Antitumor therapy by combined use of chymase inhibitor and immunotherapy

Abstract

The present invention relates to: a pharmaceutical composition which is for treating a tumor, and which includes a chymase inhibitor as an active ingredient, said pharmaceutical composition being characterized by being used in combination with at least one selected from interleukin-18 (IL-18), an anti-PD-1 / PD-L1 antibody, and interleukin-12 (IL-12); a pharmaceutical composition which is for treating a tumor, and which includes, as an active ingredient, at least one selected from interleukin-18 (IL-18), an anti-PD-1 / PD-L1 antibody, and interleukin-12 (IL-12), said pharmaceutical composition being characterized by being used in combination with a chymase inhibitor; and the like.

Description

Antitumor therapy using a combination of chymase inhibitors and immunotherapy

[0001] RELATED APPLICATIONS This application claims priority to Japanese Patent Application No. 2023-127528 (filed August 4, 2023), the contents of which are incorporated herein by reference. TECHNICAL FIELD The present invention relates to a novel antitumor therapy characterized by combining a chymase inhibitor with immunotherapy using interleukin-18 (IL-18), an anti-PD-1 / PD-L1 antibody, interleukin-12 (IL-12), or the like.

[0002] Chymase is a neutral serine protease present mainly in mast cells, and human chymase is known to be involved in hypertension and arteriosclerosis by producing angiotensin II from angiotensin I as a substrate. Although there have been reports showing that chymase correlates with angiogenesis around tumor tissues in lung cancer, colon cancer, and pancreatic cancer (Non-Patent Documents 1 to 3), there have been no reports showing the direct effect of chymase on tumors.

[0003] Interleukin-18 (IL-18) is a cytokine discovered by Okamura et al. as an interferon-γ (IFNγ) production inducer. IL-18 binds to the IL-18 receptor expressed on the cell surface of cells in which IFNγ production is induced in the presence of IL-12, enhancing IFNγ production and enhancing Th1-type immune responses. On the other hand, in the absence of IL-12, IL-18 enhances Th2-type immune responses.

[0004] IL-18 is expected to have antitumor effects, and cancer treatments using IL-18 in combination with rituximab or Herceptin (Patent Document 1), or IL-18 in combination with a molecular target antibody have been reported (Patent Document 2).Recently, it has been reported that a strong antitumor effect was achieved by using IL-18-secreting CAR-T cells to secrete IL-18 in a tumor-specific manner (Non-Patent Document 4).

[0005] IL-18 is produced as an IL-18 precursor containing a propeptide, which is cleaved by caspases and released extracellularly as active IL-18. A method for producing IL-18 has been reported in which the IL-18 precursor is cleaved with chymase instead of caspase (Patent Document 3). This report does not demonstrate the effect of chymase inhibitors on the active (mature) form of IL-18, but suggests that chymase inhibitors may inhibit the conversion of the IL-18 precursor to the active form, thereby inhibiting IL-18 activity.

[0006] PD-1 and PD-L1 are representative immune checkpoint molecules, and antibodies targeting these molecules have been shown to exhibit significant anti-cancer effects in various cancer types and are currently being developed as medicines. However, the remission rate for anti-PD-1 / PD-L1 antibody therapy in solid tumors is low, and its response rate cannot be said to be sufficient.

[0007] WO2006 / 116423WO2016 / 021720JP2007-151414

[0008] Ibaraki et al., The relationship of tryptase- and chymase-positive mast cells to angiogenesis in stage I non-small cell lung cancer. Eur J Cardiothorac Surg. 2005;28(4):617-621.Kondo et al., Expression of chymase-positive cells in gastric cancer and its correlation with the angiogenesis. J Surg Oncol. 2006;93(1):36-43.Laface et al., Chymase-positive mast cells correlate with tumor angiogenesis: first report in pancreatic cancer patients. Eur Rev Med Pharmacol Sci. 2021;25(22):6862-6873.Svoboda et al., Interleukin-18 Secreting Autologous Anti-CD19 CAR T-Cells (huCART19-IL18) in Patients with Non-Hodgkin Lymphomas Relapsed or Refractory to Prior CAR T-Cell Therapy. Blood (2022) 140 (Supplement 1): 4612-4614

[0009] An object of the present invention is to provide a novel method for treating tumors.

