Cancer therapy using interleukin-26 inhibitor

Abstract

The present invention provides a cancer microenvironment-improving agent containing an interleukin-26 (IL-26) inhibitor. Also provided is a cancer therapeutic agent comprising a combination of an IL-26 inhibitor with at least one drug having antitumor activity, preferably an immune checkpoint inhibitor.

Description

Cancer treatment using interleukin-26 inhibitors

[0001] The present invention relates to cancer treatment using interleukin-26 (IL-26) inhibitors. More specifically, the present invention relates to a therapeutic agent for cancer, preferably inflammation-related cancer, containing an IL-26 inhibitor including an anti-IL-26 antibody; a cancer microenvironment improving agent containing an IL-26 inhibitor, preferably an immune checkpoint inhibitor (ICI) resistance improving agent; and a therapeutic agent for cancer, preferably ICI-resistant cancer, comprising a combination of an IL-26 inhibitor and an ICI.

[0002] Inflammation and immune responses are biological defense mechanisms to eliminate invading pathogenic microorganisms and foreign substances from the body. However, persistent chronic inflammation is known to accumulate damage to cells, leading to autoimmune diseases, allergies, and the development and progression of cancer. Reactive oxygen species generated during the inflammatory response damage DNA and proteins, causing mutations in oncogenes and tumor suppressor genes, and promoting the cancerous transformation of cells. In chronic inflammation, the expression of cytokines, chemokines, growth factors, angiogenic factors, and proteases is increased, leading to malignant progression such as cancer growth, invasion, and metastasis. Furthermore, persistent chronic inflammation can suppress anti-tumor immunity and lead to drug resistance. In fact, patients with ulcerative colitis and Crohn's disease, which chronically experience relapses and remissions of colitis, are known to have a high risk of developing colorectal cancer. It is also known that chronic hepatitis caused by hepatitis C virus infection increases the risk of hepatocellular carcinoma, and Helicobacter pylori infection increases the risk of gastric cancer. Thus, there are cancer types that are strongly associated with chronic inflammation and infection.

[0003] IL-26 is an inflammatory cytokine mainly produced by Th17 cells, and increased expression has been reported at the sites of inflammation in various inflammatory diseases such as psoriasis, inflammatory bowel disease, and rheumatoid arthritis. It is considered that IL-26 contributes to the exacerbation of inflammation in these diseases. On the other hand, almost no reports have been made on the relationship between cancer and IL-26. The inventors of the present invention have found that IL-26 is expressed around cancer cells of refractory triple-negative breast cancer, and also that, using bacteria artificial chromosome transgenic (hIL-26BACTg) mice containing the human IL-26 gene, IL-26 activates bypass signals even in the presence of an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), and is involved in the acquisition of EGFR-TKI resistance (Patent Document 1, Non-Patent Document 1).

[0004] However, the role of IL-26 in inflammation-related cancer and its influence on tumor immunity still remain unclear.

[0005] By the way, for immune checkpoint inhibitor (ICI) therapies such as anti-PD-1 / anti-PD-L1 antibodies to exert an anti-cancer effect, it is a prerequisite that immune cells such as T cells infiltrate into the cancer local area, and such cancers are called hot tumors. However, in the state of cold tumors where immune cells do not exist in the cancer local area or exist only around the cancer tissue, the therapeutic effect of ICI alone cannot be expected. Therefore, by destroying some of the cancer cells with chemotherapeutic agents, molecular target drugs, radiation therapy, etc., it is necessary to promote the release of cancer antigens and intracellular components, etc., induce inflammation locally, promote the infiltration of immune cells into the cancer tissue, and improve the cancer microenvironment from a cold tumor to a hot tumor state. The basis of chemotherapeutic agent treatment is administration of the maximum tolerable dose that the patient can withstand. However, since the effect of ICI treatment is borne by host immune cells, especially T cells, administration of the maximum tolerable dose of anti-cancer agents is inconvenient for subsequent ICI treatment. Therefore, there is a need for a means to improve the cancer microenvironment that converts cold tumors to hot tumors while minimizing the decrease in the patient's immunity.

[0006] Furthermore, as mentioned above, chronic inflammation caused by autoimmune diseases and chronic viral infections is widely known to be a risk factor for carcinogenesis and cancer progression. On the other hand, in cancers in which cytotoxic T cells infiltrate, i.e., hot tumors, ICI treatment is highly effective. Thus, there are both harmful inflammations that induce and promote cancer and beneficial inflammations that eliminate cancer. However, it remains completely unclear whether IL-26 is involved in the inflammatory state in the tumor microenvironment, and if so, how it relates to it.

