Cancer treatment drugs

Abstract

To provide a novel and effective cancer immunotherapeutic agent exhibiting a therapeutic effect against refractory cancers, particularly against cancers resisting an immune checkpoint inhibitor as a monotherapy or a combination therapy with another drug.SOLUTION: A cancer treatment agent is prepared by combining an immune checkpoint inhibitor, interleukin-18 (IL-18), and a T-cell growth factor (e.g., interleukin-2 (IL-2)).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a cancer treatment agent comprising a combination of a T cell growth factor such as interleukin-2 (IL-2), interleukin-18 (IL-18), and an immune checkpoint inhibitor. [Background technology]

[0002] Liver cancer is primarily treated with non-pharmacological therapies such as hepatectomy, liver transplantation, local aspiration therapy, and transarterial chemoembolization (TACE). For advanced hepatocellular carcinoma that is not suitable for these therapies, drug therapy with vascular endothelial growth factor (VEGF) inhibitors such as sorafenib and lenvatinib is used, but even with the latest drug, lenvatinib, the response rate is only about 40%. Recently, the immune checkpoint inhibitor anti-PD-1 antibodies (nivolumab, pembrolizumab) have been approved in the United States as a second-line treatment for hepatocellular carcinoma with a history of sorafenib administration. However, pembrolizumab failed to meet the primary endpoints of overall survival and progression-free survival in a placebo-controlled phase III trial. Combinations with VEGF inhibitors are also being studied, but the response rate with pembrolizumab and lenvatinib combination therapy remains at only about 60%, and it cannot yet be said to be sufficiently effective.

[0003] Inflammatory cytokines strongly activate T cells and natural killer (NK) cells, which are important in cancer immunity, and various cytokine therapies have been investigated with the expectation of antitumor effects. Interleukin-18 (IL-18) significantly promotes the proliferation of NK cells, CD8-positive killer T cells (CTLs), and γδ T cells. However, IL-18 monotherapy did not show sufficient efficacy in a phase II trial for metastatic melanoma. Our research group previously reported that combining IL-18 with immune checkpoint inhibitors increases NK cells (helper NK cells) and CTLs, which are expected to enhance acquired immunity, and enhances the antitumor effect of immune checkpoint inhibitors (Patent Document 1, Non-Patent Document 1).

[0004] On the other hand, interleukin-2 (IL-2) is produced by activated T cells, NK cells, dendritic cells, etc., and causes proliferation and activation of tumor-killing cells such as CTLs and NK cells. For this reason, it is clinically used in the treatment of metastatic renal cell carcinoma and malignant melanoma, either by administration alone or in combination with NK cells activated by IL-2 (Non-Patent Literature 2). The present inventors have found that culturing peripheral blood-derived NK cells with IL-18 and IL-2 in combination significantly promotes the proliferation and activation of NK cells (Non-Patent Literature 3).

[0005] However, cytokines that exhibit immunostimulatory effects may also exhibit tumor growth-promoting effects. IL-2 induces differentiation and activation of naive T cells into Th1, Th2, Th17, and regulatory T cells (Treg), but Treg enhances the suppression of CTL activity, making it impossible to predict whether in vivo administration will reproduce the in vitro effect. IL-18 has also been reported to have a dual nature, exhibiting both antitumor and tumor growth-promoting effects (Non-Patent Literature 4). Furthermore, the expression of IL-18-binding protein (IL-18BP), which has extremely high affinity for IL-18, is increased in the tumor microenvironment, and it has been reported that treatment with IL-18 or anti-PD-L1 antibodies further increases IL-18BP expression (Non-Patent Literature 5). Therefore, the effectiveness of combining immune checkpoint inhibitors with IL-18 in actual human clinical practice remains unknown. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Patent No. 6245622 [Non-patent literature]

[0007] [Non-Patent Document 1] Ma, Z. et al., Clin. Cancer Res., 22(12): 2969-2980 (2009) [Non-Patent Document 2] Japanese Society of Clinical Oncology (ed.), "Guidelines for Cancer Immunotherapy" (Kinbara Publishing Co., Ltd.), p. 15, published December 20, 2016. [Non-Patent Document 3] El-Darawish, Y. et al., J. Leukoc. Biol., 104: 253-264 (2018) [Non-Patent Document 4] Fabbi, M. et al., J. Leukoc. Biol., 97: 665-675 (2015) [Non-Patent Document 5] Zhou, T. et al., Nature, 583(7817): 609-614 (2020) [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The objective of the present invention is to provide a novel and effective cancer immunotherapy agent that shows therapeutic effects against refractory cancers, particularly cancers that are ineffective with monotherapy or combination therapy with immune checkpoint inhibitors. [Means for solving the problem]

[0009] Spontaneous hepatocellular carcinoma is used as a model for liver cancer that is difficult to treat, but there are few effective therapeutic drugs for this model, and lenvatinib, the standard drug for first-line treatment of advanced hepatocellular carcinoma, is ineffective. The inventors used spontaneous hepatocellular carcinoma model mice lacking the mdr2 gene encoding P-glycoprotein (Mdr2 KO mice) to investigate the therapeutic effect of combining an immune checkpoint inhibitor (anti-PD-L1 antibody) with IL-18, which had shown efficacy in a peritoneal dissemination model due to colorectal cancer cell transplantation and a lung metastasis model due to melanoma cell transplantation. As a result, a tendency for tumor size to increase was observed, suggesting that the combination of immune checkpoint inhibitors and IL-18 does not produce sufficient efficacy against refractory liver cancer. Therefore, after diligent research, the inventors found that when Mdr2 KO mice were administered T-cell growth factor (e.g., IL-2) in addition to the two drugs mentioned above, tumor size tended to decrease. When only two drugs, an immune checkpoint inhibitor and IL-2, were used, tumor size tended to increase, and sufficient therapeutic effect was not obtained. With the three-drug combination, serum levels of the tumor marker α-fetoprotein (AFP) were significantly reduced compared to any of the two-drug combinations. In addition, the production of antitumor cytokines (interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α)) was significantly increased with the three-drug combination, showing an increasing trend compared to any of the two-drug combinations. The tumor reduction, AFP level decrease, and IFN-γ·TNF-α production increase effects of the triple therapy were neutralized by the administration of an anti-CD8 antibody, suggesting that at least CD8-positive T cells are important for the therapeutic effect of the triple therapy. Furthermore, the tumor reduction effect of the triple therapy was neutralized by the administration of an anti-asialoGM1 antibody, suggesting the involvement of NK cells in the therapeutic effect of the triple therapy.

[0010] When we examined the expression of IL-18BP in Mdr2 KO mice, which is suggested to be a barrier to IL-18-based cancer treatment, we found that serum IL-18BP protein levels did not change with the combination of anti-PD-L1 antibody and IL-18, but actually increased with the combination of anti-PD-L1 antibody and IL-2, while they significantly decreased with the combination of three drugs. When we examined serum IL-18BP protein levels in patients with hepatocellular carcinoma and intrahepatic cholangiocarcinoma, we found that IL-18BP levels were significantly elevated in hepatocellular carcinoma patients compared to healthy individuals, and differences of up to four times or more were observed between patients. This suggests that triple combination therapy may be effective in cancer patients with elevated serum IL-18BP levels. Based on these findings, the inventors conducted further research and, as a result, completed the present invention.

