-propyloxy, isopropyloxy, cyclopropyloxy, cyclopropylmethyloxy, n-butoxy, isobutyloxy, sec-butyloxy, tert-butyloxy, pentyloxy, hexyloxy, and cyclohexyloxy, and methoxy, ethoxy, and n-propyloxy are preferred. ５ Examples of the halogen atom represented by the formula (I) include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, with a fluorine atom and a chlorine atom being preferred.

[0092] R ６ and R ７each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms. In this specification, the term "hydrocarbon group" refers to a substituted or unsubstituted alkyl group having 1 to 28 carbon atoms, an alkenyl group having 2 to 28 carbon atoms, an alkynyl group having 2 to 28 carbon atoms, a cycloalkyl group having 3 to 28 carbon atoms, a cycloalkenyl group having 3 to 28 carbon atoms, an aryl group having 6 to 14 carbon atoms, an arylalkyl group having 7 to 28 carbon atoms (for example, C ６－１４ Aryl-C １－１４ The concept of R includes alkyl groups, and may be any of linear, branched, and cyclic, may be a saturated hydrocarbon group, or an unsaturated hydrocarbon group, and may have an unsaturated bond either in the molecule or at the terminal. ６ and R ７ As R, a substituted or unsubstituted alkyl group having 1 to 28 carbon atoms and a substituted or unsubstituted arylalkyl group having 7 to 28 carbon atoms are preferred. ５ is a hydrogen atom, R ６ is preferably a substituted or unsubstituted hydrocarbon group having 24 to 28 carbon atoms, and more preferably a substituted or unsubstituted alkyl group having 24 to 28 carbon atoms.

[0093] R ６ and R ７Examples of the substituent of the hydrocarbon group represented by the formula (I) include a halogen atom (preferably a chlorine atom or a fluorine atom); an alkoxy group (preferably having 1 to 24 carbon atoms, more preferably having 1 to 16 carbon atoms, even more preferably having 1 to 10 carbon atoms, and particularly preferably having 1 to 4 carbon atoms) such as a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, or a tert-butoxy group; an aryloxy group (preferably having 6 to 14 carbon atoms) such as a phenoxy group; a hydroxyl group; an amino group; an alkylamino group such as a methylamino group, a dimethylamino group, an ethylamino group, or a diethylamino group; a cycloalkylamino group; Examples of the alkyl group include electron-donating groups such as alkylcarbonylamino groups, acetamide groups, cycloalkylcarbonylamino groups, and arylcarbonylamino groups, such as benzoylamino groups (preferably, arylcarbonylamino groups, the aryl moiety of which is an aryl group having 6 to 14 carbon atoms), as well as electron-withdrawing groups, such as carboxyl groups, alkoxycarbonyl groups, acyl groups (acyl groups are as described below; preferably, alkyl-carbonyl groups, the alkyl moiety of which is a linear or branched alkyl group having 1 to 24 carbon atoms); carbamoyl groups, and trifluoromethyl groups.

[0094] In this specification, the term "acyl group" refers to, for example, a formyl group; an alkyl-carbonyl group (for example, an alkyl-carbonyl group in which the alkyl moiety is a straight-chain or branched alkyl group having 1 to 24 carbon atoms (preferably 1 to 12 carbon atoms) (for example, an acetyl group, propionyl group, butyryl group, isobutyryl group, valeryl group, pivaloyl group, hexanoyl group)); a cycloalkyl-carbonyl group (for example, a cycloalkyl-carbonyl group in which the cycloalkyl moiety is a cycloalkyl group having 3 to 10 carbon atoms); an alkenyl-carbonyl group (for example, an alkenyl-carbonyl group in which the alkenyl moiety is a straight-chain or branched alkenyl group having 2 to 12 carbon atoms (for example, an acryloyl group, a methacryloyl group)); an aryl-carbonyl group (for example, an aryl-carbonyl group in which the aryl moiety is an aryl group having 6 to 14 carbon atoms (for example, a benzoyl group, a naphthoyl group)); The aryl group in the aryl-carbonyl group refers to, for example, a monocyclic to tricyclic aromatic hydrocarbon group, and specific examples thereof include a phenyl group, a naphthyl group, an anthryl group, and a phenanthryl group. Among these, as the acyl group, a formyl group, an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a benzoyl group, a naphthoyl group, and the like are preferred, and an acetyl group and a benzoyl group are more preferred.

[0095] Examples of the alkyl moiety of the alkylamino group and alkylcarbonylamino group include a straight-chain or branched alkyl group (preferably having 1 to 24 carbon atoms, more preferably having 1 to 16 carbon atoms, even more preferably having 1 to 10 carbon atoms, and particularly preferably having 1 to 4 carbon atoms), such as a methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, or octadecyl group. Examples of the cycloalkyl moiety of the cycloalkylamino group and cycloalkylcarbonylamino group include a cycloalkyl group (preferably having 3 to 24 carbon atoms, more preferably having 3 to 16 carbon atoms, even more preferably having 3 to 10 carbon atoms, and particularly preferably having 3 to 6 carbon atoms), such as a cyclopentyl group or a cyclohexyl group. The alkoxy moiety of the alkoxycarbonyl group is exemplified by the same as the alkoxy group.

[0096] The above-mentioned substituents may be further substituted at substitutable positions with at least one of a halogen, an alkyl group, a cycloalkyl group, an alkenyl group, an alkynyl group, a phenyl group, an alkoxy group, a hydroxyl group, an amino group, an alkylamino group, and a cycloalkylamino group. Examples of the halogen, alkoxy group, alkylamino group, and cycloalkylamino group are the same as those described above. Examples of the alkyl group include alkyl groups (preferably having 1 to 24 carbon atoms, more preferably having 1 to 16 carbon atoms, even more preferably having 1 to 10 carbon atoms, and particularly preferably having 1 to 4 carbon atoms) such as methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, and octadecyl group. Examples of the cycloalkyl group include a cycloalkyl group such as a cyclopentyl group or a cyclohexyl group (preferably having 3 to 24 carbon atoms, more preferably having 3 to 16 carbon atoms, even more preferably having 3 to 10 carbon atoms, and particularly preferably having 3 to 6 carbon atoms). Examples of the alkenyl group include an alkenyl group such as a vinyl group, a propenyl group, or a butenyl group (preferably having 2 to 24 carbon atoms, more preferably having 2 to 16 carbon atoms, even more preferably having 2 to 10 carbon atoms, and particularly preferably having 2 to 4 carbon atoms). Examples of the alkynyl group include an alkynyl group such as an ethynyl group, a propargyl group, a butynyl group, or a pentynyl group (preferably having 2 to 24 carbon atoms, more preferably having 2 to 16 carbon atoms, even more preferably having 2 to 10 carbon atoms, and particularly preferably having 2 to 4 carbon atoms).

[0097] Among them, R ６ R is preferably a substituted or unsubstituted alkyl group, and more preferably a linear alkyl group. ６ As R, a substituted or unsubstituted arylalkyl group is also preferred. ５ is an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R ６ The number of carbon atoms in R is preferably 18 to 26, more preferably 24 to 26. ５ is a hydrogen atom, R６ The number of carbon atoms in R is preferably 24 to 26. ６ Specific examples of the group include -(CH ２ ) ２３ -CH ３ , -(CH ２ ) ２４ -CH ３ , -(CH ２ ) ２５ -CH ３ , 10-(4-fluorophenyl)decyl, etc. ７ R is preferably a substituted or unsubstituted alkyl group, and more preferably a linear alkyl group. ７ The number of carbon atoms in R is preferably 9 to 20, more preferably 12 to 18. ７ Specific examples of the group include -(CH ２ ) １１ -CH ３ , -(CH ２ ) １２ -CH ３ , -(CH ２ ) １３ -CH ３ , -(CH ２ ) １４ -CH ３ , -(CH ２ ) １５ -CH ３ , -(CH ２ ) １６ -CH ３ , -(CH ２ ) １７ -CH ３ etc.

[0098] Y is -CH ２ It represents -, -CH(OH)- or -CH=CH-, and among these, -CH(OH)- is preferred.