[0007] WO 2021 / 251340

[0008] Cell Death Dis. 2021; 12: 520.

[0009] The objective of the present invention is to identify key factors that promote immune evasion in the tumor microenvironment and to provide novel and groundbreaking cancer therapeutics that target these factors.

[0010] The inventors focused on IL-26 as a candidate factor that promotes cancer-related inflammation and created a drug-induced colitis-associated colorectal cancer model using hIL-26BACTg mice expressing human IL-26. They then compared the onset and progression of colorectal cancer in this model with that of a similar model created using wild-type mice. The results showed that hIL-26BACTg mice formed significantly more polyps than wild-type mice. Polyps in hIL-26BACTg mice showed significantly greater neutrophil infiltration, indicating strong induction of cancer-related inflammation. In hIL-26BACTg mice, the number of polyps formed by AOM / DSS was significantly reduced by monotherapy with a humanized anti-IL-26 antibody, and colitis was also reduced. Neutrophil infiltration into polyps was also significantly suppressed.

[0011] Furthermore, the inventors created a tumor-bearing model by subcutaneously transplanting a mouse colorectal cancer cell line with properties similar to human mismatch repair deficiency (MMRd) / high microsatellite instability (MSI-High) colorectal cancer, which is known to be highly effective with ICI, into mice, and analyzed the antitumor effect of the anti-PD-1 antibody. As a result, while tumor growth was significantly suppressed in wild-type mice, the tumor size was significantly increased in hIL-26BACTg mice compared to wild-type mice. When comparing the proportion of neutrophils and CD8-positive T cells infiltrating the tumors in hIL-26BACTg mice with that of wild-type mice, neutrophil infiltration was significantly higher in hIL-26BACTg mice, while CD8-positive T cell infiltration was very low. Therefore, when a humanized anti-IL-26 antibody was administered along with an anti-PD-1 antibody to the hIL-26BACTg mouse subcutaneous tumor model, tumor growth was significantly suppressed and neutrophil infiltration into the tumor was greatly inhibited compared to ICI monotherapy, while CD8-positive T cell infiltration was significantly increased. Furthermore, no adverse events, including immune-related adverse events which are typical side effects of ICI, were observed with concomitant administration.

[0012] Based on the above, the inventors have revealed that IL-26 is deeply involved in the process of carcinogenesis and progression of inflammation-related cancers, and that IL-26 promotes neutrophil infiltration into tumors, and that infiltrated neutrophils in the tumor microenvironment reduce tumor-attacking immune cells, suppress their activation, and promote immune evasion, thereby promoting tumor growth and inducing resistance to ICIs. In other words, IL-26 changes the tumor microenvironment from a hot tumor state to a cold tumor state. Furthermore, they demonstrated that even monotherapy with an IL-26 inhibitor can produce a therapeutic effect by suppressing the process leading to carcinogenesis and cancer progression in inflammation-related cancers, and that inhibiting IL-26 and changing the tumor microenvironment from a cold tumor state to a hot tumor state can increase the ICI sensitivity of cancers that are resistant to ICIs. Based on these findings, the inventors conducted further studies and have now completed the present invention.

[0013] In other words, the present invention provides the following: [Claim 1] A tumor microenvironment improving agent containing an interleukin-26 (IL-26) inhibitor. [Claim 2] The agent according to Claim 1, wherein the improvement of the tumor microenvironment is an increase in the cancer's sensitivity to immune checkpoint inhibitors (ICIs). [Claim 3] The agent according to Claim 1 or 2, wherein the cancer is an ICI-resistant cancer. [Claim 4] The agent according to any one of Claims 1 to 3, wherein the cancer is an inflammation-related cancer. [Claim 5] The agent according to any one of Claims 1 to 4, wherein the cancer is colorectal cancer. [Claim 6] The agent according to any one of Claims 1 to 5, wherein the IL-26 inhibitor is a substance that inhibits the interaction between IL-26 and molecules involved in downstream signaling. [Claim 7] The agent according to Claim 6, wherein the IL-26 inhibitor is an anti-IL-26 antibody. [Claim 8] The agent according to any one of Claims 1 to 7, administered alone as a cancer treatment agent. [Clause 9] The agent according to any one of Claims 1 to 7, used in combination with at least one drug having antitumor activity. [Clause 10] The agent according to Claim 9, wherein the drug is an ICI. [Clause 11] A cancer treatment agent comprising an interleukin-26 (IL-26) inhibitor and at least one drug having antitumor activity. [Clause 12] The agent according to Claim 11, wherein the IL-26 inhibitor is an anti-IL-26 antibody. [Clause 13] The agent according to Claim 11 or 12, wherein the drug is an ICI. [Clause 14] The agent according to Claim 13, wherein the ICI is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. [Clause 15] The agent according to any one of Claims 11 to 14, wherein the IL-26 inhibitor and at least one drug having antitumor activity are formulated separately. [Item 16] The agent according to any one of items 11 to 14, wherein the IL-26 inhibitor and the drug having antitumor activity are in the form of a combination agent.