[0011] In other words, the present invention provides the following: [Section 1] A cancer therapeutic agent comprising an immune checkpoint inhibitor, interleukin-18 (IL-18), and a T cell growth factor. [Item 2] The agent according to [Item 1], wherein the T cell growth factor is one or more cytokines selected from the group consisting of interleukin-2 (IL-2), interleukin-15 (IL-15), and interleukin-7 (IL-7). [Item 3] The agent according to [Item 2], comprising IL-2 as the T cell growth factor. [Item 4] The agent according to any one of [Item 1] to [Item 3], wherein the immune checkpoint inhibitor is one or more antibodies selected from the group consisting of an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CTLA4 antibody, an anti-LAG-3 antibody, and an anti-TIM-3 antibody. [Item 5] The agent according to any one of [Item 1] to [Item 4] for cancer resistant to monotherapy with an immune checkpoint inhibitor or combined therapy with an immune checkpoint inhibitor and IL-18. [Item 6] The agent according to any one of [Item 1] to [Item 5], which is administered to a subject having a serum IL-18 binding protein (IL-18BP) level of 10 pg / mL or more. [Item 7] The agent according to any one of [Item 1] to [Item 6], wherein the cancer is an inflammation-related cancer. [Item 8] The agent according to [Item 7], wherein the inflammation-related cancer is a digestive organ cancer.

Advantages of the Invention

[0012] According to the present invention, there is provided a combined cancer immunotherapy agent effective against refractory cancers such as liver cancer, particularly cancers ineffective to monotherapy with an immune checkpoint inhibitor or combined use with other agents.

Brief Description of the Drawings

[0013] [Figure 1] It is a diagram schematically showing the schedule of drug administration and tests for Mdr2 KO mice. [Figure 2-1] These are representative CT images showing tumor size at baseline and 4 weeks after the start of administration (4W) in Mdr2 KO mice in the control (PBS administration) group. The lower right image is a photograph of the liver resected 4 weeks after the start of administration. [Figure 2-2] This is a representative example of hematoxylin-eosin (HE) staining of the liver of Mdr2 KO mice in the control (PBS administration) group 4 weeks after the start of administration. [Figure 3-1] This is a representative CT image showing tumor size reduction in Mdr2 KO mice treated with three agents (anti-PD-L1 antibody, IL-18, and IL-2). Baseline: at the start of administration, 4W: 4 weeks after the start of administration. The lower right image is a photograph of the liver resected 4 weeks after the start of administration. [Figure 3-2] This is a representative example of HE staining of the liver four weeks after the start of administration in a group receiving three drugs (anti-PD-L1 antibody, IL-18, and IL-2). [Figure 4] This is a representative CT image showing increased tumor size in Mdr2 KO mice treated with two drugs (anti-PD-L1 antibody and IL-2). Baseline: at the start of administration, 4W: 4 weeks after the start of administration. The lower right image is a photograph of the liver resected 4 weeks after the start of administration. [Figure 5] This is a representative CT image showing increased tumor size in Mdr2 KO mice treated with two drugs (anti-PD-L1 antibody and IL-18). Baseline: at the start of administration, 4W: 4 weeks after the start of administration. The lower right image is a photograph of the liver resected 4 weeks after the start of administration. [Figure 6] (A) This figure shows the serum AFP levels (ng / mL) of Mdr2 KO mice in each treatment group 4 weeks after the start of administration. (B) This figure shows the serum AFP levels (ng / mL) at the start of administration (baseline) and 4 weeks after the start of administration (4W) in Mdr2 KO mice in the 3-agent (anti-PD-L1 antibody, IL-18, and IL-2) treatment group and the control (PBS administration) group. [Figure 7]This figure shows the serum IFN-γ and TNF-α levels (ng / mL) in Mdr2 KO mice in each treatment group 4 weeks after the start of administration, as well as the serum IFN-γ and TNF-α levels (ng / mL) at the start of administration (baseline) and 4 weeks after the start of administration (4W) in Mdr2 KO mice in the 3-agent (anti-PD-L1 antibody, IL-18, and IL-2) treatment group and the control (PBS administration) group. [Figure 8] This is a representative CT image showing that the tumor reduction effect of the three-drug (anti-PD-L1 antibody, IL-18, and IL-2) administration in Mdr2 KO mice is nullified by the removal of NK cells. Baseline: at the start of administration, 4W: 4 weeks after the start of administration. The lower right image is a photograph of the liver resected 4 weeks after the start of administration. [Figure 9-1] This is a representative CT image showing that the tumor reduction effect of the three-drug (anti-PD-L1 antibody, IL-18, and IL-2) administration in Mdr2 KO mice is nullified by the removal of CD8-positive T cells. Baseline: at the start of administration, 4W: 4 weeks after the start of administration. The lower right image is a photograph of the liver resected 4 weeks after the start of administration. [Figure 9-2] This figure shows that the effects of administering three drugs (anti-PD-L1 antibody, IL-18, and IL-2) on reducing serum AFP levels and promoting IFN-γ and TNF-α production in Mdr2 KO mice are neutralized by the removal of CD8-positive T cells. [Figure 10-1] This figure shows the serum IL-18BP levels (pg / mg) of Mdr2 KO mice in each treatment group 4 weeks after the start of administration, as well as the serum IL-18BP levels (pg / mg) at the start of administration (baseline) and 4 weeks after the start of administration (4W) in Mdr2 KO mice in the 3-agent (anti-PD-L1 antibody, IL-18, and IL-2) treatment group and the control (PBS administration) group. [Figure 10-2] This figure shows that the reduction in serum IL-18BP levels in Mdr2 KO mice induced by administration of three agents (anti-PD-L1 antibody, IL-18, and IL-2) is neutralized by the removal of CD8-positive T cells. [Figure 11-1]This figure shows serum IL-18BP levels (pg / mL) in patients with hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (CCC), and healthy volunteers. [Figure 11-2] This figure shows serum IL-18 levels (pg / mL) in patients with hepatitis C virus (HCV)-positive hepatocellular carcinoma (HCC), chronic hepatitis (CH), and cirrhosis (LC), as well as in healthy volunteers. [Figure 11-3] This figure shows the results of an investigation into the correlation between serum IL-18 levels and IL-18BP levels in patients with hepatitis C virus (HCV)-positive hepatocellular carcinoma. [Modes for carrying out the invention]

[0014] The present invention provides a cancer treatment agent (hereinafter also referred to as "the combination agent of the present invention") comprising an immune checkpoint inhibitor, IL-18, and T cell growth factor. That is, the combination agent of the present invention is a combination cancer immunotherapy agent comprising (a) an immune checkpoint inhibitor, (b) IL-18, and (c) T cell growth factor as active ingredients.

[0015] (I) Active ingredients (a) Immune checkpoint inhibitors In this specification, "immune checkpoint inhibitor" means a drug that inhibits the binding of immunosuppressive costimulatory molecules (immune checkpoint molecules) expressed on T cells or NK cells to their ligands expressed on cancer cells or antigen-presenting cells, thereby blocking the transmission of immunosuppressive signals and releasing the suppression of T cell or NK cell activation.

[0016] The immune checkpoint inhibitor used as the active ingredient in the combination agent of the present invention is not particularly limited as long as it is a substance that binds to inhibitory costimulatory molecules expressed on T cells or NK cells (e.g., PD-1, CTLA4, TIM-3, LAG-3, TIGIT, CD96, BTLA, VISTA, KIR, etc.) and inhibits their binding to ligands (e.g., PD-L1, PD-L2, CD80 / 86, CEACAM1, Galectin-9, MHC class-II molecules, LSECtin, Galectin-3, CD155, CD112, CD113, CD111, HVEM, VSIG3, etc.) or a substance that binds to such ligands and inhibits their binding to immune checkpoint molecules. However, in one preferred embodiment, a blocking antibody against an immune checkpoint molecule or its ligand can be mentioned. Preferably, the antibodies include anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA4 antibodies, anti-PD-L2 antibodies, anti-TIM-3 antibodies, anti-LAG-3 antibodies, anti-KIR antibodies, and more preferably, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA4 antibodies, etc.