[0010] The inventors have found that the anti-tumor effect of immunotherapy using IL-18 or an anti-PD-1 / PD-L1 antibody (anti-PD-1 antibody or anti-PD-L1 antibody) can be enhanced by combining the immunotherapy with a chymase inhibitor, which does not have an anti-tumor effect alone. Based on this finding, the present application provides the following items [1] to [8] as a first aspect: [1] A pharmaceutical composition for treating tumors, comprising a chymase inhibitor as an active ingredient, wherein the pharmaceutical composition is used in combination with one or more selected from interleukin-18 (IL-18), an anti-PD-1 / PD-L1 antibody, and interleukin-12 (IL-12). [2] A pharmaceutical composition for treating tumors, comprising a chymase inhibitor in combination with one or more selected from interleukin-18 (IL-18), a PD-1 / PD-L1 antibody, and interleukin-12 (IL-12). [3] A pharmaceutical composition for treating a tumor, comprising as an active ingredient one or more selected from interleukin-18 (IL-18), an anti-PD-1 / PD-L1 antibody, and interleukin-12 (IL-12), wherein the pharmaceutical composition is used in combination with a chymase inhibitor.[4] Chymase inhibitors include Suc-Val-Pro-PheP(OPh)2, Chymostatin, Fulacimstat, 2-[4-[[(5-Fluoro-3-methylbenzo[b]thien-2-yl)sulfonyl]amino]-3-(methylsulfonyl)phenyl]-4-thiazolecarboxylic acid, N-[1-Benzyl-2,3-dioxo-6-(2-pyridyloxy)hexyl]-2-[2-phenyl-5-(formylamino)-6-oxo-1,6-dihydropyrimidine-1-yl]acetamide, and (2-[5-amino-2-(4-fluorop henyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide), SUN-C8257, SUN13834, BCEAB, Compound 17, TEI-E548, RO5066852, JNJ-10311795 (RWJ-355871), Y-40079, ASB17061, ONO-8020435, and ONO-2220445. [5] The pharmaceutical composition of any of [1] to [4], wherein any one or more selected from IL-18, anti-PD-1 / PD-L1 antibody, and IL-12 are of human origin. [6] The pharmaceutical composition of any of [1] to [5], wherein any one or more selected from IL-18, anti-PD-1 / PD-L1 antibody, and IL-12 and a chymase inhibitor are administered simultaneously or sequentially. [7] A pharmaceutical composition for enhancing the effect of any one or more selected from interleukin-18 (IL-18), anti-PD-1 / PD-L1 antibody, and IL-12 in tumor treatment, comprising a chymase inhibitor as an active ingredient.[8] Chymase inhibitors include Suc-Val-Pro-PheP(OPh)2, Chymostatin, Fulacimstat, 2-[4-[[(5-Fluoro-3-methylbenzo[b]thien-2-yl)sulfonyl]amino]-3-(methylsulfonyl)phenyl]-4-thiazolecarboxylic acid, N-[1-Benzyl-2,3-dioxo-6-(2-pyridyloxy)hexyl]-2-[2-phenyl-5-(formylamino)-6-oxo-1,6-dihydropyrimidine-1-yl]acetamide, (2-[5-am ino-2-(4-fluorophenyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide) The pharmaceutical composition according to [7], which is any one selected from SUN-C8257, SUN13834, BCEAB, Compound 17, TEI-E548, RO5066852, JNJ-10311795 (RWJ-355871), ASB17061, ONO-8020435, and ONO-2220445.

[0011] In a second aspect, the present application provides the following [1] to [6]: [1] A method for treating a tumor, comprising administering to a subject a combination of a chymase inhibitor and one or more selected from interleukin-18 (IL-18), an anti-PD-1 / PD-L1 antibody, and interleukin-12 (IL-12). [2] Chymase inhibitors include Suc-Val-Pro-PheP(OPh)2, Chymostatin, Fulacimstat, 2-[4-[[(5-Fluoro-3-methylbenzo[b]thien-2-yl)sulfonyl]amino]-3-(methylsulfonyl)phenyl]-4-thiazolecarboxylic acid, N-[1-Benzyl-2,3-dioxo-6-(2-pyridyloxy)hexyl]-2-[2-phenyl-5-(formylamino)-6-oxo-1,6-dihydropyrimidine-1-yl]acetamide, (2-[5-am ino-2-(4-fluorophenyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide) [1] The method according to [1], wherein the chymase inhibitor is any one selected from SUN-C8257, SUN13834, BCEAB, Compound 17, TEI-E548, RO5066852, JNJ-10311795 (RWJ-355871), ASB17061, ONO-8020435, and ONO-2220445. [3] The method according to [1], wherein the chymase inhibitor is administered simultaneously with any one or more selected from IL-18, an anti-PD-1 / PD-L1 antibody, and IL-12. [4] The method according to [1], wherein the chymase inhibitor is administered sequentially with any one or more selected from IL-18, an anti-PD-1 / PD-L1 antibody, and IL-12.[5] A method for enhancing the antitumor effect of any one or more selected from interleukin-18 (IL-18), an anti-PD-1 / PD-L1 antibody, and interleukin-12 (IL-12), the method comprising administering a chymase inhibitor to a subject to be administered with or to a subject that has been administered with IL-18, an anti-PD-1 / PD-L1 antibody, and IL-12. [6] The chymase inhibitor is selected from the group consisting of Suc-Val-Pro-PheP(OPh)2, Chymostatin, Fulacimstat, and 2-[4-[[(5-Fluoro-3-methylbenzo[b]thien-2-yl)sulfonyl]amino]-3-(methylsulfonyl)phenyl]-4-thiazolecarboxylic acid. acid, N-[1-Benzyl-2,3-dioxo-6-(2-pyridyloxy)hexyl]-2-[2-phenyl-5-(formylamino)-6-oxo-1,6-dihydropyrimidine-1-yl]acetamide, (2-[5-amino-2-(4-fluoroph enyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide), SUN-C8257, SUN13834, BCEAB, Compound 17, TEI-E548, RO5066852, JNJ-10311795 (RWJ-355871), ASB17061, ONO-8020435, and ONO-2220445.