[0099] When compound (II) has stereoisomers, all isomers are encompassed in the present invention, and a mixture of two or more isomers in any ratio (including a racemate) may also be included. In particular, compound (II) has optical isomers derived from the asymmetric carbon of the lipid moiety, and in the present invention, it may be a single optically active substance or a mixture of two or more optically active substances in any ratio (including a racemate). -NHCOR ６ The asymmetric carbon to which is bonded is preferably in the S configuration. ６ The asymmetric carbon atom having —OH adjacent to the asymmetric carbon atom to which —NHCOR is bonded is ６ When Y is —CH(OH)—, the asymmetric carbon in —CH(OH)— represented by Y preferably has the R configuration.

[0100] The lipid moiety of compound (II) may be:

[0101]

[0102] (wherein each symbol has the same meaning as defined above), etc.

[0103] The salt of compound (II) is preferably a pharmacologically acceptable salt, and examples thereof include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, and phosphate; organic acid salts such as succinate, fumarate, acetate, methanesulfonate, and toluenesulfonate; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; and ammonium salts such as ammonium salt and alkylammonium salt.

[0104] Specific examples of the compound (II) suitable for use in the present invention are shown in Table 2, but the present invention is not limited to these.

[0105]

[0106] Among these, the following compound is preferred: A compound represented by Compound No. 2-9′ in Table 2, which has a stereostructure represented by the following formula (RCAI-61):

[0107]

[0108] Compounds represented by Compound No. 3-4' in Table 2, which have a stereostructure represented by the following formula (RCAI-64):

[0109]

[0110] A compound represented by Compound No. 3-13′ in Table 2, which has a stereostructure represented by the following formula (RCAI-85):

[0111]

[0112] A compound represented by Compound No. 165' in Table 2, which has a stereostructure represented by the following formula (RCAI-165):

[0113]

[0114] The compound represented by formula (II) or a salt thereof can be produced by the methods disclosed in WO2009 / 119692A1, US Patent No. 8,551,959, and the like.

[0115] [2. Liposome] The liposome composition of the present invention contains the glycolipid in the liposome.

[0116] In this specification, liposomes refer to substances that have a closed vesicle structure (water-soluble particles obtained by aggregation of amphipathic molecules containing hydrophilic and hydrophobic regions) whose main component is lipid or the like. Recently, they have also been called lipid nanoparticles (LNPs), but in this specification, the concept of liposomes also includes those called lipid nanoparticles as long as they have the above structure. In the present invention, the liposome component may be any amphipathic molecule that can form a lipid-closed vesicle structure by known methods, and lipids are preferred.Examples of lipids in the present invention include phosphatidylcholines (e.g., dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine), phosphatidylglycerols (e.g., dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol), and ol), phosphatidylethanolamines (e.g., dioleoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidiethanolamine), phosphatidylserine, phosphatidylinositol, phosphatidic acid, phospholipids such as cardiolipin, glycosphingolipids, glyceroglycolipids, cationic lipids (e.g., DOTAP, DLin-MC3-DMA ((6Z, Examples include (9Z, 28Z, 31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino) butanoate), ALC-0315 ([(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), and SM-102 (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate)), and these can be used alone or in combination with other substances, such as cholesterol or lipid derivatives in which water-soluble polymers such as polyethylene glycol are bound to lipids, to prepare liposomes.

[0117] The liposome in the present invention is preferably a liposome having affinity for antigen-presenting cells. Suitable examples of the liposome in the present invention (examples of liposomes having affinity for antigen-presenting cells) include the following liposomes (#2) to (#8).

[0118] (#2) Liposomes Comprising DOTAP, DOPE, and Cholesterol In the liposomes of (#2), the weight ratio of DOTAP, DOPE, and cholesterol (DOTAP:DOPE:cholesterol) is, for example, 1:0.1-10:0.1-10, preferably 1:0.3-3:0.2-2. When the liposomes of (#2) are used in the present invention, the weight ratio of the "NKT cell ligand of the present invention" to the "total of DOTAP, DOPE, and cholesterol" is, for example, 1:1-10,000, preferably 1:1-1,000.

[0119] (#3) Liposomes Comprising DOTAP, DOPE, MPEG2000-DMPE, Na Salt, and Cholesterol In the liposomes of (#3), the weight ratio of DOTAP, DOPE, MPEG2000-DMPE, Na salt, and cholesterol (DOTAP:DOPE:MPEG2000-DMPE, Na salt:cholesterol) is, for example, 1:0.1-10:0.1-10:0.1-10, preferably 1:0.3-3:0.2-2:0.2-2. When the liposomes of (#3) are used in the present invention, the weight ratio of the "NKT cell ligand of the present invention" to the "total of DOTAP, DOPE, MPEG2000-DMPE, Na salt, and cholesterol" is, for example, 1:1-10,000, preferably 1:1-1,000.

[0120] (#4) Liposomes Comprising DOTAP, DOPE, MPEG2000-DSPE, Na Salt, and Cholesterol In the liposomes of (#4), the weight ratio of DOTAP, DOPE, MPEG2000-DSPE, Na salt, and cholesterol (DOTAP:DOPE:MPEG2000-DSPE, Na salt:cholesterol) is, for example, 1:0.1-10:0.1-10:0.1-10, preferably 1:0.3-3:0.2-2:0.2-2. When the liposomes of (#4) are used in the present invention, the weight ratio of the "NKT cell ligand of the present invention" to the "total of DOTAP, DOPE, MPEG2000-DSPE, Na salt, and cholesterol" is, for example, 1:1-10,000, preferably 1:1-1,000.

[0121] (#5) Liposomes Comprising HSPC, DSPG, and Cholesterol In the liposomes of (#5), the weight ratio of HSPC, DSPG, and cholesterol (HSPC:DSPG:cholesterol) is, for example, 1:0.1-10:0.1-10, preferably 1:0.2-2:0.2-2. When the liposomes of (#5) are used in the present invention, the weight ratio of the "NKT cell ligand of the present invention" to the "total of HSPC, DSPG, and cholesterol" is, for example, 1:1-10,000, preferably 1:1-1,000.

[0122] (#6) Liposomes Comprising HSPC, DOPS, and Cholesterol Liposomes (#6) and liposomes (#7) below have the same composition, but when used to produce NKT cell ligand-containing liposomes, the former is an example of an embodiment in which a relatively large amount is added relative to the NKT cell ligand, while the latter is an example of an embodiment in which a relatively small amount is added relative to the NKT cell ligand. In liposomes (#6), the weight ratio of HSPC, DOPS, and cholesterol (HSPC:DOPS:cholesterol) is, for example, 1:0.1-10:0.1-10, preferably 1:0.2-2:0.2-2. When liposomes (#6) are used in the present invention, the weight ratio of the "NKT cell ligand of the present invention" to the "total of HSPC, DOPS, and cholesterol" is, for example, 1:1-100,000, preferably 1:1-10,000.

[0123] (#7) Liposomes Comprising HSPC, DOPS, and Cholesterol In liposomes (#7), the weight ratio of HSPC, DOPS, and cholesterol (HSPC:DOPS:cholesterol) is, for example, 1:0.1-10:0.1-10, preferably 1:0.2-2:0.2-2. When liposomes (#7) are used in the present invention, the weight ratio of the "NKT cell ligand of the present invention" to the "total of HSPC, DOPS, and cholesterol" is, for example, 1:1-10,000, preferably 1:1-1,000. Examples of liposome compositions of the present invention using liposomes (#7) include the RK-163-containing liposome composition, RCAI-61-containing liposome composition, and RCAI-137-containing liposome composition described in Examples 1 and 2 below.

[0124] (#8) Liposomes Comprising HSPC, MPEG2000-DSPE, Na Salt, and Cholesterol In the liposomes of (#8), the weight ratio of HSPC, MPEG2000-DSPE, Na salt, and cholesterol (HSPC:MPEG2000-DSPE, Na salt:cholesterol) is, for example, 1:0.1-10:0.1-10, preferably 1:0.2-2:0.2-2. When the liposomes of (#8) are used in the present invention, the weight ratio of the "NKT cell ligand of the present invention" to the "total of HSPC, MPEG2000-DSPE, Na salt, and cholesterol" is, for example, 1:1-10,000, preferably 1:1-1,000.