[0017] Antibodies against immune checkpoint molecules or their ligands may be polyclonal or monoclonal antibodies, but monoclonal antibodies are preferred. These antibodies can be manufactured according to known antibody or antiserum manufacturing methods. The antibody isotype is not particularly limited, but IgG, IgM, or IgA are preferred, with IgG being particularly preferred. The Fc region of IgG1 and IgG2 has effector functions such as ADCC, ADCP, and CDC, and can therefore be preferably used in antibodies targeting ligands expressed on cancer cells. On the other hand, IgG4 has low effector function, and can therefore be preferably used in antibodies targeting immune checkpoint molecules expressed on T cells and NK cells. Furthermore, since CTLA4 is strongly expressed on regulatory T cells (Tregs), IgG1 or IgG2 subtypes can also be used as anti-CTLA4 antibodies with the expectation of Treg removal.

[0018] Antibodies against immune checkpoint molecules or their ligands are not particularly limited as long as they have at least a complementarity-determining region (CDR) for specifically recognizing and binding to the target antigen. In addition to complete antibody molecules, they may also be fragments such as Fab, Fab', F(ab')2, genetically engineered conjugate molecules such as scFv, scFv-Fc, mini-bodies, and diabodies, or derivatives thereof modified with molecules that have protein-stabilizing effects, such as polyethylene glycol (PEG).

[0019] In one preferred embodiment, since the antibody against an immune checkpoint molecule or its ligand is used as a pharmaceutical product intended for human administration, the antibody (preferably a monoclonal antibody) is one that has a reduced risk of exhibiting antigenicity when administered to humans. Specifically, it is a fully human antibody, a humanized antibody, a mouse-human chimeric antibody, and is particularly preferred to be a fully human antibody. Humanized antibodies and chimeric antibodies can be produced genetically engineered according to conventional methods. While fully human antibodies can also be produced from human-human (or mouse) hybridomas, it is desirable to produce them using human antibody-producing mice or phage display methods in order to provide large quantities of antibodies stably and at low cost.

[0020] Some monoclonal antibodies against immune checkpoint molecules or their ligands are already on the market as pharmaceuticals or are in clinical trials, and can be used. For example, anti-PD-1 antibodies include nivolumab (Opdivo®), pembrolizumab (Keytruda®), AMP-514 (MEDI0680), vizilizumab (CT-011), etc.; anti-PD-L1 antibodies include atezolizumab (RG7446, MPDL3280A), durvalumab (MEDI4736), avelumab (PF-06834635, MSB0010718C), BMS-936559 (MDX1105), etc.; anti-CTLA4 antibodies include ipilimumab (Yervoy®), tremelimumab, etc.; anti-TIM-3 antibodies include MBG453, etc.; anti-LAG-3 antibodies include BMS-986016, LAG525, etc.; and anti-KIR antibodies include lirilumab, etc.

[0021] Alternatively, as a substance that binds to an immune checkpoint molecule and inhibits its binding to its ligand expressed on antigen-presenting cells such as cancer cells and tumor-infiltrating macrophages, a fragment can be used that contains the portion necessary for the ligand's binding to the immune checkpoint molecule (e.g., the extracellular domain) and does not have the ability to transmit immunosuppressive signals. Furthermore, a substance can be used in which a molecule capable of removing immune-exhausted effector cells is conjugated to the fragment. An example of the latter is a fusion protein of the extracellular domain of PD-L1 or PD-L2 and the Fc region of IgG1 or IgG2 antibodies (e.g., AMP-224).

[0022] A substance that binds to an immune checkpoint molecule and inhibits its binding to its ligand may also be an antagonist that competitively binds to the immune checkpoint molecule with the ligand. Such antagonists can be obtained by constructing a competitive assay system using the immune checkpoint molecule and its ligand and screening a compound library.

[0023] The combination agent of the present invention can be used in combination with one or more of the above-mentioned immune checkpoint inhibitors.

[0024] (b) IL-18 IL-18 is a pro-inflammatory cytokine belonging to the IL-1 family that promotes IFN-γ production by T cells and NK cells. Human IL-18 is produced as an inactive precursor (pro-IL-18) consisting of 192 amino acids. However, when a protein complex called the inflammasome is activated by PAMPs and DAMPs, caspase-1 is activated, and pro-IL-18 is processed by its enzymatic activity to produce active, mature IL-18 consisting of 157 amino acids. The amino acid sequence information for human IL-18 can be found, for example, in UniProtKB (accession number: Q14116). The amino acid sequence of mature human IL-18 is shown in Sequence ID No. 2.

[0025] The IL-18 used as the active ingredient in the combination agent of the present invention is a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence shown in Sequence ID No. 2. IL-18 may be a protein isolated and purified from IL-18-producing cells (e.g., macrophages, dendritic cells, microglia, synovial fibroblasts, epithelial cells, etc.) or tissues containing IL-18 from humans or other mammals (e.g., rats, mice, monkeys, dogs, cattle, rabbits, pigs, sheep, etc.). It may also be a protein synthesized chemically or biochemically in a cell-free translation system, or a recombinant protein produced from a transformant into which a nucleic acid having the base sequence encoding the above amino acid sequence has been introduced.

[0026] An amino acid sequence substantially identical to the amino acid sequence shown in Sequence ID No. 2 is an amino acid sequence having 80% or more identity, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, and most preferably 98% or more identity with the amino acid sequence shown in Sequence ID No. 2. Here, "identity" means the percentage (%) of identical amino acid residues to the total overlapping amino acid residues in the optimal alignment (preferably, the algorithm may consider introducing gaps into one or both of the sequences for optimal alignment) when the two amino acid sequences are aligned using a mathematical algorithm known in the art. The identity of amino acid sequences in this specification can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gaps allowed; matrix = BLOSUM62; filtering = OFF).

[0027] IL-18 is a protein that contains substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 and has the same activity as the protein containing the amino acid sequence shown in SEQ ID NO: 2. Here, "activity" refers to any activity that contributes to cancer suppression, such as receptor binding activity or activity that promotes the proliferation and activation of T cells and NK cells. Here, "similar" means that these activities are qualitatively the same. Therefore, it is preferable that the activity of IL-18 is equivalent to or greater than that of wild-type IL-18, but the degree of these activities may differ.

[0028] Alternatively, IL-18 used in the present invention may include, for example, (i) an amino acid sequence in which one or more amino acids (for example, about 1 to 30, preferably about 1 to 10, more preferably 1 to several (5, 4, 3, or 2)) are deleted from the amino acid sequence shown in SEQ ID NO: 2, (ii) an amino acid sequence in which one or more amino acids (for example, about 1 to 30, preferably about 1 to 10, more preferably 1 to several (5, 4, 3, or 2)) are added to the amino acid sequence shown in SEQ ID NO: 2, or (iii) an amino acid sequence in which SEQ ID NO: 2 This also includes (iv) an amino acid sequence in which one or more amino acids (for example, about 1 to 30, more preferably about 1 to 10, and more preferably 1 to several (5, 4, 3, or 2)) are inserted into the shown amino acid sequence, (iv) an amino acid sequence in which one or more amino acids (for example, about 1 to 30, more preferably about 1 to 10, and more preferably 1 to several (5, 4, 3, or 2)) in the amino acid sequence shown in Sequence ID No. 2 are replaced with other amino acids, or (v) a protein containing an amino acid sequence that combines these.