[0012] In a third aspect, the present application provides the following [1] to [7]. [1] An agent for use in tumor treatment, comprising a chymase inhibitor, characterized in that the agent is used in combination with one or more selected from interleukin-18 (IL-18), an anti-PD-1 / PD-L1 antibody, and interleukin-12 (IL-12). [2] An agent for use in tumor treatment, comprising a chymase inhibitor in combination with one or more selected from interleukin-18 (IL-18), a PD-1 / PD-L1 antibody, and interleukin-12 (IL-12). [3] An agent for use in tumor treatment, comprising one or more selected from interleukin-18 (IL-18), an anti-PD-1 / PD-L1 antibody, and interleukin-12 (IL-12), characterized in that the agent is used in combination with a chymase inhibitor. [4] Chymase inhibitors include Suc-Val-Pro-PheP(OPh)2, Chymostatin, Fulacimstat, 2-[4-[[(5-Fluoro-3-methylbenzo[b]thien-2-yl)sulfonyl]amino]-3-(methylsulfonyl)phenyl]-4-thiazolecarboxylic acid, N-[1-Benzyl-2,3-dioxo-6-(2-pyridyloxy)hexyl]-2-[2-phenyl-5-(formylamino)-6-oxo-1,6-dihydropyrimidine-1-yl]acetamide, and (2-[5-amino-2-(4-fluorop henyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide), SUN-C8257, SUN13834, BCEAB, Compound The drug for use according to any one of [1] to [3], which is selected from the group consisting of 17, TEI-E548, RO5066852, JNJ-10311795 (RWJ-355871), Y-40079, ASB17061, ONO-8020435, and ONO-2220445.[5] The composition for use according to any one of [1] to [4], wherein IL-18, anti-PD-1 / PD-L1 antibody, and IL-12 are of human origin. [6] The drug for use according to any one of [1] to [5], wherein one or more selected from IL-18, anti-PD-1 / PD-L1 antibody, and IL-12 are administered simultaneously or sequentially with a chymase inhibitor. [7] The drug for use according to any one of [1] to [6], wherein the chymase inhibitor enhances the anti-tumor effect of one or more selected from IL-18, anti-PD-1 / PD-L1 antibody, and IL-12.

[0013] In a fourth aspect, the present application provides the following [1] to [7]. [1] Use of a chymase inhibitor, together with any one or more selected from interleukin-18 (IL-18), an anti-PD-1 / PD-L1 antibody, and interleukin-12 (IL-12), in the manufacture of a medicament for treating a tumor. [2] Use of a chymase inhibitor, in the manufacture of a medicament for treating a tumor, wherein the medicament is used in combination with any one or more selected from interleukin-18 (IL-18), an anti-PD-1 / PD-L1 antibody, and interleukin-12 (IL-12). [3] Use of an chymase inhibitor, together with any one or more selected from interleukin-18 (IL-18), an anti-PD-1 / PD-L1 antibody, and interleukin-12 (IL-12), in the manufacture of a medicament for treating a tumor, wherein the medicament is used in combination with a chymase inhibitor. [4] Chymase inhibitors include Suc-Val-Pro-PheP(OPh)2, Chymostatin, Fulacimstat, 2-[4-[[(5-Fluoro-3-methylbenzo[b]thien-2-yl)sulfonyl]amino]-3-(methylsulfonyl)phenyl]-4-thiazolecarboxylic acid, N-[1-Benzyl-2,3-dioxo-6-(2-pyridyloxy)hexyl]-2-[2-phenyl-5-(formylamino)-6-oxo-1,6-dihydropyrimidine-1-yl]acetamide, and (2-[5-amino-2-(4-fluorop henyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide), SUN-C8257, SUN13834, BCEAB, Compound The use according to any one of [1] to [3], wherein the compound is selected from the group consisting of 17, TEI-E548, RO5066852, JNJ-10311795 (RWJ-355871), Y-40079, ASB17061, ONO-8020435, and ONO-2220445.[5] The use according to any one of [1] to [4], wherein IL-18, anti-PD-1 / PD-L1 antibody, and IL-12 are of human origin. [6] The use according to any one of [1] to [5], wherein one or more selected from IL-18, anti-PD-1 / PD-L1 antibody, and IL-12 are administered simultaneously or sequentially with a chymase inhibitor. [7] The use according to any one of [1] to [6], wherein the chymase inhibitor enhances the anti-tumor effect of one or more selected from IL-18, anti-PD-1 / PD-L1 antibody, and IL-12.

[0014] The present invention provides novel clinical uses for chymase inhibitors and immunotherapeutic agents such as IL-18, anti-PD-1 / PD-L1 antibodies, and L-12.