[0014] According to the present invention, the use of IL-26 inhibitors can suppress the process leading to cancer development and the progression of inflammation-related cancers, and therapeutic effects can be achieved even with monotherapy. Furthermore, IL-26 inhibitors can change the tumor microenvironment from a cold tumor state to a hot tumor state, and can also suppress the change from a hot tumor state to a cold tumor state. In other words, the infiltration of tumor immunosuppressive neutrophils into the tumor site is suppressed, while the number of tumor-attacking immune cells such as infiltrating T cells increases and their activation is promoted. As a result, the sensitivity of cancer to ICIs is increased by using IL-26 inhibitors, and the antitumor activity can be further enhanced by combining IL-26 inhibitors with ICIs.

[0015] This figure shows the schedule for a colitis-associated colorectal cancer model. Azoximethane (AOM) was administered intraperitoneally to 200 μg per mouse each to bacterial artificial chromosome transgenic mice containing the human IL-26 gene (hIL-26BACTg) (abbreviated as "hIL-26Tg" in the figures) and wild-type (WT) mice lacking the IL-26 gene. Seven days later, the mice were given 2.0% dextran sulfate sodium (DSS) water ad libitum for 5 days, then switched to normal drinking water for 14 days. This cycle of 5 days of 2.0% DSS water intake was repeated for 3 cycles. The number of polyps throughout the entire colon and the number of immune cells infiltrating the tumors were then analyzed. This figure shows the number of polyps throughout the entire colon in the colitis-associated colorectal cancer model. Following the schedule shown in Figure 1A, after 3 cycles of AOM administration and DSS water / normal water intake, the colons of the mice were collected and the number of polyps was counted (n=12 in each group). The data are shown as the mean ± standard deviation of the results for each individual in each group (*p<0.01 between groups shown in the figure). This figure shows the percentage of neutrophils infiltrating tumors in a colitis-associated colorectal cancer model. Following the schedule shown in Figure 1A, AOM administration and DSS water / normal water intake were repeated for 3 cycles, then polyps were collected from the colon of mice, and the percentage of neutrophils (CD11b-positive, Ly6G-positive) among CD45-positive leukocytes was analyzed using flow cytometry (n=5 for each). The data are shown as the mean ± standard deviation of the results for each individual in each group (*p<0.01 between groups shown in the figure). This figure shows the administration schedule of humanized anti-IL-26 antibody in a colitis-associated colorectal cancer model. Colorectal cancer was induced by repeating AOM administration and DSS water / normal water intake for 3 cycles, similar to Figure 1A. According to the schedule shown in the figure, 100 μg each of humanized anti-IL-26 antibody or control human IgG was administered intraperitoneally to bacterial artificial chromosome transgenic (hIL-26Tg) mice containing the human IL-26 gene. The number of polyps throughout the entire length of the colon was counted, and immune cells infiltrating the tumors were analyzed. The figure shows the antitumor effect of humanized anti-IL-26 antibody in a colitis-associated colorectal cancer model.In the schedule shown in Figure 1D, antibody administration was performed during a period of three cycles of AOM administration and intake of DSS water / normal water. The colon of each mouse was collected and the number of polyps was counted (n=8 in each group). The data are shown as the mean ± standard deviation of the results for each individual in each group (*p<0.01 between groups shown in the figure). This figure shows the effect of humanized anti-IL-26 antibody administration on the proportion of tumor-infiltrating neutrophils in a colitis-associated colorectal cancer model. In the schedule shown in Figure 1D, antibody administration was performed during a period of three cycles of AOM administration and intake of DSS water / normal water. Polyps were collected from the colon of each mouse, and the proportion of neutrophils (CD11b-positive, Ly6G-positive) among CD45-positive leukocytes was analyzed using flow cytometry (n=5 in each group). The data are shown as the mean ± standard deviation of the results for each individual in each group (*p<0.01 between groups shown in the figure). This figure shows the time course of tumor size in a mouse colorectal cancer cell line-bearing model of