[0029] As described above, when an amino acid sequence is inserted, deleted, or substituted, the location of the insertion, deletion, or substitution is not particularly limited. However, for example, as an IL-18 mutant, in which at least one of the cysteine ​​(Cys) residues at positions 38, 68, 76, and 127 in the amino acid sequence shown in Sequence ID No. 2 is replaced with another amino acid (e.g., Ser, Ala, Asp, Thr, Val, Leu, etc.), a mutant with improved stability is obtained (e.g., Patent No. 40243). (See Patent No. 66), a variant having the amino acid substitution L144C or D157C in addition to C38S, C68S or C68D, which alters the affinity to the IL-18 receptor and / or IL-18BP (see, for example, Patent No. 4753867), an amino acid residue important for binding to IL-18BP, for example, Glu at position 42, Ile at position 85, Met at position 87, Lys at position 89, Met at position 96, and As at position 130 in the amino acid sequence shown in Sequence ID No. 2 mutants in which at least one amino acid residue of p, Lys at position 132, Pro at position 143, Met at position 149, and Leu at position 189, preferably G42 and / or K89, is substituted with another amino acid, resulting in reduced affinity with IL-18BP (see, for example, Japanese Patent Publication No. 2004-530432), Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D1 Examples of IL-18BP low-affinity mutants (see, for example, Japanese Patent Publication No. 2020-533301) include those having at least one substitution from among 10X, N111X, M113X, V153X, and N155X (where X represents an amino acid different from the amino acid residue of wild-type IL-18), preferably M51X, M60X, S105X, D110X, and N111X, or substitutions of M51X, K53X, Q56X, S105X, and N111X.Alternatively, IL-18 variants created by the present inventors (i.e., C38 / 68 / 76 / 127S-E6A-K53A, C68 / 76 / 127S-E6A-K53A-C38M, C38 / 68 / 76 / 127S-E6A-K53A-G3Y, C38 / 68 / 76 / 127S-E6A-K53A-G3L, C38 / 68 / 76 / 127S-E6A-K53A-S72Y, C38 / 68 / 76 / 127S-E6A-K53A-S72M, C38 / 68 / 76 / 127S-E6A-K53A-S72F) can also be preferably used.

[0030] The IL-18 used in the present invention may be in free form or as a salt. Such salts may be salts with physiologically acceptable acids (e.g., inorganic acids, organic acids) or bases (e.g., alkali metals, alkaline earth metals), with physiologically acceptable acid addition salts being particularly preferred. Such salts may include, for example, salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or salts with organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

[0031] IL-18 can be isolated from the extracellular matrix or culture supernatant of human or other mammalian IL-18-producing cells or tissues using known protein purification methods, such as reverse-phase chromatography, ion-exchange chromatography, affinity chromatography, and other chromatography techniques. IL-18 can also be produced according to known peptide synthesis methods. These methods may include solid-phase or liquid-phase synthesis. Specifically, IL-18 can be produced by condensing a partial peptide or amino acid with the remainder, and removing any protecting groups from the resulting product.

[0032] In a preferred embodiment, IL-18 can be produced by culturing a transformant containing the nucleic acid encoding it, and then separating and purifying the resulting culture. Here, the nucleic acid may be DNA or RNA. In the case of DNA, double-stranded DNA is preferred. The DNA encoding IL-18 can be cloned, for example, by synthesizing oligo DNA primers based on its cDNA sequence information, and amplifying it by RT-PCR using a total RNA or mRNA fraction prepared from IL-18-producing cells as a template. Using the obtained cDNA as a template, the various mutations described above can be introduced using known site-directed mutagenesis methods.

[0033] Examples of DNA encoding IL-18 include DNA containing the base sequence shown in Sequence ID No. 1, or DNA containing a base sequence that hybridizes with the base sequence shown in Sequence ID No. 1 under stringent conditions, and encoding a protein having the same activity as the wild-type IL-18 described above. Examples of DNA that can hybridize with the base sequence shown in Sequence ID No. 1 under stringent conditions include DNA containing a base sequence that has 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, and most preferably 98% or more identity with the base sequence shown in Sequence ID No. 1. In this specification, the identity of base sequences can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3). The DNA encoding IL-18 is preferably DNA encoding wild-type human IL-18 having the base sequence shown in Sequence ID No. 1, or DNA encoding one of the various human IL-18 mutant proteins described above.

[0034] The DNA encoding IL-18 can be constructed by chemically synthesizing the DNA strand, or by joining partially overlapping oligoDNA short chains using PCR or Gibson Assembly. The advantage of constructing full-length DNA using chemical synthesis or a combination of PCR or Gibson Assembly is that the codons used can be designed to match the host organism into which the DNA is introduced, across the entire CDS sequence. When expressing heterologous DNA, converting the DNA sequence to codons that are frequently used in the host organism can be expected to increase protein expression levels. Data on codon usage frequency in the host organism can be obtained, for example, from the Genetic Code Usage Frequency Database published on the Kazusa DNA Research Institute website (http: / / www.kazusa.or.jp / codon / index.html), or by referring to literature that lists codon usage frequency in various hosts.

[0035] The cloned DNA can be used as is, or, depending on the purpose, after being digested with restriction enzymes or after linker addition as desired. The DNA may have an ATG translation start codon at its 5' end and a TAA, TGA, or TAG translation stop codon at its 3' end. These translation start and stop codons can be added using a suitable synthetic DNA adapter.

[0036] Expression vectors containing DNA encoding IL-18 can be produced, for example, by excising a target DNA fragment from the IL-18 encoding DNA and ligating the DNA fragment downstream of a promoter in a suitable expression vector. Examples of expression vectors include plasmids derived from E. coli (e.g., pBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, pC194); plasmids derived from yeast (e.g., pSH19, pSH15); insect cell expression plasmids (e.g., pFast-Bac); animal cell expression plasmids (e.g., pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophages such as lambda phage; insect virus vectors such as baculoviruses (e.g., BmNPV, AcNPV); and animal virus vectors such as retroviruses, lentiviruses, vaccinia viruses, adenoviruses, adeno-associated viruses, and herpesviruses. Any promoter can be used as long as it is appropriate for the host used for gene expression. For example, when the host is an animal cell, the SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Molony mouse leukemia virus) LTR, and HSV-TK (herpes simplex virus thymidine kinase) promoter can be used. For other hosts, any well-known promoter can be selected as appropriate.

[0037] In addition to the above, expression vectors containing enhancers, splicing signals, poly(A) addition signals, selection markers, and SV40 replication origins (hereinafter sometimes abbreviated as SV40 ori) can be used as desired. Examples of selection markers include dihydrofolate reductase genes, ampicillin resistance genes, and neomycin resistance genes.

[0038] IL-18 can be produced by transforming a host with an expression vector containing the DNA encoding IL-18 as described above, and then culturing the resulting transformant. Suitable hosts include, for example, Escherichia species, Bacillus species, yeast, insect cells, insects, and animal cells. Suitable mammalian cells include, for example, monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), and dhfr gene-deficient CHO cells (hereinafter abbreviated as CHO(dhfr - Cells such as mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, human FL cells, HeLa cells, HepG2 cells, and HEK293 cells are used. For other hosts, known cells can be appropriately selected.