[0125] In the present invention, among the above liposomes, (#5) to (#8) are preferred, (#7) and (#8) are more preferred, and (#7) is even more preferred.

[0126] 3. NKT Cell Ligand-Containing Liposome Composition The liposomes constituting the NKT cell ligand-containing liposome composition of the present invention can be prepared according to known methods. For example, the method described in Liposome Technology, vol. 1, 2nd edition (by Gregory Gregoriadis (CRC Press, Boca Raton, Ann Arbor, London, Tokyo), Chapter 4, pp. 67-80, Chapter 10, pp. 167-184, and Chapter 17, pp. 261-276 (1993)) can be used. More specifically, examples of such methods include, but are not limited to, ultrasonication, ethanol injection, French press, ether injection, cholate method, calcium fusion, freeze-thaw method, and reverse-phase evaporation. Specifically, as shown in the Examples, the liposome components and the NKT cell ligand are each dissolved in an organic solvent in a glass container, nitrogen gas is sprayed onto the solution to evaporate the organic solvent, and the solution is completely dried in a desiccator (under reduced pressure). An isotonic aqueous solvent (e.g., 9% sucrose aqueous solution, 5% glucose aqueous solution, phosphate-buffered saline, etc.) is added to the dried material, and emulsified using a homogenizer or the like to obtain NKT cell ligand-containing liposomes. Furthermore, liposomes with a uniform particle size can be obtained by passing the dried material through a polycarbonate membrane filter (pore size 0.4 μm or 0.2 μm) under heat and pressure. Finally, to ensure sterility, the material can also be passed through a standard sterilizing filter (pore size 0.22 μm; for example, a PVDF membrane filter).

[0127] In the present invention, the NKT cell ligand-containing liposome composition is preferably an RK-163-containing liposome composition, an RCAI-61-containing liposome composition, or an RCAI-137-containing liposome composition, and more preferably an RK-163-containing liposome composition, an RCAI-61-containing liposome composition, or an RCAI-137-containing liposome composition, in which the liposome is the aforementioned liposome (#7).

[0128] In the NKT cell ligand-containing liposome composition of the present invention, the weight ratio of "a glycolipid (the NKT cell ligand of the present invention) selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and a salt thereof" to "the total of other lipids constituting the liposome (other than the NKT cell ligand as the main drug)" is, for example, 1:1 to 100,000, preferably 1:1 to 10,000, more preferably 1:1 to 1,000, and even more preferably 1:1 to 100.

[0129] The average particle size of the NKT cell ligand-containing liposome composition of the present invention is not particularly limited, but is, for example, 10 to 1,000 nm, preferably 10 to 500 nm, and more preferably 50 to 500 nm. The average particle size can be measured by a conventional quasi-elastic light scattering method.

[0130] The NKT cell ligand-containing liposome composition of the present invention can be used alone or after being formulated by known methods together with commonly used pharmaceutically acceptable additives (e.g., solvents, stabilizers, isotonicity agents, soothing agents, buffers, pH adjusters). When the liposome composition of the present invention is a liquid preparation such as an injection, it may be stored frozen or after removing water by freeze-drying or the like. When using a freeze-dried preparation, it is redissolved by adding distilled water for injection or the like.

[0131] [4. Immune Checkpoint Inhibitors] The immune checkpoint inhibitors used in the present invention are drugs that inhibit the function of immune checkpoint molecules or their ligands, including, but not limited to, anti-PD-1 antibodies and their ligand antibodies, anti-PD-L1 antibodies. Commercially available anti-PD-1 antibodies can be used, including nivolumab (trade name Opdivo) and pembrolizumab (trade name Keytruda). Nivolumab is a recombinant human IgG4 monoclonal antibody against human PD-1, in which the 221st amino acid residue in the heavy chain is substituted with Pro. It is a glycoprotein produced by Chinese hamster ovary cells and is composed of two heavy chains of 440 amino acid residues and two light chains of 214 amino acid residues (see the package insert "Opdivo (registered trademark) Intravenous Infusion 20 mg," revised June 2023). Commercially available anti-PD-L1 antibodies include atezolizumab (trade name Tecentriq), avelumab (trade name Bavencio), and durvalumab (trade name Imfinzi). Decentriq is a protein consisting of two light chains of 214 amino acids and two heavy chains of 448 amino acids (see the package insert "Tecentriq (registered trademark) Intravenous Infusion 1200 mg," revised May 2022).

[0132] [5. Combination of immune checkpoint inhibitor and NKT cell ligand-containing liposome composition] In the present invention, a synergistic antitumor effect can be expected by administering an immune checkpoint inhibitor and an NKT cell ligand-containing liposome in combination.

[0133] Tumors (malignant tumors) for which a therapeutic effect is expected from a combination of an immune checkpoint inhibitor and an NKT cell ligand-containing liposome composition include, but are not limited to, breast cancer, colon cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, ovarian cancer, skin cancer, melanoma, and blood tumors. Furthermore, the agent is expected to be effective even in cases where multiple cancers occur simultaneously.

[0134] Examples of administration methods include, but are not limited to, intravenous administration, intraarterial administration, intramucosal administration, intralymph node administration, and intra-affected tissue administration. The immune checkpoint inhibitor and the NKT cell ligand-containing liposome composition may be administered simultaneously or separately at different times. They may also be administered via different routes.

[0135] The amount of NKT cell ligand in the NKT cell ligand-containing liposome composition administered at one time may be sufficient to activate NKT cells in vivo and treat the target disease. For example, in the case of an NKT cell ligand-containing liposome composition, the amount is typically 400 ng / kg to 4 mg / kg, but is not limited to this range. Administration may be performed multiple times depending on the condition of the subject, the severity of the disease, and other factors. Furthermore, the anti-PD-1 antibody was administered at 80 mg / mouse (3.2 mg / kg: 192 mg equivalent to 60 kg), an amount that was not effective when administered alone in preliminary experiments. For administration to humans, the NKT cell ligand-containing liposome composition can be administered at a dose of 480 mg every four weeks, or at a dose of 240 mg every two weeks, as determined by the standard adult administration method.

[0136] The antitumor agent of the present invention can be safely administered to humans and non-human mammals (for example, mice, rats, rabbits, dogs, cats, cows, horses, monkeys, pigs, etc.).

[0137] The present invention also relates to the following (1) and (2). (1) An NKT cell ligand-containing liposome composition for enhancing the therapeutic effect of an immune checkpoint inhibitor. (2) An immune checkpoint inhibitor for enhancing the therapeutic effect of an NKT ligand-containing liposome composition. In (1) and (2), the definitions, examples, and manufacturing methods (methods of obtaining) of the "NKT cell ligand," "liposome," "NKT ligand-containing liposome composition," and "immune checkpoint inhibitor" are the same as the definitions, examples, and manufacturing methods (methods of obtaining) of the "NKT cell ligand," "liposome," "NKT ligand-containing liposome composition," and "immune checkpoint inhibitor" explained above for the combination agent of the present invention. In (1) and (2), the administration method, dosage, administration target, and therapeutic effect of the combination of the "NKT ligand-containing liposome composition" and the "immune checkpoint inhibitor" can be appropriately determined based on the administration method, dosage, administration target, and therapeutic effect of the combination explained above in [5. Combination of an immune checkpoint inhibitor and an NKT cell ligand-containing liposome composition] for the combination agent of the present invention.

[0138] The present invention also relates to the following (3) and (4). (3) An agent for enhancing the expression of immune checkpoint molecules by activating a cancer immune cell population, comprising an NKT cell ligand-containing liposome composition. (4) An agent for enhancing the expression of immune checkpoint ligand molecules on cancer cells by artificially activating NKT cells, comprising an NKT cell ligand-containing liposome composition. In (3) and (4), the definitions, examples, and production methods (methods of obtaining) of "NKT cell ligand," "liposome," and "NKT ligand-containing liposome composition" are the same as the definitions, examples, and production methods (methods of obtaining) of "NKT cell ligand," "liposome," and "NKT ligand-containing liposome composition" explained above for the combination drug of the present invention.