[0015] Figure 1 shows the survival curves for the placebo group, IL-18 administration group, chymase inhibitor administration group, and IL-18 / chymase inhibitor combination group at 100 days after tumor (CT26) cell transplantation. Figure 2 shows photographs of tumor-inoculated areas of mice that survived at 100 days after tumor (CT26) cell transplantation (top: IL-18, bottom: IL-18 and chymase inhibitor combination). Figure 3 shows the survival curves for the placebo group, IL-18 administration group, chymase inhibitor administration group, and IL-18 / chymase inhibitor combination group at 110 days after tumor (CT26) cell transplantation. Figure 4 shows the percentage (A), absolute number (B), cell size (C), and size of intracellular granules (D) of mast cells in ascites after tumor (CT26) cell transplantation. Figure 5 shows the CD4 counts after tumor (CT26) cell transplantation. + T cell count (A), CD8 + T cell count (B), CD4 + T cells / CD8 + Figure 6 shows the percentage of NK cells in the ascites after tumor (CT26) cell transplantation (A), the absolute number of NK cells (B), and the percentage of T cells (C). + B220 + The percentage of NK cells (C) is shown. Figure 7 shows the percentage of immune cells (mast cells, CD8 + T cells, NK cells, DX5 + B220 +Figure 8 shows the transition of NK cell counts. Figure 8 shows the results of comparing T cells in ascites in the placebo group, IL-18 administration group, chymase inhibitor administration group, and IL-18 / chymase inhibitor combination group. + T cells / CD8 + T cell percentage (A), CD4 + T cell count (B), CD8 + T cell count (C), CD44high CD8 + T cell count (D) Figure 9 shows the results of comparing the number of NK cells in ascites in the placebo group, IL-18 administration group, chymase inhibitor administration group, and IL-18 / chymase inhibitor combination group. + NK cell count (A), DX5 + B220 + NK cell count (B) Figure 10 shows the survival curves for the placebo group, anti-PD-1 antibody administration group, chymase inhibitor administration group, and anti-PD-1 antibody / chymase inhibitor combination group 34 days after tumor (CT26) cell transplantation.

[0016] 1. Chymase Inhibitors As used herein, a chymase inhibitor refers to a drug capable of inhibiting the activity of chymase. Chymase is a neutral serine protease, and human chymase is known to be involved in hypertension and arteriosclerosis by producing angiotensin II from angiotensin I. The chymase inhibitor of the present invention is preferably a human chymase inhibitor.

[0017] Known chymase inhibitors can be used as the chymase inhibitor. Examples of known chymase inhibitors include compounds that have already been reported to be clinically applicable or verified in preclinical models: SUN-C8257, SUN13834, BCEAB, Compound 17, NK3201, TEI-E548, RO5066852, JNJ-10311795 (RWJ-355871), Suc-Val-Pro-Phe PExamples of such anti-inflammatory drugs include (OPh)2, Y-40613, Y-40079, TY-51469, chymostatin, fulacimstat (BAY1142524), ASB17061, ONO-8020435, and ONO-2220445 (see, for example, Heuston and Hyland, Br J Pharmacol. 2012 Oct; 167(4): 732-740. doi: 10.1111 / j.1476-5381.2012.02055.x).

[0018] Alternatively, WO2016 / 132483, JP 2016-150915, JP 2016-535095, JP 2012-516335, JP 2012-502034, JP 2012-121898, JP 2010-536768, WO2010 / 147133, JP 2010-515699, JP 2010-505900, WO2 Chymase inhibitors disclosed in WO 005 / 018672, WO 03 / 018061, JP 2004-507539, WO 01 / 062294, WO 01 / 062293, WO 01 / 062292, WO 00 / 005204, WO 99 / 032459, JP 11-001479, and JP 10-087493 can also be used.

[0019]

[0020] 2. Interleukin-18 (IL-18) Interleukin-18 (IL-18) is a cytokine discovered as an IFN-γ production inducer. IL-18, along with IL-12, is a cytokine that stimulates CD4 ＋ CD8 ＋ It acts on T cells and NK cells to induce the production of IFN-γ.

[0021] IL-18 is produced as an inactive precursor containing a propeptide (pro-IL-18), and is released as active IL-18 upon cleavage of the propeptide by caspase 1, caspase 4, or the like. In the present invention, the source of IL-18 is not particularly limited, but IL-18 derived from the same species as the subject to be administered or a closely related species is preferred. Therefore, when administered to humans, mammalian IL-18, particularly human IL-18, is preferred.

[0022] Human IL-18 precursor consists of 193 amino acids (24 kDa), and mature (active) human IL-18, which does not contain the propeptide, consists of 157 amino acids (18 kDa). The amino acid sequence of human IL-18 is known, and an example of the amino acid sequence of human IL-18 is the amino acid sequence set forth in SEQ ID NO: 1. Human IL-18 having an amino acid sequence that shares 80%, 85%, 90%, 95%, 96%, 98%, or 99% or more sequence identity with this amino acid sequence can also be used as the IL-18 of the present invention, as long as it has the activity of IL-18. IL-18 activity refers to the physiological activity of IL-18, such as the activity of inducing IFNγ production.