[0039] Transformation can be carried out according to known methods, depending on the host type. Animal cells can be transformed, for example, according to the methods described in Cell Engineering Supplement 8 New Cell Engineering Experimental Protocols, 263-267 (1995) (published by Shujunsha) and Virology, Vol. 52, 456 (1973).

[0040] The culture of the transformed organism can be carried out according to known methods, depending on the type of host. For example, when culturing transformants whose host is animal cells, suitable culture media include, for instance, minimal essential medium (MEM) containing approximately 5-20% fetal bovine serum, Dulbecco's modified Eagle medium (DMEM), RPMI 1640 medium, 199 medium, and Ham's F-12 medium. The pH of the medium is preferably approximately 6-8. Culturing is usually carried out at approximately 30°C-40°C for approximately 15-60 hours. Aeration and stirring may be performed as needed. In this way, IL-18 can be produced either inside or outside the cells of the transformed organism.

[0041] IL-18 can be separated and purified from the culture obtained by culturing the aforementioned transformants according to known methods. Such methods include methods utilizing solubility, such as salting out and solvent precipitation; methods mainly utilizing differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis; methods utilizing differences in charge, such as ion exchange chromatography; methods utilizing specific affinity, such as affinity chromatography; methods utilizing differences in hydrophobicity, such as reverse-phase high-performance liquid chromatography; and methods utilizing differences in isoelectric point, such as isoelectric focusing electrophoresis. These methods can also be combined as appropriate.

[0042] If the IL-18 thus obtained is in the form of a free substance, it can be converted to a salt by a method known to the present day or a similar method, and if the IL-18 is obtained as a salt, it can be converted to a free substance or another salt by a method known to the present day or a similar method.

[0043] (c) T cell growth factor The T cell growth factors used in the combination agent of the present invention are not particularly limited as long as they are humoral factors that cause proliferation and activation of T cells, including CTLs and Th1 cells. Examples include cytokines such as IL-2, IL-15, IL-7, IL-9, and IL-21, but IL-2, IL-15, and IL-7 are preferred, and IL-2 is more preferred. These T cell growth factors may be used individually or in combination of two or more.

[0044] IL-2 is produced by activated T cells, NK cells, and dendritic cells, and induces the proliferation and activation of tumor-killing cells such as CTLs and NK cells. Human IL-2 is produced as a precursor consisting of 153 amino acids, and the 20-amino acid signal sequence at the N-terminus is cleaved to secrete mature IL-2 consisting of 133 amino acids. The amino acid sequence information of human IL-2 can be found, for example, in UniProtKB (accession number: P60568). The amino acid sequence of mature human IL-2 is shown in Sequence ID No. 4.

[0045] IL-15 is produced by monocytes, macrophages, dendritic cells, etc., and exhibits effects similar to IL-2 on CTLs and NK cells. Human IL-15 is produced as a precursor consisting of 162 amino acids, and the 29-amino acid signal sequence at the N-terminus is cleaved to secrete an inactive pro-isomer. Further cleavage of the 19-amino acid propeptide results in mature IL-15 (active form) consisting of 114 amino acids. The amino acid sequence information for human IL-15 can be found, for example, in UniProtKB (accession number: P40933). The amino acid sequence of mature human IL-15 is shown in Sequence ID No. 6.

[0046] IL-7 is produced by stromal cells, reticular fibroblasts, thymic epithelial cells, etc., and contributes to promoting the early differentiation of T cells and maintaining the quantitative level of peripheral T cells. Human IL-7 is produced as a precursor consisting of 177 amino acids, and the 25-amino acid signal sequence at the N-terminus is cleaved to secrete mature IL-7 consisting of 152 amino acids. The amino acid sequence information of human IL-7 can be found, for example, in UniProtKB (accession number: P13232). The amino acid sequence of mature human IL-7 is shown in Sequence ID No. 8.

[0047] IL-2, IL-15, and IL-7 used as active ingredients in the combination agent of the present invention are proteins containing amino acid sequences identical or substantially identical to the amino acid sequences shown in SEQ ID NOs: 4, 6, and 8, respectively. IL-2, IL-15, and IL-7 may be proteins isolated and purified from cytokine-producing cells (e.g., IL-2-producing cells include T cells, NK cells, NKT cells, activated dendritic cells, mast cells, etc.; IL-15-producing cells include monocytes, macrophages, dendritic cells, etc.; IL-7-producing cells include stromal cells, reticular fibroblasts, thymic epithelial cells, etc.) or tissues containing such cells of humans or other mammals (e.g., rats, mice, monkeys, dogs, cattle, rabbits, pigs, sheep, etc.) or tissues containing such cells. Alternatively, they may be proteins synthesized chemically or biochemically using a cell-free translation system, or recombinant proteins produced from transformants into which nucleic acids having the base sequences encoding the above-mentioned amino acid sequences have been introduced.

[0048] Examples of amino acid sequences that are substantially identical to the amino acid sequences shown in SEQ ID NOs: 4, 6, or 8 include amino acid sequences that have 80% or more identity, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, and most preferably 98% or more, with the amino acid sequences shown in SEQ ID NOs: 4, 6, or 8. Here, "identity" has the same meaning as described above for IL-18.

[0049] IL-2, IL-15, and IL-7 are proteins that contain substantially the same amino acid sequences as those shown in SEQ ID NOs: 4, 6, and 8, respectively, and that have the same activity as the proteins containing the amino acid sequences shown in SEQ ID NOs: 4, 6, and 8. Here, "activity" refers to any activity that contributes to cancer suppression, such as receptor binding activity or activity that promotes the differentiation, proliferation, and activation of T cells and NK cells. Here, "homogeneous" means that their activities are qualitatively the same. Therefore, it is preferable that the activity of IL-2, IL-15, and IL-7 is equivalent to or greater than that of wild-type IL-2, wild-type IL-15, and wild-type IL-7, respectively, but the degree of their activity may differ.

[0050] Alternatively, the IL-2, IL-15, and IL-7 used in the present invention include, for example, (i) amino acid sequences in which one or more amino acids (for example, about 1 to 30, preferably about 1 to 10, more preferably 1 to several (5, 4, 3, or 2)) are deleted from each amino acid sequence shown in SEQ ID NOs: 4, 6, and 8; (ii) amino acid sequences in which one or more amino acids (for example, about 1 to 30, preferably about 1 to 10, more preferably 1 to several (5, 4, 3, or 2)) are added to each amino acid sequence shown in SEQ ID NOs: 4, 6, and 8; and (iii) This also includes amino acid sequences in which one or more amino acids (for example, about 1 to 30, preferably about 1 to 10, and more preferably 1 to several (5, 4, 3, or 2)) are inserted into each amino acid sequence shown in sequence numbers 4, 6, and 8; (iv) amino acid sequences in which one or more amino acids (for example, about 1 to 30, more preferably about 1 to 10, and more preferably 1 to several (5, 4, 3, or 2)) in each amino acid sequence shown in sequence numbers 4, 6, and 8 are replaced with other amino acids; or (v) proteins containing amino acid sequences that combine these.