[0139] 2. Invention-2 The present invention (Invention-2) is described in detail below. The present invention relates to a liposome composition containing an NKT cell ligand and an antigen.

[0140] [1. NKT cell ligand] The NKT cell ligand used in the present invention can be the same as the NKT cell ligand explained in Invention-1.

[0141] [2. Antigen] In the present invention, an antigen is a substance that can be recognized by the immune system, and examples thereof include self-components such as cancer antigens and foreign components such as pathogen antigens. In the present invention, a cancer antigen is a self-component produced by cancer cells or a modified self-component that can be recognized by the immune system. Cancer antigens can be produced by peptide synthesis based on gene sequences. WT-1, described in a literature (Jpn J Clin Oncol 2010 40:(5) 377-387), can also be used as a cancer antigen.

[0142] [3. Liposome] The liposome composition of the present invention contains the glycolipid and the antigen in a liposome. The liposome used in the present invention can be the same as the liposome described in Invention-1.

[0143] [4. Liposome composition containing an NKT cell ligand and an antigen] The liposome composition of the present invention containing an NKT cell ligand and an antigen (e.g., a cancer antigen) may be a liposome containing an NKT cell ligand and an antigen (e.g., a cancer antigen).

[0144] Liposomes constituting the liposome composition of the present invention containing an NKT cell ligand and an antigen can be prepared according to known methods. For example, the method described in Liposome Technology, vol. 1, 2nd edition (by Gregory Gregoriadis (CRC Press, Boca Raton, Ann Arbor, London, Tokyo), Chapter 4, pp. 67-80, Chapter 10, pp. 167-184, and Chapter 17, pp. 261-276 (1993)) can be used. More specifically, examples of such methods include, but are not limited to, ultrasonication, ethanol injection, French press, ether injection, cholate method, calcium fusion, freeze-thaw method, and reverse-phase evaporation. Specifically, for example, when the antigen is a cancer antigen, as shown in the Examples, the liposome components and the NKT cell ligand are each dissolved in an organic solvent in a glass container, nitrogen gas is sprayed onto the solution to evaporate the organic solvent, and the mixture is completely dried in a desiccator (under reduced pressure). A cancer antigen solution dissolved in an isotonic aqueous solvent (e.g., 9% sucrose aqueous solution, 5% glucose aqueous solution, phosphate-buffered saline, etc.) is added to the dried product, and the mixture is emulsified using a homogenizer or the like to obtain liposomes containing an NKT cell ligand and a cancer antigen (the liposome composition of the present invention). Furthermore, liposomes with a uniform particle size can be obtained by passing the mixture through a polycarbonate membrane filter (pore size 0.4 μm or 0.2 μm) under heat and pressure. Finally, to ensure sterility, the mixture can also be passed through a standard sterilizing filter (pore size 0.22 μm; for example, a PVDF membrane filter).

[0145] The liposome composition of the present invention containing an NKT cell ligand and an antigen is preferably a liposome composition containing RK-163 and an antigen, a liposome composition containing RCAI-61 and an antigen, or a liposome composition containing RCAI-137 and an antigen, and more preferably a liposome composition containing RK-163 and an antigen, a liposome composition containing RCAI-61 and an antigen, or a liposome composition containing RCAI-137 and an antigen, in which the liposome is the aforementioned liposome (#7). The liposome composition of the present invention containing an NKT cell ligand and a cancer antigen is preferably a liposome composition containing RK-163 and a cancer antigen, a liposome composition containing RCAI-61 and a cancer antigen, or a liposome composition containing RCAI-137 and a cancer antigen, and more preferably a liposome composition containing RK-163 and a cancer antigen, a liposome composition containing RCAI-61 and a cancer antigen, or a liposome composition containing RCAI-137 and a cancer antigen, in which the liposome is the aforementioned liposome (#7).

[0146] In the liposome composition of the present invention containing an NKT cell ligand and an antigen, the weight ratio of "a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and a salt thereof (the NKT cell ligand of the present invention)" to "the total of other lipids constituting the liposome (other than the NKT cell ligand as the main drug)" is, for example, 1:1 to 100,000, preferably 1:1 to 10,000, more preferably 1:1 to 1,000, and even more preferably 1:1 to 100.

[0147] The average particle size of the liposome composition of the present invention containing an NKT cell ligand and an antigen is not particularly limited, but is, for example, 10 to 1,000 nm, preferably 10 to 500 nm, and more preferably 50 to 500 nm. The average particle size can be measured by a conventional quasi-elastic light scattering method.

[0148] The liposome composition of the present invention containing an NKT cell ligand and an antigen can be used alone or after being formulated by known methods together with commonly used pharmaceutically acceptable additives (e.g., solvents, stabilizers, isotonicity agents, soothing agents, buffers, pH adjusters). When the liposome composition of the present invention is a liquid preparation such as an injection, it may be stored frozen or after removing water by freeze-drying or the like. When using a lyophilized preparation, it is redissolved by adding distilled water for injection or the like.

[0149] The liposome composition of the present invention containing an NKT cell ligand and an antigen (e.g., a cancer antigen) is useful as a pharmaceutical for inducing immunological memory. The liposome composition of the present invention containing an NKT cell ligand and a cancer antigen is useful as an antitumor agent. Furthermore, the liposome composition of the present invention containing an NKT cell ligand and a cancer antigen is expected to prophylactically induce the activation and proliferation of immune cell populations that recognize cancer antigens, even in the absence of cancer. Furthermore, the liposome composition of the present invention containing an NKT cell ligand and a cancer antigen is expected to prophylactically induce immunological memory against cancer, even in the absence of cancer. Furthermore, the liposome composition of the present invention containing an NKT cell ligand and a cancer antigen is expected to have an enhanced effect in suppressing the onset, recurrence, and metastasis of cancer.

[0150] Cancers for which a preventive effect can be expected from a liposome composition containing an NKT cell ligand and a cancer antigen include, for example, hereditary cancers and similar main examples such as breast cancer, colon cancer, ovarian cancer, bone and soft tissue tumors, skin cancer, urinary tract cancer, brain tumors, and endocrine tumors, but the agent can also be applied to other types of cancer, such as lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumors, skin cancer, melanoma, and blood tumors, without being limited to these. The agent is also expected to be effective in cases where multiple cancers have developed simultaneously.

[0151] Examples of administration methods include, but are not limited to, intravenous administration, intraarterial administration, intramucosal administration, intralymph node administration, and intra-tissue administration at the affected site.

[0152] The amount of NKT cell ligand in a liposome composition containing an NKT cell ligand and an antigen administered at one time should be sufficient to activate NKT cells and prevent disease by activating and proliferating antigen-specific CD8 T cells and forming long-term memory. Typically, one course of treatment is administered by first administering 400 ng / kg to 4 mg / kg of NKT cell ligand and 2 mg / kg to 20 mg / kg of antigen, followed by a second administration of the same doses four weeks later. Since the establishment and maintenance of immune memory differs depending on the type of antigen, the number of CD8 immune memory T cells is subsequently monitored through follow-up, and "administrations at four-week intervals" are repeated on an ongoing basis, aiming to maintain the number of memory cells at or above pre-treatment levels. Multiple administrations may also be administered depending on the subject's condition, the severity of the disease, and other factors.

[0153] The liposome composition of the present invention containing an NKT cell ligand and a cancer antigen can activate various immune cell populations and induce cancer immune memory before cancer cells have yet to develop in the body, making it possible to prevent the onset, recurrence, and metastasis of cancer. Therefore, the liposome of the present invention containing an NKT cell ligand and an antigen may be applicable to the prevention of not only cancer but also infectious diseases, simply by changing the type of antigen.

[0154] The liposome composition of the present invention containing an NKT cell ligand and an antigen can be safely administered to humans and non-human mammals (e.g., mice, rats, rabbits, dogs, cats, cows, horses, monkeys, pigs, etc.).