[0023] Examples of human IL-18 include the following proteins (1) and (2): (1) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, or (2) a protein having 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and having IL-18 activity (such as IFNγ production-inducing activity).

[0024] IL-18 exerts its physiological activity by binding to receptors on the cell membrane. However, because IL-18-specific IL-18-binding proteins exist in excess in vivo, much of the secreted IL-18 binds to the IL-18-binding proteins and loses its activity. Recently, it has been reported that the antitumor effect of IL-18 is enhanced by using modified IL-18 with increased stability or by secreting IL-18 from Car-T cells. These modified IL-18 and new delivery systems are also within the scope of the present invention, provided they do not conflict with the objectives of the present invention.

[0025] 3. Anti-PD-1 / PD-L1 Antibody (Anti-PD-1 Antibody, Anti-PD-L1 Antibody) In the present invention, the term "anti-PD-1 / PD-L1 antibody" refers to an antibody that specifically inhibits PD-1 or PD-L1, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

[0026] The "anti-PD-1 antibody" used in the present invention is not particularly limited as long as it can bind to PD-1 and inhibit its function as an immune checkpoint. Anti-PD-1 antibodies include those already approved and sold as pharmaceuticals and those under development, and these can be suitably used. Examples of such "anti-PD-1 antibodies" include, but are not limited to, nivolumab (Opdivo (GSK / Ono Pharmaceutical)), the humanized IgG4 antibody pembrolizumab (Merck), and dipirizumab.

[0027] The "anti-PD-L1 antibody" used in the present invention is not particularly limited as long as it can bind to PD-L1 and inhibit its function as an immune checkpoint. PD-L1 antibodies include those already approved (sold) as pharmaceuticals and those under development, and these can be suitably used. Examples of such "anti-PD-L1 antibodies" include, but are not limited to, atezolizumab (Roche / Chugai), durvalumab (AstraZeneca), avelumab (Merck), and MED10680 / AMP-514.

[0028] The antibody may be produced according to conventional methods. In this case, the antibody is preferably a chimeric antibody, a humanized antibody, or a fully human antibody in order to reduce heterologous antigenicity to humans. The antibody may also be an antibody fragment, so long as it functions as an immune checkpoint inhibitor. Examples of antibody fragments include F(Ab')2, Fab', Fab, Fv, and scFv.

[0029] 4. Interleukin-12 (IL-12) Interleukin-12 (IL-12) is a cytokine produced mainly by macrophages, polymorphonuclear leukocytes, and dendritic cells, and is known to induce the production of IFNγ in NK cells and T cells and to enhance the activity of NK cells and cytotoxic T cells.

[0030] It has been reported that IL-12 preferentially activates Th1 cells and activates cellular immunity by suppressing Th2 cells. Therefore, it is expected to be used in the treatment of Th2-driven immune disorders and in the treatment of cancer and infectious diseases by activating cellular immune function. As mentioned above, IL-12, along with IL-18, activates CD4 ＋ CD8 ＋ IL-12 and IL-18 act on T cells and NK cells to induce the production of IFN-γ. Therefore, IL-12 and IL-18 may be used in combination in the treatment of cancer and infectious diseases.

[0031] 5. Pharmaceutical Composition (Medicine, Drug) The pharmaceutical composition of the present invention is characterized by effectively treating tumors by combining one or more selected from IL-18, an anti-PD-1 / PD-L1 antibody, and IL-12 with a chymase inhibitor that does not exhibit anti-tumor activity alone.

[0032] In a first embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for treating a tumor, comprising a chymase inhibitor as an active ingredient, which is used in combination with one or more selected from IL-18, an anti-PD-1 / PD-L1 antibody, and IL-12.

[0033] In a second embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for treating tumors, comprising a chymase inhibitor in combination with one or more selected from IL-18, anti-PD-1 / PD-L1 antibody, and IL-12. In this case, the chymase inhibitor and one or more selected from IL-18, anti-PD-1 / PD-L1 antibody, and IL-12 may be formulated together or separately as a kit preparation.

[0034] In a third embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for treating tumors, comprising as an active ingredient any one or more selected from IL-18, an anti-PD-1 / PD-L1 antibody, and IL-12, and is characterized in that it is used in combination with a chymase inhibitor.

[0035] In a fourth embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for enhancing the effect of any one or more selected from IL-18, anti-PD-1 / PD-L1 antibody, and IL-12 in tumor treatment, comprising a chymase inhibitor as an active ingredient.

[0036] The pharmaceutical composition of the present invention may contain pharmacologically acceptable carriers and additives, such as excipients, binders, lubricants, solvents, disintegrants, solubilizers, suspending agents, emulsifiers, isotonicity agents, stabilizers, soothing agents, preservatives, antioxidants, flavoring agents, colorants, buffers, and flow enhancers, but is not limited to these, and other commonly used carriers and additives may also be used as appropriate.