[0051] As described above, when amino acid sequences are inserted, deleted, or substituted, the location of such insertion, deletion, or substitution is not particularly limited. For example, an IL-2 mutant in which at least one of the residues at position 3 (Thr), position 42 (Phe), position 45 (Tyr), and position 72 (Leu) in the amino acid sequence shown in Sequence ID No. 4 is substituted with another amino acid (e.g., Ala, Ser, Val, etc.), resulting in a mutant in which the affinity of the high-affinity αβγc receptor expressed on Treg cells for the IL-2Rα chain is reduced (see, for example, Japanese Patent Publication No. 2020-33362). For IL-15 and IL-7, known mutants having activity equivalent to or greater than that of the wild type can also be used.

[0052] The IL-2, IL-15, and IL-7 used in this invention may be in free form or as salts. Examples of such salts include those similar to those described above for IL-18.

[0053] IL-2, IL-15, and IL-7 can be isolated from the extracellular matrix or culture supernatant of human and other mammalian cytokine-producing cells or tissues, using known protein purification methods. IL-2, IL-15, and IL-7 can also be produced according to known peptide synthesis methods. The same methods used for protein purification and peptide synthesis as those described above for IL-18 can be used.

[0054] In a preferred embodiment, IL-2, IL-15, and IL-7 can be produced by culturing transformants containing the nucleic acids encoding them and then separating and purifying them from the resulting culture.

[0055] Examples of DNA encoding IL-2, IL-15, and IL-7 include DNA containing the base sequences shown in SEQ ID NOs: 3, 5, and 7, respectively, or DNA containing base sequences that hybridize with the base sequences shown in SEQ ID NOs: 3, 5, and 7 under stringent conditions, and which encodes proteins having the same activity as the wild-type IL-2, IL-15, and IL-7. Examples of DNA that can hybridize with the base sequences shown in SEQ ID NOs: 3, 5, and 7 under stringent conditions include DNA containing base sequences that have 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, and most preferably 98% or more identity with the base sequences shown in SEQ ID NOs: 3, 5, and 7. The identity of the base sequence in this specification can be calculated in the same manner as in the case of DNA encoding IL-18. The DNA encoding IL-2, IL-15, and IL-7 is preferably DNA encoding wild-type human IL-2, IL-15, and IL-7 having the respective base sequences shown in SEQ ID NOs: 3, 5, and 7, or DNA encoding the various human IL-2, IL-15, and IL-7 mutant proteins described above.

[0056] The DNA encoding IL-2, IL-15, and IL-7 can be constructed either by chemically synthesizing the DNA strands, or by connecting partially overlapping oligoDNA short chains using PCR or Gibson Assembly to create the full-length DNA encoding these molecules.

[0057] The cloned DNA can be used as is, or, depending on the purpose, after being digested with restriction enzymes or after linker addition as desired. The DNA may have an ATG translation start codon at its 5' end and a TAA, TGA, or TAG translation stop codon at its 3' end. These translation start and stop codons can be added using a suitable synthetic DNA adapter.

[0058] Expression vectors containing DNA encoding IL-2, IL-15, or IL-7 can be prepared, for example, by excising a target DNA fragment from the DNA encoding IL-2, IL-15, or IL-7, and ligating the DNA fragment downstream of a promoter in a suitable expression vector. For IL-18, the expression vectors and promoters described above are similarly preferred. The expression vector may also optionally contain enhancers, splicing signals, poly(A) addition signals, selection markers, SV40 ori, etc. Examples of selection markers include dihydrofolate reductase genes, ampicillin resistance genes, and neomycin resistance genes.

[0059] IL-2, IL-15, or IL-7 can be produced by transforming a host with an expression vector containing DNA encoding IL-2, IL-15, or IL-7 as described above, and culturing the resulting transformant. The host, transformation method, transformant culture method, purification method for the obtained IL-2 protein, and method for converting from free form to salt or salt to free form are similarly preferred to those described above or the same methods used for IL-18.

[0060] While IL-2, IL-15, and IL-7 have been given as examples of T cell growth factors, those skilled in the art can easily obtain other T cell growth factors based on the descriptions herein.

[0061] (II) The combination agent of the present invention As shown in the examples described below, by using a combination of three agents—an immune checkpoint inhibitor, IL-18, and T-cell growth factor—tumor growth can be significantly suppressed in a spontaneously occurring cancer model mouse, which is a model of refractory cancer for which there are few other drugs that produce therapeutic effects, compared to the combination of an immune checkpoint inhibitor and IL-18, or an immune checkpoint inhibitor and T-cell growth factor. Therefore, the combination agent of the present invention can be used as a cancer treatment agent. Here, "cancer treatment" is used as a concept that encompasses all of the following: suppression of tumor growth, cancer progression, invasion and metastasis, delay of cancer onset, and suppression of recurrence. The combination agent of the present invention can be preferably used in subjects (humans or other mammals, preferably humans) with cancer that is resistant to or unsuitable for existing cancer treatments, including immune checkpoint inhibitor monotherapy or combination therapy of an immune checkpoint inhibitor and IL-18.

[0062] The types of cancers in which the combination agents of the present invention can be used are not particularly limited, and any cancer can be mentioned. For example, it may be cancer of epithelial cell origin, but it may also be non-epithelial sarcoma or hematological cancer. More specifically, it includes, but is not limited to, cancers of the digestive system (e.g., liver cancer (hepatocellular carcinoma, cholangiocarcinoma), gallbladder cancer, bile duct cancer, pancreatic cancer, duodenal cancer, esophageal cancer, stomach cancer, colorectal cancer (colon cancer, rectal cancer), anal cancer), cancers of the urinary system (e.g., kidney cancer, ureteral cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer), cancers of the chest (e.g., breast cancer, lung cancer (non-small cell lung cancer, small cell lung cancer)), cancers of the reproductive system (e.g., uterine cancer (cervical cancer, endometrial cancer), ovarian cancer, vulvar cancer, vaginal cancer), cancers of the head and neck (e.g., maxillary cancer, pharyngeal cancer, laryngeal cancer, tongue cancer, thyroid cancer), and cancers of the skin (e.g., basal cell carcinoma, squamous cell carcinoma).

[0063] The Mdr2 KO mice used in the examples described later develop chronic pericholangiocarcinoma and cholestatic liver disease due to a deficiency in the iAbc4 protein, a member of the ATP-binding cassette (ABC) transporter superfamily encoded by the mdr2 gene, and spontaneously develop hepatocellular carcinoma (Katzenellengoben et al., Mol. Cancer Res. 2007, 5(11): 1159-1170). Therefore, in a preferred embodiment of the present invention, the cancers targeted by the combination agent of the present invention may be inflammation-related cancers, preferably gastrointestinal cancers such as liver cancer, pancreatic cancer, bile duct cancer, gallbladder cancer, and duodenal cancer, which may be caused by chronic inflammation due to digestive fluids such as bile and pancreatic juice, or liver cancer (including metastatic cancers originating from these) resulting from inflammation, fibrosis, and cirrhosis due to viral infections such as hepatitis B and C viruses.

[0064] Immune checkpoint inhibitors, IL-18, and T cell growth factors are low-toxicity and can be administered orally or parenterally (e.g., intravascular (intravenous, intra-arterial, etc.), subcutaneous, intradermal, intraperitoneal, intramuscular, local, etc.) to humans or other mammals as liquid preparations or as pharmaceutical compositions in appropriate dosage forms.

[0065] The combination agent of the present invention may be formulated by separately developing each of the three active ingredients, or two or more of these ingredients may be formulated as single agents.