[0155] The present invention will be described in more detail below based on examples and test examples, but the present invention is not limited to these.

[0156] RK-163, RCAI-61, and RCAI-137 used in the examples are compounds represented by the chemical structural formulas described above. In the examples, the pseudo-cancer antigen ovalbumin (OVA) was used instead of a cancer antigen. Pseudo-cancer antigens do not exist in the body, but are foreign artificial substances to which the immune system can react, and can be expressed in cancer cell lines to behave like cancer antigens. The abbreviations used in this specification have the following meanings: HSPC: Hydrogenated soybean phosphatidycholine DOPS: 1,2-dioleoyl-sn-glycero-3-phospho-L-serine OVA: Ovalbumin PBS: phosphate buffered saline

[0157] [Example 1] [Preparation of NKT cell ligand-containing liposomes] Samples for safety testing and main pharmacological testing of NKT cell ligand-containing liposome compositions (liposome compositions containing an NKT cell ligand (RK-163, RCAI-61, or RCAI-137) in the aforementioned liposome (#7)) were prepared using the reagents and ingredients shown in Table 3 according to the preparation method described below.

[0158] (Composition of prepared samples) - For safety testing: 15 ng / mL RK-163 (or RCAI-61, and RCAI-137), 220.8 ng / mL total lipid in 9% sucrose aqueous solution. Lipid composition: HSPC 103 ng / mL, DOPS 79.7 ng / mL, cholesterol 38.1 ng / mL - For main pharmacology testing: 1.5 ng / mL RK-163 (or RCAI-61, and RCAI-137), 22.08 ng / mL total lipid in 9% sucrose aqueous solution. Lipid composition: HSPC 10.3 ng / mL, DOPS 7.97 ng / mL, cholesterol 3.81 ng / mL. Particle size: 145.7 nm. PDI: 0.06813

[0159]

[0160] (Preparation Method) - Safety Test: 10.3 mg of HSPC, 7.97 mg of DOPS, and 3.81 mg of cholesterol were placed in one flask and dissolved in 10 mL of a 1:2 (volume ratio) methanol:chloroform solution. 1.0 mL of this solution was mixed with 1.5 mL of a 100 μg / mL RK-163 (or RCAI-61 or RCAI-137) solution in a 1:2 (volume ratio) methanol:chloroform solution. The mixture was dried by heating and blowing with nitrogen, then placed in a desiccator. 10 mL of 9% sucrose solution was added to the dried mixture, emulsified using a Teflon homogenizer, and sieved using an extruder (heated to 50°C, 0.2 μm pore size polycarbonate membrane filter, one sheet). The prepared liposome dispersion was further diluted 1000-fold with a 9% sucrose solution, filtered through a 0.22 μm pore size PVDF membrane filter for sterilization, placed in a glass bottle with a screw cap, and stored at 4° C. This solution was used as the sample (for safety testing).

[0161] - Primary pharmacological test: 1 mL of the sample (for safety testing) was placed in a separate glass bottle, and 9 mL of 9% sucrose aqueous solution was added to it and mixed for dilution. The resulting liposome dispersion was filtered through a 0.22 μm pore size PVDF membrane filter for sterilization, placed in a glass bottle with a screw cap, and stored at 4°C. This solution was used as the sample (for primary pharmacological testing). This sample was used as the NKT cell ligand-containing liposome composition in the following Test Examples 1 to 3 and 5.

[0162] Test Example 1 Activation of immune cell populations and PD-1 expression by administration of a liposome composition containing an NKT cell ligand (RK-163, RCAI-61, or RCAI-137) An effective dose (1 ng / mouse) of a liposome composition containing an NKT cell ligand (RK-163-containing liposome composition (hereinafter referred to as RK-163-LIPO), RCAI-61-containing liposome composition (hereinafter referred to as RCAI-61-LIPO), or RCAI-137-containing liposome composition (hereinafter referred to as RCAI-137-LIPO)) and 2 x 10 OVA-pulsed splenocytes as a pseudotumor antigen were administered. 7The adjuvant effect of activated NKT cells in the liver was analyzed using PD-1 expression as an indicator of innate and adaptive immune cell populations by administering RK-163-LIPO, RCAI-61-LIPO, and RCAI-137-LIPO via the tail vein of C57BL / 6 mice (10 weeks old, 5 mice per group). A control group received the same volume of PBS instead of the NKT cell ligand-containing liposome composition. The results are shown in Figure 1. Following activation of NKT cells in the liver with RK-163-LIPO, RCAI-61-LIPO, and RCAI-137-LIPO, adaptive immune CD4 T and CD8 T cells and innate immune NK cells were activated, and the number of cells expressing PD-1, an indicator of activation, was confirmed. Administration of the NKT cell ligand-containing liposome composition to a mouse tumor-bearing model (subcutaneous tumor model), was confirmed to enhance PD-1 expression in various immune cell populations.

[0163] Test Example 2 Enhancement of Expression of Immune Checkpoint Ligand Molecules (PD-1 Ligand Molecules: PD-L1) on Tumor Cells in Vivo by Administration of Liposome Compositions Containing NKT Cell Ligands (RK-163, RCAI-61, or RCAI-137)

[0123] An effective dose (1 ng / mouse) of a liposome composition containing an NKT cell ligand (RK-163-containing liposome composition (RK-163-LIPO), RCAI-61-containing liposome composition (RCAI-61-LIPO), or RCAI-137-containing liposome composition (RCAI-137-LIPO)) and 1 x 10 X-ray irradiated MO4 tumor cells (B16 mouse melanoma cells) were administered. 6 were administered into the tail vein of mice (C57BL / 6J, 10-week-old) (5 mice per group), and then 3x10 live MO4 tumor cells were injected into the tail vein of mice (C57BL / 6J, 10-week-old) (5 mice per group). 5The NKT cell ligand-containing liposome composition was subcutaneously implanted into the tumor cells (MO4) and analyzed for immune checkpoint ligand molecule (PD-1 ligand molecule: PD-L1) expression 17 days later. A control group received the same amount of PBS instead of the NKT cell ligand-containing liposome composition. The results are shown in Figure 2. As a result, the number of cells expressing immune checkpoint ligand molecules (PD-1 ligand molecule: PD-L1) on the membrane of tumor cells (MO4) in vivo was significantly increased in mice treated with RK-163-LIPO, RCAI-61-LIPO, and RCAI-137-LIPO. Administration of the NKT cell ligand-containing liposome composition to a mouse tumor-bearing model confirmed enhanced expression of immune checkpoint ligand molecules (PD-1 ligand molecule: PD-L1) on tumor cells.

[0164] Test Example 3 Enhancement of PD-1 Ligand Molecule (PD-L1) Expression on Tumor Cells (MO4) by Artificially Activated NKT Cells An effective dose of 1 ng of a liposome composition containing an NKT cell ligand (RK-163, RCAI-61, or RCAI-137) (RK-163-containing liposome composition (RK-163-LIPO), RCAI-61-containing liposome composition (RCAI-61-LIPO), or RCAI-137-containing liposome composition (RCAI-137-LIPO)) was administered to 3 x 10 cells of the NKT cell line 2E10. 5 After 24 hours of co-culture with 2E10 cells (3x10 5 ) and MO4 cells (3x10 5The MO4 cells were co-cultured with RK-163-LIPO, RCAI-61-LIPO, and RCAI-137-LIPO, and the expression of immune checkpoint ligand molecules (PD-1 ligand molecules: PD-L1) was measured after 4, 24, and 48 hours. The results are shown in Figure 3. The number of cells expressing immune checkpoint ligand molecules (PD-1 ligand molecules: PD-L1) on tumor cells (MO4) significantly increased between 24 and 48 hours after co-culture in all groups treated with RK-163-LIPO, RCAI-61-LIPO, and RCAI-137-LIPO. To confirm the mechanism behind increased PD-L1 expression on tumor cells, an NKT cell line (2E10) activated with the NKT cell ligand-containing liposome composition was co-cultured with tumor cells (MO4), and PD-L1 expression on tumor cells was measured over time. It was confirmed that the expression of immune checkpoint ligand molecules (PD-L1) reached its peak between 24 and 48 hours after the start of co-culture.