[0037] Examples of excipients include organic excipients such as sugars such as lactose, glucose, and D-mannitol, starches, and celluloses such as crystalline cellulose, and inorganic excipients such as calcium carbonate and kaolin.

[0038] Examples of binders include pregelatinized starch, gelatin, gum arabic, methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, crystalline cellulose, D-mannitol, trehalose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, and polyvinyl alcohol.

[0039] Examples of lubricants include stearic acid, fatty acid salts such as stearates, talc, silicates, etc. Examples of solvents include purified water, physiological saline, phosphate buffer solution, etc.

[0040] Disintegrants include low-substituted hydroxypropyl cellulose, chemically modified celluloses and starches.

[0041] Examples of solubilizing agents include polyethylene glycol, propylene glycol, trehalose, benzyl benzoate, ethanol, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate.

[0042] Examples of suspending agents or emulsifying agents include sodium lauryl sulfate, gum arabic, gelatin, lecithin, glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, celluloses such as sodium carboxymethylcellulose, polysorbates, and polyoxyethylene hydrogenated castor oil.

[0043] Examples of isotonic agents include sodium chloride, potassium chloride, sugars, glycerin, and urea.

[0044] Stabilizers include polyethylene glycol, dextran sodium sulfate, other amino acids, etc. Soothing agents include glucose, calcium gluconate, procaine hydrochloride, etc.

[0045] Examples of preservatives include parahydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0046] Antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0047] Flavoring agents include sweeteners, fragrances, and the like that are commonly used in the pharmaceutical field, and coloring agents include coloring agents that are commonly used in the pharmaceutical field.

[0048] The route of administration of the pharmaceutical composition of the present invention is not particularly limited, and may be oral or parenteral. Specific examples of parenteral administration include injection, nasal administration, pulmonary administration, and transdermal administration. Examples of injection include intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intramedullary injection, intrathecal injection, and intradermal injection. The administration method can be appropriately selected depending on the age and symptoms of the patient.

[0049] The pharmaceutical composition of the present invention is administered to a subject in a therapeutically effective amount. The term "therapeutically effective amount" refers to an amount of a therapeutically active substance that is effective in alleviating a selected condition. The therapeutically effective amount is determined appropriately depending on the purpose of use, the route of administration, the age and symptoms of the patient, etc.

[0050] Tumors to which the pharmaceutical composition of the present invention can be applied include, for example, leukemias such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia, malignant lymphomas such as Hodgkin's lymphoma and non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, head and neck cancer, tongue cancer, pharyngeal cancer, oral cancer, esophageal cancer, esophageal adenocarcinoma, gastric cancer, colorectal cancer, colon cancer, rectal cancer, liver cancer, gallbladder / bile duct cancer, biliary tract cancer, pancreatic cancer, Examples of malignant tumors include, but are not limited to, thyroid cancer, lung cancer such as non-small cell lung cancer and small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, kidney cancer, adenocarcinoma, urothelial carcinoma, prostate cancer, testicular tumor, bone and soft tissue sarcoma, uveal malignant melanoma, malignant melanoma, skin cancer such as Merkel cell carcinoma, neuroblastoma, glioblastoma, brain tumor, and pleural mesothelioma, and benign tumors such as angiomyolipoma. Preferably, the tumor is a malignant tumor such as colon cancer.

[0051] The pharmaceutical composition of the present invention may be used in combination with other drugs or treatment methods as long as the object of the present invention is not impaired.

[0052] 6. Treatment Method The present application also provides a method for treating a tumor, comprising administering to a subject a chymase inhibitor in combination with one or more selected from IL-18, an anti-PD-1 / PD-L1 antibody, and IL-12.

[0053] The present application also provides a method for enhancing the anti-tumor effect of any one or more selected from IL-18, an anti-PD-1 / PD-L1 antibody, and IL-12, comprising administering a chymase inhibitor to a subject to which any one or more selected from IL-18, an anti-PD-1 / PD-L1 antibody, and IL-12 are administered or which has been administered to a subject.

[0054] In the above method, the chymase inhibitor and one or more selected from IL-18, anti-PD-1 / PD-L1 antibody, and IL-12 may be administered simultaneously or sequentially. The administration route for each is not particularly limited, and the methods described in the previous section can be applied.

[0055] The present invention will be specifically described below using examples, but the present invention is not limited to these examples.

[0056] Example 1: Antitumor effect of combined use of peptide chymase inhibitor and IL-18 Method: Colon cancer cells (CT26: 5 x 10) were injected into the abdominal cavity of 7-week-old male BALB-c mice (purchased from Japan SLC). 4 The placebo group (n=8) received intraperitoneal administration of 0.17% dimethyl sulfoxide (drug solvent: placebo) once daily. The IL-18 administration group (n=8) received intraperitoneal administration of 2 μg / body of IL-18 (mouse IL-18: obtained from GSK) once every two days. The chymase inhibitor administration group (n=8) received intraperitoneal administration of 3 mg / kg of Suc-Val-Pro-Phe p (OPh)2 (peptide chymase inhibitor, synthesized by Peptide Institute) was administered intraperitoneally once daily. The IL-18 / chymase inhibitor combination group (n=8) received 2 μg / mouse IL-18 once every two days and 3 mg / kg chymase inhibitor once daily intraperitoneally. Medication was discontinued 30 days after cell transplantation, and analysis was performed at 31 and 100 days after cell transplantation.