[0066] For parenteral administration, compositions such as injections and suppositories may be used, and injections may include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip infusions. Such injections can be prepared according to known methods. As a method for preparing injections, for example, one or more active ingredients can be dissolved, suspended, or emulsified in a sterile aqueous solution or oily solution commonly used for injections. As aqueous solutions for injection, for example, physiological saline, isotonic solutions containing glucose or other adjuvants may be used, and may be used in combination with appropriate solubilizers, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduc of hydrogenated castor oil)]. As oily solutions, for example, sesame oil and soybean oil may be used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The prepared injection solution is preferably filled into a suitable ampoule. Suppositories used for rectal administration may be prepared by mixing the active ingredient with a standard suppository base.

[0067] Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. Such compositions may be manufactured by known methods and may contain carriers, diluents, or excipients commonly used in the pharmaceutical field. Examples of carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate.

[0068] The above parenteral or oral pharmaceutical composition is preferably prepared in a dosage unit form suitable for the dosage of the active ingredient. Examples of such dosage unit forms include tablets, pills, capsules, injections (ampoules), and suppositories. The immune checkpoint inhibitor is usually contained at 10 to 10,000 mg, preferably 50 to 1,000 mg, more preferably 100 to 1,000 mg per dosage unit form. IL-18 is usually contained at 1 to 100 mg, preferably 5 to 50 mg per dosage unit form. IL-2 is usually contained at 10 4 ~10 6 units, preferably 5×10 4 ~5×10 5 units per dosage unit form.

[0069] The dosage of each active ingredient also varies depending on the administration subject, target disease, symptoms, administration route, etc. For example, when used for the treatment of adult liver cancer, the immune checkpoint inhibitor is usually about 0.5 to 500 mg / kg body weight, preferably about 2.5 to 50 mg / kg body weight, more preferably about 5 to 50 mg / kg body weight as a single dose, IL-18 is usually about 0.05 to 1 mg / kg body weight, preferably about 0.25 to 2.5 mg / kg body weight as a single dose, and IL-2 is usually 5×10 2 ~5×10 4 units / kg body weight, preferably 2.5×10 3 ~2.5×10 4 units / kg body weight, and it is convenient to administer once a day to once a week, preferably about 2 to 3 times a week, by intravenous or intraperitoneal administration. In the case of other parenteral and oral administrations, amounts corresponding thereto can be administered. When the symptoms are particularly severe, the dosage may be increased according to the symptoms.

[0070] The ratio of active ingredients in the combination agent of the present invention is not particularly limited as long as the dosage of each active ingredient is within the above range. For example, if the dosage of IL-18 is 1, the dosage of the immune checkpoint inhibitor may be about 5 to 200, preferably about 10 to 50. In addition, the dosage of T cell growth factor may be about 0.1 to 1, preferably about 0.2 to 0.5, when calculated at 1 unit = 0.5 ng, relative to the dosage of IL-18.

[0071] If the combination agent of the present invention is provided in the form of separate compositions for each active ingredient, or if any two agents are provided in the form of a single composition and the remaining agent is provided in the form of another composition, each composition can be administered to the subject simultaneously or with a time difference, via the same route or via different routes.

[0072] IL-18BP is expressed in an elevated manner in the tumor microenvironment and can function as a soluble immune checkpoint molecule that binds to IL-18 with high affinity, blocking its binding to IL-18 receptors and signal transduction on effector cells such as T cells and NK cells, thereby suppressing the proliferation and activation of these effector cells. Furthermore, it has been reported that IL-18BP levels are significantly elevated in the serum of cancer patients who have received cytokine therapy with IL-18. As shown in the examples described below, IL-18BP levels are elevated in the serum of spontaneously occurring liver cancer model mice. While two-drug combinations of immune checkpoint inhibitors and IL-18 or IL-2 cannot lower IL-BP levels, the combination agent of the present invention, which combines three drugs, can significantly lower IL-18BP levels. In addition, serum IL-18BP levels in hepatocellular carcinoma patients who have not received IL-18 therapy are significantly higher than those in healthy individuals, but it has become clear that these levels vary considerably among patients. High serum IL-18BP levels are thought to reflect the establishment of an immunosuppressive state by IL-18BP in the tumor microenvironment. Therefore, the combination agent of the present invention, which can reduce IL-18BP expression, is thought to be particularly useful for cancer patients with particularly high serum IL-18BP levels, as it may alleviate IL-18-induced immunosuppression (immune exhaustion of T cells, NK cells, etc.) in such patients. In fact, in the examples described later, although the number of individuals was small, a significant decrease in IL-18BP levels was observed after administration of the three agents in spontaneously developing liver cancer model mice with particularly high serum IL-18BP levels at the start of administration, strongly suggesting the possibility of identifying targets for which the combination agent of the present invention is highly effective using serum IL-18BP levels as an indicator.

[0073] IL-18BP is constitutively expressed even in healthy individuals, and normal serum levels are said to be around a few pg / mL. Therefore, in a preferred embodiment, the combination agent of the present invention can be administered, for example, to cancer patients with serum IL-18BP levels of 10 pg / mL or higher, preferably 20 pg / mL or higher. Serum IL-18BP levels can be measured, for example, by an immunochemical assay (e.g., ELISA) using an anti-IL-18BP antibody, and such ELISA kits are commercially available.

[0074] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0075] Example 1: Mdr2 - / - Combination therapy of immune checkpoint inhibitors and IL-18 and / or IL-2 in knockout mice MDR2 patients who spontaneously developed hepatocellular carcinoma - / - We divided the KO mice (60-64 weeks old, female; obtained from The Jackson Laboratory) into four groups. 1) Anti-mouse PD-L1 antibody (clone 10F.9G2; purchased from BioXcell) 200 μg / mouse, IL-18 (recombinant mouse IL-18; GlaxoSmithKline) 10 μg / mouse, and IL-2 (recombinant IL-2 (Imunase Injection 35); Shionogi & Co., Ltd.) 5,000 units / kg (IL-2 + IL-18 + PD-L1 administration group); 2) Anti-mouse PD-L1 antibody 200 μg / mouse, and IL-2 5,000 units / kg (IL-2 + PD-L1 administration group); 3) Anti-mouse PD-L1 antibody 200 μg / mouse, and IL-18 10 μg / mouse (IL-18 + PD-L1 administration group); or 4) Physiological saline (Control (PBS administration) group); The drug was administered intraperitoneally twice a week for four weeks. The effects of each concomitant drug were examined four weeks after the start of drug administration. The schedule of this experiment is shown in Figure 1.

[0076] (1) Inhibitory effect on tumor growth CT scans (3D micro X-ray CT scanner for experimental animals (R_mCT2-SP2): Rigaku Corporation) were used to acquire CT images of the liver at the start of drug administration and 4 weeks after the start of administration. As a result, the control group showed a tendency for tumor size to increase (Figure 2-1), while the IL-2 + IL-18 + PD-L1 administration group showed a tendency for tumor size to decrease (Figure 3-1). On the other hand, the IL-2 + PD-L1 administration group (Figure 4) and the IL-18 + PD-L1 administration group (Figure 5) showed a tendency for tumor size to increase, making it clear that the combination of an immune checkpoint inhibitor and one of the cytokines does not produce a tumor growth inhibitory effect.

[0077] HE staining of liver tissue sections revealed that in the control group, hepatocellular carcinoma with glandular structures within the liver parenchyma was observed, but inflammatory cell infiltration around the tumor was not evident (Figure 2-2, A-C). On the other hand, in the IL-2+IL-18+PD-L1 administration group, hepatocellular carcinoma with glandular structures and necrotic tissue were observed within the liver parenchyma. Furthermore, inflammatory cell infiltration around the tumor was significant, with lymphocytes and mononuclear cells being observed. In addition, inflammatory cell infiltration was observed within the tumor, and hepatocyte regeneration and lymphocyte infiltration were observed around the necrotic tissue (Figure 3-2, A-C).