[0165] [Test Example 4] Enhancement of immune checkpoint ligand molecule PD-L1 expression on tumor cells (MO4) by IFNγ Tumor cells (MO4) 3 x 10 5 1 ng of IFNγ recombinant protein was added to each cell and cultured. After 0, 4, 24, and 48 hours, the MO4 cells were harvested and the expression of immune checkpoint ligand molecules (PD-1 ligand molecules: PD-L1) was measured. The results are shown in Figure 4. As a result, the expression of immune checkpoint ligand molecules (PD-1 ligand molecules: PD-L1) on tumor cells was significantly increased 24 and 48 hours after culture, demonstrating that IFNγ is involved in the expression of immune checkpoint ligand molecules (PD-1 ligand molecules: PD-L1) on tumor cells. Tumor cells (MO4) were cultured with 1 ng / mL of IFNγ recombinant protein, and the tumor cells MO4 were recovered over time to analyze PD-L1 expression. PD-L1 ligand molecule expression on the tumor cells significantly increased 24-48 hours after the start of culture, confirming that the increased expression of immune checkpoint ligand molecules (PD-1 ligand molecules: PD-L1) on tumor cells in Test Example 2 (Figure 2) and Test Example 3 (Figure 3) was due to NKT cell-derived IFNγ.

[0166] Test Example 5: Induction of a strong antitumor effect by combined treatment with an anti-PD-1 antibody and a liposome composition containing an NKT cell ligand (RK-163, RCAI-61, or RCAI-137) NKT cell ligand-containing liposome compositions (RK-163-containing liposome composition (RK-163-LIPO), RCAI-61-containing liposome composition (RCAI-61-LIPO), or RCAI-137-containing liposome composition (RCAI-137-LIPO)) (administered at a dose of 1 ng / 25 g body weight equivalent to the amount of RK-163, RCAI-61, or RCAI-137)) and 1 x 10 B16 mouse melanoma (MO4) cells irradiated with X-rays (50 Gy) were administered. 6 were administered intravenously to C57BL / 6 mice (10 weeks old), followed by the administration of 3x10 live B16 mouse melanoma cells (MO4). 5 The MO4 cells were then subcutaneously transplanted into the same mice, and tumor growth was monitored over time. Furthermore, 80 mg of anti-PD-1 antibody was administered intravenously on days 3 and 14 after transplantation. After subcutaneous transplantation of MO4 cells, tumor diameter was monitored on days 7, 10, 14, and 17. All measurements were performed using a digital caliper. Tumor volume (mm 3 ) = major axis (mm) x minor axis (mm) x height (mm). The results are shown in Figures 5-1, 5-2, and 5-3. As a result, the combined administration of an NKT cell ligand-containing liposome composition (RK-163-LIPO, RCAI-61-LIPO, or RCAI-137-LIPO) and an anti-PD-1 antibody demonstrated a stronger antitumor effect than either composition administered alone. It was confirmed that the combined administration of an NKT cell ligand-containing liposome composition and an anti-PD-1 antibody to tumor-bearing mouse models (subcutaneous tumor models) resulted in a more effective antitumor effect than the administration of either an NKT cell ligand-containing liposome formulation or an anti-PD-1 antibody alone.

[0167] [Example 2] [Preparation of liposomes containing an NKT cell ligand and a cancer antigen] Samples for safety testing and main pharmacological testing of a liposome composition containing an NKT cell ligand and a cancer antigen (a liposome composition comprising the aforementioned liposome (#7) containing an NKT ligand (RK-163, RCAI-61, or RCAI-137) and a pseudo-cancer antigen (OVA)) were prepared using the reagents and ingredients shown in Table 4 according to the preparation method described below.

[0168] (Composition of prepared samples) - For safety testing: 15 ng / mL RK-163 (or RCAI-61, RCAI-137), 221 ng / mL lipid in 9% sucrose solution. Lipid composition: HSPC 103 ng / mL, DOPS 79.7 ng / mL, cholesterol 38.1 ng / mL OVA: 100 mg / mL in 9% sucrose solution - For main pharmacology testing: 1.5 ng / mL RK-163 (or RCAI-61, RCAI-137), 22.08 ng / mL lipid in 9% sucrose solution. Lipid composition: HSPC 10.3 ng / mL, DOPS 7.97 ng / mL, cholesterol 3.81 ng / mL OVA: 10 mg / mL in 9% sucrose solution. Particle size: 145.7 nm, PDI: 0.06813

[0169]

[0170] (Preparation Method) - For safety testing: 10.3 mg of HSPC, 7.97 mg of DOPS, and 3.81 mg of cholesterol were placed in one flask and dissolved in 10 mL of a 1:2 (volume) methanol:chloroform solution. 1.5 mL of this solution was mixed with 1 mL of a 100 μg / mL RK-163 (or RCAI-61 or RCAI-137) solution in a 1:2 (volume) methanol:chloroform solution. The mixture was heated and dried under nitrogen to form a lipid film, which was then placed in a desiccator. 2.208 mL of a 100 mg / mL OVA solution (in 9% sucrose water) was added to the dried mixture (lipid concentration: 5 mg / mL). The mixture was sized using an extruder (heated to 40°C, pore size: 0.4 μm → 0.2 μm). Unencapsulated OVA was removed by stirring ultrafiltration (membrane molecular weight cutoff: 300 kDa, external solution: 9% sucrose aqueous solution). The solution was filtered through a 0.22 μm pore size PVDF membrane filter for sterilization in a clean bench to adjust the particle size. The prepared solution was placed in a glass bottle with a screw cap. This solution was further diluted 1000-fold with 9% sucrose aqueous solution, filtered through a 0.22 μm filter, placed in a glass bottle with a screw cap, and stored at 4°C. This solution was used as a liposome sample (for safety testing) containing an NKT cell ligand (RK-163 (or RCAI-61, RCAI-137)) and a cancer antigen (OVA).

[0171] - For main pharmacological testing: 1 mL of liposomes containing an NKT cell ligand (RK-163 (or RCAI-61, RCAI-137)) and a cancer antigen (OVA) (for safety testing) was transferred to a separate glass vial, and 9 mL of sucrose solution was added and mixed. The resulting solution was filtered through a 0.2 μm polycarbonate membrane filter, placed in a glass vial with a screw cap, and stored at 4°C. This solution was used as a liposome sample containing an NKT cell ligand (RK-163 (or RCAI-61, RCAI-137)) and a cancer antigen (OVA) (for main pharmacological testing). This sample was used as a liposome composition containing an NKT cell ligand and a cancer antigen in the following Test Examples 6 to 10.

[0172] Test Example 6 Antitumor activity (dose) and ED50 of a liposome composition containing an NKT cell ligand (RK-163) and a pseudo-cancer antigen (OVA) (hereinafter referred to as RK-LIPO-OVA) RK-LIPO-OVA (administered at 0.1 ng-10 ng / 25 g body weight in terms of RK-163 amount) was intravenously administered to unsensitized normal C57BL / 6 mice (10 weeks old), and then 3x10 B16 mouse melanoma cells (MO4) expressing the pseudo-cancer antigen OVA were cultured. 5 3x10 cells were injected subcutaneously into RK-LIPO-OVA-treated mice. 5 The tumor diameter was observed on days 7, 11, 14, and 17 after transplantation. All measurements were performed using a digital caliper. Tumor volume (mm 3 ) = major axis (mm) x minor axis (mm) x height (mm). A control group received the same amount of PBS instead of the NKT cell ligand-containing liposome composition. The results are shown in Figure 6. RK-LIPO-OVA demonstrated a strong antitumor effect, inhibiting melanoma tumor cell growth in proportion to the amount of RK-163. The effective dose, calculated as RK-163, was 1 ng / 25 g to 10 ng / 25 g of mouse body weight, with an ED50 of 0.7797. RK-LIPO-OVA was intravenously administered to normal C57BL / 6 mice, followed by subcutaneous transplantation of B16 mouse melanoma cells (MO4) expressing the cancer-mimicking antigen OVA into the RK-LIPO-OVA-treated mice. RK-LIPO-OVA demonstrated a strong antitumor effect, inhibiting MO4 tumor cell growth in proportion to the amount of NKT cell ligand.