[0057] Observation: Mice were observed twice a day, morning and evening, for survival.Analysis method: The significance of the survival curves of each group was tested using the Kaplan-Meier method, with a P value of 5% or less considered significant.

[0058] Results: At 31 days after cell transplantation, there was no significant difference in survival time between the placebo group and the IL-18-treated group (P = 0.5024) or chymase inhibitor group (P = 0.2068). However, there was a significant difference between the placebo group and the IL-18 / chymase inhibitor combination group (P = 0.0125), demonstrating that the IL-18 / chymase inhibitor combination group had a significantly longer survival time than the placebo group (Figure 1). At 100 days after cell transplantation, there were no surviving mice in the chymase inhibitor group, and one surviving mouse in the IL-18-treated group. However, there was no significant difference in survival time between the placebo group and the IL-18-treated group (P = 0.2916) or chymase inhibitor group (P = 0.2507) (Figure 1). However, a significant difference was observed between the survival curves of the placebo group and the IL-18 / chymase inhibitor combination group (P = 0.0126, Figure 1). Furthermore, the tumors in surviving mice were macroscopically clear (Figure 2).

[0059] In this experiment, administration of a chymase inhibitor alone did not extend survival time after cancer transplantation, but administration of IL-18 in combination with a chymase inhibitor extended survival time compared to IL-18 alone.

[0060] Example 2: Antitumor effect of a commercially available chymase inhibitor in combination with IL-18 Method: Colon cancer cells (CT26: 5 x 10) were injected into the abdominal cavity of 7-week-old male BALB-c mice (purchased from Japan SLC). 4Mice were administered 100 μg / body of IL-18 (mouse IL-18) intraperitoneally every other day. The chymase inhibitor group (n=10) received 5 mg / kg (100 μg / body) of fulacimstat (chymase inhibitor; MedChemExpress) intraperitoneally every other day. The IL-18 / chymase inhibitor combination group (n=10) received 2 μg / body of IL-18 every other day and 5 mg / kg of chymase inhibitor intraperitoneally every other day. Treatment ended 30 days after cell transplantation, and analysis was continued up to 110 days.

[0061] After the administration, the mice were checked daily for survival.Analysis method: The significance of the survival curves of each group was tested using the Kaplan-Meier method, and a P value of 5% or less was considered significant.

[0062] Results: There was no difference in survival rate between the placebo group and the chymase inhibitor group, and all mice died before day 40 in both groups. However, the IL-18 group and the IL-18 / chymase inhibitor combination group showed a clear improvement in survival rate compared to the placebo group, and at 110 days after cell transplantation, the IL-18 / chymase inhibitor combination group showed a higher survival rate than the IL-18 group (Figure 3).

[0063] Even when different chymase inhibitors were used, the overall trend was similar to that observed with peptidic chymase inhibitors: administration of a chymase inhibitor alone did not extend survival time after cancer transplantation, but the combination of IL-18 and a chymase inhibitor extended survival time compared to IL-18 alone.

[0064] Example 3: Changes in intraperitoneal lymphocytes due to tumor antigen stimulation Method: Colon cancer cells (CT26: 5 x 10) were injected into the abdominal cavity of 7-week-old male BALB-c mice (purchased from Japan SLC). 4 Ascites was collected on days 3, 5, 7, 11, 14, 17, and 21 after cell transplantation (n=4 each time).

[0065] Ascites was collected from mice anesthetized with isoflurane and cooled PBS was injected intraperitoneally (8 ml total). The collected ascites was centrifuged and washed twice, and the number of cells was counted using a trypan blue exclusion assay test. The collected cells were plated on a chamber and subjected to HE and TB staining. Cell subsets were also analyzed by flow cytometry.

[0066] Results: Mast cells tended to increase from 7 days after cell transplantation, but granular mast cells decreased (Fig. 4). Mast cells also tended to gather around the tumor tissue (data not shown). Tumor cell transplantation increased CD4 + T cells, CD8 + T cells increased, and CD4 + T cells / CD8 + The proportion of T cells decreased (Fig. 5). + This is presumed to be due to an increase in killer cells. NK cells, especially activated killer NK cells (DX5 + B220 + NK cells also increased after tumor cell transplantation (Figure 6).

[0067] Analysis of immune cell subsets in the peritoneal cavity showed an increase in NK cells early after transplantation up to 10 days, and CD8 + T cells increased, and mast cells gradually increased after 7 days (Fig. 7).