[0078] (2) Decreased expression of tumor markers Blood samples were collected from mice in each group four weeks after the start of administration, and serum AFP levels were measured using a standard method. As a result, the IL-2+IL-18+PD-L1 administration group showed a significantly lower AFP level compared to the IL-2+PD-L1 administration group and the IL-18+PD-L1 administration group (Figure 6A). Comparing the data before and after drug administration, the IL-2+IL-18+PD-L1 administration group showed a tendency for AFP levels to decrease with concomitant administration (Figure 6B).

[0079] (3) Enhancement of cytokine production Serum levels of IFN-γ and TNF-α were measured and compared in each drug administration group four weeks after the start of treatment. As a result, while the combination of two drugs (IL-2 + PD-L1 administration group and IL-18 + PD-L1 administration group) did not significantly increase the production of either IFN-γ or TNF-α, the combination of three drugs (IL-2 + IL-18 + PD-L1 administration group) significantly increased the production of both IFN-γ and TNF-α (Figure 7, left). Comparing before and after drug administration, serum levels of both IFN-γ and TNF-α were significantly elevated in the IL-2 + IL-18 + PD-L1 administration group with concomitant administration (Figure 7, right).

[0080] (4) Contribution of T cells and NK cells to the therapeutic effect of triple therapy In addition to IL-2, IL-18, and PD-L1, an antibody against the glycolipid asialoGM1 expressed on the surface of NK cells was administered to eliminate NK cells. Imaging studies before and after drug administration revealed an increase in tumor size (Figure 8). This suggests that NK cells play a role in the tumor growth inhibitory effect of the triple drug combination. Furthermore, when antibodies against CD8 expressed on the surface of killer T cells (CTLs) were administered in addition to IL-2+IL-18+PD-L1 to remove CTLs, it was revealed that tumor size increased as measured by imaging before and after drug administration (Figure 9-1). In addition, when comparing serum AFP, IFN-γ, and TNF-α levels four weeks after the start of administration, the IL-2+IL-18+PD-L1+anti-CD8 antibody administration group showed a significant increase in AFP levels and a significant decrease in IFN-γ and TNF-α levels compared to the IL-2+IL-18+PD-L1 administration group (Figure 9-2). This indicates that CTLs play an important role in the tumor growth inhibitory effect of the triple drug combination, at least through their ability to enhance the production of inflammatory cytokines.

[0081] (5) Relationship with IL-18BP Serum samples were collected from mice in each drug administration group four weeks after the start of administration, and serum IL-18BP levels were measured and compared. The results showed elevated IL-18BP levels in the serum of Mdr2 KO mice, suggesting high expression of IL-18BP in the tumor microenvironment. In combination therapy with two drugs (IL-2+PD-L1 and IL-18+PD-L1), IL-18BP levels could not be reduced. Although the sample size was small, IL-18BP levels actually increased in the IL-2+PD-L1 group. In contrast, IL-18BP levels were significantly reduced in the IL-2+IL-18+PD-L1 group (Figure 10-1, left). Comparing pre- and post-administration data, IL-18BP levels were significantly reduced in the IL-2+IL-18+PD-L1 group with combination therapy (Figure 10-1, right). The IL-18BP expression-reducing effect of the triple therapy was neutralized by the administration of an anti-CD8 antibody (Figure 10-2). This suggests that CTLs play an important role in the IL-18BP expression reduction effect.

[0082] IL-18BP levels were measured in serum collected with informed consent from 69 hepatitis C virus (HCV)-positive hepatocellular carcinoma patients, 8 intrahepatic cholangiocarcinoma patients, and 24 healthy volunteers who visited Tokyo Metropolitan Komagome Hospital. The results revealed that serum IL-18BP levels were significantly elevated in hepatocellular carcinoma patients compared to healthy individuals and intrahepatic cholangiocarcinoma patients (Figure 11-1). Furthermore, differences of up to four times or more in IL-18BP levels were observed among hepatocellular carcinoma patients. On the other hand, when serum IL-18 levels were measured from 69 hepatocellular carcinoma patients and 24 healthy volunteers, serum IL-18 levels were significantly elevated in hepatocellular carcinoma patients compared to healthy individuals (Figure 11-2; results for serum IL-18 levels from 30 chronic hepatitis patients and 15 cirrhosis patients are also shown). However, no correlation was found between serum IL-18 levels and serum IL-18BP levels in hepatocellular carcinoma patients (Figure 11-3). [Industrial applicability]

[0083] The combination agent of the present invention is extremely useful as an effective combination cancer immunotherapy agent for refractory cancers such as liver cancer, particularly cancers for which monotherapy with immune checkpoint inhibitors or combination therapy with other agents is ineffective or unsuitable. Furthermore, by performing companion diagnostics that measure serum IL-18BP levels, it becomes possible to predict cancer patients for whom this combination agent will be more effective.

Claims

1. A cancer treatment agent for cancer resistant to monotherapy with an immune checkpoint inhibitor or combination therapy with an immune checkpoint inhibitor and IL-18, comprising an immune checkpoint inhibitor, interleukin-18 (IL-18), and interleukin-2 (IL-2), wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-PD-1 antibody.

2. The agent according to claim 1, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody.

3. The agent according to claim 1 or 2, administered to subjects whose serum IL-18 binding protein (IL-18BP) level is 10 pg / mL or higher.

4. The agent according to any one of claims 1 to 3, wherein the cancer is an inflammation-related cancer.

5. The agent according to claim 4, wherein the inflammation-related cancer is a gastrointestinal cancer.

6. The agent according to any one of claims 1 to 5, wherein the cancer is a spontaneously occurring type of cancer.

7. The agent according to any one of claims 1 to 6, wherein the cancer is liver cancer.

8. The agent according to any one of claims 1 to 7, wherein the immune checkpoint inhibitor, IL-18, and IL-2 are each formulated separately.

9. The agent according to any one of claims 1 to 7, wherein the agent is formulated by compounding two or more of the immune checkpoint inhibitor, IL-18, and IL-2 as single agents.

10. The agent according to claim 8 or 9, characterized in that the immune checkpoint inhibitor, IL-18, and IL-2 are administered simultaneously or with a time difference.

11. A cancer treatment agent comprising an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is administered simultaneously or with a time delay in combination with interleukin-18 (IL-18) and interleukin-2 (IL-2), characterized in that the immune checkpoint inhibitor is administered monotherapy with an immune checkpoint inhibitor or combination therapy with an immune checkpoint inhibitor and IL-18, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-PD-1 antibody.

12. A cancer treatment agent comprising interleukin-18 (IL-18), characterized in that the interleukin-18 (IL-18) is administered simultaneously or with a time delay in combination with an immune checkpoint inhibitor and interleukin-2 (IL-2), for cancers resistant to monotherapy with an immune checkpoint inhibitor or combination therapy with an immune checkpoint inhibitor and IL-18, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-PD-1 antibody.

13. A cancer treatment agent comprising interleukin-2 (IL-2), characterized in that the interleukin-2 (IL-2) is administered simultaneously or with a time delay in combination with an immune checkpoint inhibitor and interleukin-18 (IL-18), for cancers resistant to monotherapy with an immune checkpoint inhibitor or combination therapy with an immune checkpoint inhibitor and IL-18, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-PD-1 antibody.

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