[0173] Test Example 7: NKT Cell Activation and Proliferation by a Liposome Composition Containing an NKT Cell Ligand (RK-163) and a Cancer Mimetic Antigen (OVA) (hereafter referred to as RK-LIPO-OVA). An effective dose of RK-LIPO-OVA (10 ng RK163 equivalent / mouse) was administered via the tail vein of normal C57BL / 6 mice (10 weeks old, 5 mice per group). RK-LIPO-OVA was taken up by antigen-presenting cells (e.g., Kupffer cells) in the liver, and NKT cells in the liver were artificially activated by the RK-163 ligand presented on CD1d molecules. As a result, NKT cells expressed the activation marker KLRG1 on their surface and produced IFN-γ, an adjuvant that activates other immune cell populations. A control group received the same amount of PBS instead of the NKT cell ligand-containing liposome composition. The results are shown in Figure 7 (Figure 7 shows data from 3 days after RK-LIPO-OVA administration). NKT cells were activated by RK-LIPO-OVA, increasing their numbers and inducing the expression of the activation marker KLRG1 and the production of IFNγ. It was confirmed that administration of RK-LIPO-OVA into the tail vein of normal C57BL / 6 mice artificially activated and proliferated NKT cells in the liver.

[0174] Test Example 8: Activation of innate and adaptive immune cell populations by artificially activated NKT cells after administration of liposomes containing an NKT cell ligand (RK-163) and a pseudocancer antigen (OVA) (hereinafter referred to as RK-LIPO-OVA). An effective dose of RK-LIPO-OVA (10 ng RK163 equivalent / mouse) was administered via the tail vein of C57BL / 6 mice (10 weeks old) (5 mice per group) to artificially activate NKT cells in the liver. The adjuvant effect of IFNγ produced by the activated NKT cells on innate and adaptive immune cell populations was analyzed. A control group received the same amount of PBS instead of the NKT cell ligand-containing liposome composition. The results are shown in Figure 8. As a result, by inducing artificially activated NKT cells through administration of RK-LIPO-OVA, the antigen (OVA) contained in RK-LIPO-OVA induced the proliferation of antigen (OVA)-specific CD4T and CD8T cells of the adaptive immune system in normal mice, and the artificially activated NKT cells also activated innate immune system NK cells, confirming an increase in cell numbers.By administering RK-LIPO-OVA intravenously to C57BL / 6 mice, NKT cells in the liver were artificially activated, and as a result, it was confirmed that, in normal mice, the proliferation of antigen (OVA)-specific CD4T and CD8T cells of the adaptive immune system was induced, and innate immune system NK cells were also proliferated.

[0175] Test Example 9: Establishment of Long-Term Immune Memory by Liposomes Containing an NKT Cell Ligand (RK-163) and a Cancer Antigen (OVA) (hereafter referred to as RK-LIPO-OVA). C57BL / 6 mice (10 weeks old, 5 mice per group) were intravenously administered an effective dose of RK-LIPO-OVA (10 ng / mouse) and sacrificed 60 days after RK-LIPO-OVA administration, and spleen cells were analyzed. The results are shown in Figure 9. As a result, proliferation of OVA-specific CD8 T cells and formation of long-term immune memory CD8 T cells (OVA-specific memory CD8 T cells) were confirmed 60 days after RK-LIPO-OVA administration, confirming the formation and maintenance of long-term cancer immune memory. In C57BL / 6 mice intravenously administered with RK-LIPO-OVA, formation of pseudoantigen OVA-specific memory CD8 T cells was confirmed 60 days after RK-LIPO-OVA administration, confirming the formation and maintenance of long-term cancer immune memory.

[0176] Test Example 10: Antitumor effect 60 days after long-term immune memory establishment using liposomes containing an NKT cell ligand (RK-163) and a cancer antigen (OVA) (hereinafter referred to as RK-LIPO-OVA) Normal mice were immunized with an effective dose (10 ng / mouse) of RK-LIPO-OVA, and then 60 days were allowed to pass without treatment. These C57BL / 6 mice, which had undergone long-term memory induction specific to the cancer antigen OVA, were subcutaneously injected with 3x10 B16 mouse melanoma cells (MO4) expressing the cancer antigen OVA. 5 The tumor diameter was observed on days 7, 11, 14, and 17. All measurements were performed using a digital caliper. Tumor volume (mm 3) = major axis (mm) x minor axis (mm) x height (mm). A control group received the same amount of PBS instead of the NKT cell ligand-containing liposome composition. The results are shown in Figure 10. As a result, in RK-LIPO-OVA-treated mice 60 days after RK-LIPO-OVA administration, the growth of B16 mouse melanoma cells (MO4) expressing the pseudo-cancer antigen OVA, which were transplanted on day 60, was significantly suppressed due to the induction of immunological memory against the pseudo-cancer antigen OVA. Furthermore, liposomes RCAI-61-OVA and RCAI-137-OVA, which contain NKT cell ligands (RCAI-61 and RCAI-137) and cancer antigen (OVA), also significantly suppressed the growth of B16 mouse melanoma cells expressing the pseudo-cancer antigen OVA (MO4). This confirmed the usefulness of the antitumor effect of RK-LIPO-OVA in normal mice. RK-LIPO-OVA was administered to normal mice, and 60 days later, B16 mouse melanoma cells (MO4) expressing the pseudo-cancer antigen OVA were transplanted. As a result, the growth of the pseudo-cancer antigen OVA-expressing B16 mouse melanoma cells transplanted into RK-LIPO-OVA-administered mice 60 days after RK-LIPO-OVA administration was significantly suppressed, confirming the usefulness of the anti-tumor effect due to the establishment of pseudo-cancer antigen OVA-specific immune memory in normal mice.

[0177] According to the present invention (Invention-1), a preparation obtained by combining an immune checkpoint inhibitor with an NKT cell ligand-containing liposome composition can be useful as a preparation for treating any type of cancer, including cancers that have low expression of PD-L1, a PD-1 ligand molecule, and are difficult to treat alone, and that have been resistant to immune checkpoint inhibitors, and cancers that are resistant to NKT cell ligand-containing liposome compositions.

[0178] According to the present invention (Invention-2), a liposome composition containing an NKT cell ligand and an antigen (e.g., a cancer antigen, a pathogen antigen) activates a specific immune cell population by an NKT cell ligand-containing substance having the function of artificially activating NKT cells and an antigen (e.g., a cancer antigen, a pathogen antigen), and induces long-term immune memory. The resulting preparation can be useful as a preparation for preventing diseases such as cancer and infectious diseases.

[0179] This application is based on patent application No. 2023-132368 filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. An antitumor agent comprising a combination of an immune checkpoint inhibitor and a liposome composition containing an NKT cell ligand.

2. The antitumor agent according to claim 1, wherein the NKT cell ligand is a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and a salt thereof. Formula (I): [In the formula, X represents an alkylene group or -NH-; １ and R ２ are the same or different and each represents a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group, or an aryl group which may have a substituent; R １ and R ２ may form a 5- or 6-membered ring together with the adjacent nitrogen atom; R ３ represents a hydrocarbon group having 1 to 20 carbon atoms; R ４ represents a hydrocarbon group having 1 to 30 carbon atoms.] Formula (II): [In the formula, R ５ represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom; R ６ and R ７ each independently represents a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms; Y is -CH ２ -, -CH(OH)- or -CH=CH-.] 3. The antitumor agent according to claim 2, wherein the glycolipid is a compound represented by formula (I) or a salt thereof.

4. The glycolipid has the following formula: The antitumor agent according to claim 3, which is a compound represented by the following formula (I):

5. An agent for enhancing the expression of immune checkpoint molecules by activating cancer immune cell populations, comprising a liposome composition containing an NKT cell ligand.

6. An agent for enhancing the expression of immune checkpoint ligand molecules on cancer cells by artificial activation of NKT cells, comprising an NKT cell ligand-containing liposome composition.