[0068] Example 4: Changes in intraperitoneal antitumor lymphocytes by combined use of a peptide chymase inhibitor and IL-18 Method: Colon cancer cells (CT26: 5 x 10) were injected into the abdominal cavity of 7-week-old male BALB-c mice (purchased from Japan SLC). 4 The control group received intraperitoneal administration of 0.17% dimethyl sulfoxide (drug solvent: placebo) once a day. The IL-18 alone administration group received intraperitoneal administration of 2 μg / body of IL-18 (mouse IL-18 mentioned above) once every two days. The chymase inhibitor alone administration group received intraperitoneal administration of 3 mg / kg (100 μg / body) of Suc-Val-Pro-Phe p(OPh)2 was administered intraperitoneally once daily. The IL-18 / chymase inhibitor combination group received 2 μg / body of IL-18 once every two days and 3 mg / kg of chymase inhibitor once daily intraperitoneally. Medication was discontinued 28 days after cell transplantation.

[0069] According to Example 3, ascites fluid was collected from the mice 21 to 28 days after cell transplantation, and cell subsets in the ascites fluid were analyzed.

[0070] Results: In both the IL-18 administration group and the IL-18 / chymase inhibitor combination group, CD4 + T cells / CD8 + The proportion of T cells was significantly lower than that of placebo, and CD8 + T cells, especially CD44highCD8 + The number of T cells increased (Figure 8). In the IL-18 administration group and the IL-18 / chymase inhibitor combination group, NK cells, especially B220 + The number of NK cells was significantly higher than that of the placebo group (Figure 9).

[0071] The composition of immune cell subsets in the ascites also suggested that the combined use of IL-18 and chymase enhanced the antitumor effect compared to IL-18 alone.

[0072] Example 5: Antitumor effect of combined use of chymase inhibitor and anti-PD-1 antibody Method: Colon cancer cells (CT26: 5 x 10) were injected into the abdominal cavity of 7-week-old male BALB-c mice (purchased from Japan SLC). 4Patients were administered 100μg / body of fulacimstat (a chymase inhibitor; MedChemExpress) intraperitoneally once a day. The placebo group (n=10) received 0.17% dimethyl sulfoxide (drug solvent: placebo) intraperitoneally once a day. The anti-PD-1 antibody group (n=10) received 200μg / body of anti-PD-1 antibody (BioXCell) intraperitoneally three times a week (12 times in total). The chymase inhibitor group (n=10) received 100μg / body of fulacimstat (a chymase inhibitor; MedChemExpress) intraperitoneally once a day (30 times in total). The anti-PD-1 antibody / chymase inhibitor combination group (n=10) received intraperitoneal administration of 200μg / body of anti-PD-1 antibody three times a week (12 times in total) and 100μg / body of chymase inhibitor once a day (30 times in total). Administration ended 30 days after cell transplantation, and analysis was continued up to 34 days thereafter.

[0073] Observation: Mice were observed twice a day, morning and evening, for survival.Analysis method: The significance of the survival curves of each group was tested using the Kaplan-Meier method, with a P value of 5% or less considered significant.

[0074] Results: 34 days after cancer cell transplantation, the group receiving the combination of anti-PD-1 antibody and chymase inhibitor showed a higher survival rate (50%) than not only the placebo group (20%) and the chymase inhibitor alone group (20%), but also the anti-PD-1 antibody alone group (10%), demonstrating the combined effect of adding a chymase inhibitor to an anti-PD-1 antibody (Figure 10).

[0075] In this experiment, administration of a chymase inhibitor alone did not extend survival time after cancer transplantation, but administration of an anti-PD-1 antibody in combination with a chymase inhibitor extended survival time compared to anti-PD-1 antibody alone.

[0076] The present invention is useful in the treatment of tumors.

[0077] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A pharmaceutical composition for treating tumors, comprising a chymase inhibitor as an active ingredient, said pharmaceutical composition being used in combination with one or more selected from interleukin-18 (IL-18), anti-PD-1 / PD-L1 antibody, and interleukin-12 (IL-12).

2. A pharmaceutical composition for treating a tumor, comprising a combination of one or more selected from interleukin-18 (IL-18), anti-PD-1 / PD-L1 antibody, and interleukin-12 (IL-12) with a chymase inhibitor.

3. A pharmaceutical composition for treating a tumor, comprising as an active ingredient one or more selected from interleukin-18 (IL-18), anti-PD-1 / PD-L1 antibody, and interleukin-12 (IL-12), said pharmaceutical composition being used in combination with a chymase inhibitor.

4. Chymase inhibitors include Suc-Val-Pro-PheP(OPh)2, Chymostatin, Fulacimstat, 2-[4-[[(5-Fluoro-3-methylbenzo[b]thien-2-yl)sulfonyl]amino]-3-(methylsulfonyl)phenyl]-4-thiazolecarboxylic The pharmaceutical composition according to any one of claims 1 to 3, wherein the compound is any one selected from the group consisting of (2-[5-amino-2-(4-fluorophenyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide), (2-[5-amino-2-(4-fluorophenyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide).

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein IL-18, anti-PD-1 / PD-L1 antibody, and IL-12 are of human origin.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein one or more selected from IL-18, anti-PD-1 / PD-L1 antibody, and interleukin-12 (IL-12) and a chymase inhibitor are administered simultaneously or sequentially.

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