7. A liposome composition containing an NKT cell ligand for enhancing the therapeutic effect of an immune checkpoint inhibitor.

8. An immune checkpoint inhibitor for enhancing the therapeutic effect of an NKT ligand-containing liposome composition.

9. A method for treating a malignant tumor, comprising administering to a subject an immune checkpoint inhibitor and an NKT cell ligand-containing liposome composition.

10. The method of claim 9, wherein the NKT cell ligand is a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and a salt thereof. Formula (I): [In the formula, X represents an alkylene group or -NH-; １ and R ２ are the same or different and each represents a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group, or an aryl group which may have a substituent; R １ and R ２ may form a 5- or 6-membered ring together with the adjacent nitrogen atom; R ３ represents a hydrocarbon group having 1 to 20 carbon atoms; R ４ represents a hydrocarbon group having 1 to 30 carbon atoms.] Formula (II): [In the formula, R ５ represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom; R ６ and R ７ each independently represents a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms; Y is -CH ２ -, -CH(OH)- or -CH=CH-.] 11. The method of treatment according to claim 10, wherein the glycolipid is a compound represented by formula (I) or a salt thereof.

12. The glycolipid has the following formula: The method for treatment according to claim 11, wherein the compound is represented by the following formula (I):

13. A method for enhancing expression of immune checkpoint molecules by activating cancer immune cell populations in a subject, comprising administering to the subject an NKT cell ligand-containing liposome composition.

14. A method for enhancing expression of immune checkpoint ligand molecules on cancer cells in a subject by artificially activating NKT cells, comprising administering to the subject a liposome composition containing an NKT cell ligand.

15. A pharmaceutical comprising a combination of an immune checkpoint inhibitor and a liposome composition containing an NKT cell ligand for use in treating a malignant tumor.

16. The pharmaceutical composition for use according to claim 15, wherein the NKT cell ligand is a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof. Formula (I): [In the formula, X represents an alkylene group or -NH-; １ and R ２ are the same or different and each represents a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group, or an aryl group which may have a substituent; R １ and R ２ may form a 5- or 6-membered ring together with the adjacent nitrogen atom; R ３ represents a hydrocarbon group having 1 to 20 carbon atoms; R ４ represents a hydrocarbon group having 1 to 30 carbon atoms.] Formula (II): [In the formula, R ５ represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom; R ６ and R ７ each independently represents a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms; Y is -CH ２ -, -CH(OH)- or -CH=CH-.] 17. The method for use according to claim 16, wherein the glycolipid is a compound represented by formula (I) or a salt thereof.

18. The glycolipid has the following formula: The pharmaceutical composition for use according to claim 17, which is a compound represented by the formula:

19. A liposome composition containing an NKT cell ligand for use in enhancing expression of immune checkpoint molecules by activating cancer immune cell populations.

20. A liposome composition containing an NKT cell ligand for use in enhancing the expression of immune checkpoint ligand molecules on cancer cells by artificial activation of NKT cells.

21. A liposome composition containing an NKT cell ligand for use in improving the therapeutic effect of an immune checkpoint inhibitor.

22. An immune checkpoint inhibitor for use in improving the therapeutic effect of an NKT ligand-containing liposome composition.

23. A liposome composition containing an immune checkpoint inhibitor and an NKT cell ligand for use as a pharmaceutical.

24. Use of a combination of an immune checkpoint inhibitor and a liposome composition containing an NKT cell ligand in the manufacture of an anti-tumor agent.

25. The use according to claim 24, wherein the NKT cell ligand is a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof. Formula (I): [In the formula, X represents an alkylene group or -NH-; １ and R ２ are the same or different and each represents a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group, or an aryl group which may have a substituent; R １ and R ２ may form a 5- or 6-membered ring together with the adjacent nitrogen atom; R ３ represents a hydrocarbon group having 1 to 20 carbon atoms; R ４ represents a hydrocarbon group having 1 to 30 carbon atoms.] Formula (II): [In the formula, R ５ represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom; R ６ and R ７ each independently represents a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms; Y is -CH ２ -, -CH(OH)- or -CH=CH-.] 26. The use according to claim 25, wherein the glycolipid is a compound represented by formula (I) or a salt thereof.

27. The glycolipid has the following formula: The use according to claim 26, wherein the compound is represented by the formula:

28. Use of a liposome composition containing an NKT cell ligand in the manufacture of an agent for enhancing the expression of immune checkpoint molecules by activating cancer immune cell populations.

29. Use of an NKT cell ligand-containing liposome composition in the manufacture of an agent for enhancing the expression of immune checkpoint ligand molecules on cancer cells by artificial activation of NKT cells.

30. Use of a liposome composition containing an NKT cell ligand in the manufacture of an agent for improving the therapeutic effect of an immune checkpoint inhibitor.

31. Use of an immune checkpoint inhibitor in the manufacture of an agent for improving the therapeutic effect of an NKT ligand-containing liposome composition.

32. A pharmaceutical composition comprising a combination of an immune checkpoint inhibitor and an NKT cell ligand-containing liposome composition.

33. A liposome composition containing an NKT cell ligand and an antigen.

34. The composition of claim 33, wherein the NKT cell ligand is a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof. Formula (I): [In the formula, X represents an alkylene group or -NH-; １ and R ２ are the same or different and each represents a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group, or an aryl group which may have a substituent; R １ and R ２ may form a 5- or 6-membered ring together with the adjacent nitrogen atom; R ３ represents a hydrocarbon group having 1 to 20 carbon atoms; R ４ represents a hydrocarbon group having 1 to 30 carbon atoms.] Formula (II): [In the formula, R ５ represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom; R ６ and R ７ each independently represents a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms; Y is -CH ２ -, -CH(OH)- or -CH=CH-.] 35. The composition according to claim 34, wherein the glycolipid is a compound represented by formula (I) or a salt thereof.

36. The glycolipid has the following formula: The composition according to claim 35, wherein the compound is represented by the formula:

37. A composition according to any one of claims 33 to 36 for inducing immunological memory.

38. The composition of any one of claims 33 to 36, wherein the antigen is a cancer antigen.

39. The composition according to claim 38, for prophylactically inducing activation and proliferation of immune cell populations that recognize cancer antigens even in the absence of cancer.

40. The composition of claim 38 for prophylactically inducing immune memory against cancer even in the absence of cancer.

41. The composition according to claim 38 for enhancing the inhibitory effect on the onset, recurrence, and metastasis of cancer.

42. A method for inducing immunological memory, comprising administering to a subject a composition according to any one of claims 33 to 36.

43. A method for prophylactically inducing activation and proliferation of immune cell populations that recognize cancer antigens, even in the absence of cancer in a subject, comprising administering to the subject the composition described in claim 38.

44. A method for prophylactically inducing immune memory against cancer in a subject, even in the absence of cancer, comprising administering to the subject the composition of claim 38.

45. A method for suppressing the onset, recurrence, and metastasis of cancer, comprising administering to a subject the composition according to claim 38.

46. ​​A composition according to any one of claims 33 to 36 for use in inducing immunological memory.

47. The composition of claim 38 for use in prophylactically activating and inducing proliferation of immune cell populations that recognize cancer antigens even in the absence of cancer.

48. The composition of claim 38 for use in prophylactically inducing immune memory against cancer in the absence of cancer.

49. The composition according to claim 38 for use in improving the inhibitory effect on the onset, recurrence, and metastasis of cancer.

50. A composition according to any one of claims 33 to 36 for use as a medicament.

51. Use of a composition according to any one of claims 33 to 36 for the manufacture of an agent for inducing immunological memory.

52. Use of the composition according to claim 38 for the manufacture of an agent for prophylactically inducing activation and proliferation of immune cell populations that recognize cancer antigens even in the absence of cancer.

53. Use of the composition according to claim 38 for the manufacture of an agent for prophylactically inducing immune memory against cancer, even in the absence of cancer.

54. Use of the composition according to claim 38 for the manufacture of an agent for suppressing the onset, recurrence, and metastasis of cancer.

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