mice, comparing bacterial artificial chromosome transgenic (hIL-26Tg) mice containing the human IL-26 gene with wild-type (WT) mice lacking the IL-26 gene. MC38 cells, a mouse colorectal cancer cell line, were seeded subcutaneously in the flank of each mouse, and tumor size was measured over time (n=8 in each group). Data are shown as the mean ± standard deviation of the results for each individual in each group (NS indicates no significant difference between the groups shown in the figure). This figure also shows the difference in the antitumor effect of anti-PD-1 antibodies against hIL-26Tg mice and WT mice in a mouse colorectal cancer cell line-bearing model. MC38 cells were seeded subcutaneously in the flank of each mouse (day 0), and 100 μg of anti-mouse PD-1 antibody was administered intraperitoneally to each mouse at 7, 10, and 14 days later. Tumor size was measured over time (n=5 in each group). The data are shown as the mean ± standard deviation of the results for each individual in each group (*p<0.01 between groups shown in the figure). This figure shows the difference in the effect of anti-PD-1 antibody on tumor-infiltrating immune cells in hIL-26Tg mice and WT mice in a mouse colorectal cancer cell line tumor model. Similar to Figure 2B, MC38 cells were seeded subcutaneously in the flank of each mouse, and then anti-mouse PD-1 antibody was administered intraperitoneally.Tumors were collected from the flank 17 days after seeding (day 17), and the proportion of neutrophils (CD11b-positive, Ly6G-positive) among CD45-positive leukocytes was analyzed using flow cytometry (n=5 for each group). The data are shown as the mean ± standard deviation of the results for each individual in each group (*p<0.01 between groups shown in the figure). This figure shows the difference in the effect of anti-PD-1 antibody on tumor-infiltrating immune cells in hIL-26Tg mice and WT mice in a mouse colorectal cancer cell line-bearing tumor model. Similar to Figure 2B, MC38 cells were seeded subcutaneously in the flank of each mouse, and then anti-mouse PD-1 antibody was administered intraperitoneally. Tumors were collected from the flank 17 days after seeding (day 17), and the proportion of CD8 T cells (TCRβ-positive, CD4-negative, CD8a-positive) among CD45-positive leukocytes was analyzed using flow cytometry (n=5 for each group). The data are shown as the mean ± standard deviation of the results for each individual in each group (*p<0.01 between groups shown in the figure). This figure shows the combined effect of anti-PD-1 antibody and humanized anti-IL-26 antibody against hIL-26Tg mice in a mouse colorectal cancer cell line tumor model. MC38 cells were seeded subcutaneously in the flank of hIL-26Tg mice (day 0), and at 7, 10, and 14 days thereafter, 100 μg of anti-mouse PD-1 antibody was administered intraperitoneally to each mouse, along with 100 μg each of humanized anti-IL-26 antibody or control human IgG. Tumor size was measured over time (n=6 in each group). The data are shown as the mean ± standard deviation of the results for each individual in each group (*p<0.01 between groups shown in the figure). This figure shows the effects of co-administration of anti-PD-1 antibody and humanized anti-IL-26 antibody on tumor-infiltrating immune cells in hIL-26Tg mice in a mouse colorectal cancer cell line tumor model. Similar to Figure 2E, MC38 cells were seeded subcutaneously in the flank of hIL-26Tg mice, and then anti-mouse PD-1 antibody and humanized anti-IL-26 antibody or control human IgG were administered intraperitoneally in combination. Tumors were collected from the flank 21 days after seeding (day 21), and the percentage of neutrophils (CD11b-positive, Ly6G-positive) among CD45-positive leukocytes was analyzed using flow cytometry (n=6 for each).The data are shown as the mean ± standard deviation of the results for each individual in each group (*p<0.01 between groups shown in the figure). This figure shows the effect of co-administration of anti-PD-1 antibody and humanized anti-IL-26 antibody on tumor-infiltrating immune cells in hIL-26Tg mice in a mouse colorectal cancer cell line tumor model. Similar to Figure 2E, MC38 cells were seeded subcutaneously in the flank of hIL-26Tg mice, and then anti-mouse PD-1 antibody and humanized anti-IL-26 antibody or control human IgG were administered intraperitoneally in combination. Tumors were collected from the flank 21 days after seeding (day 21), and the proportion of CD8 T cells (TCRβ-positive, CD4-negative, CD8a-positive) among CD45-positive leukocytes was analyzed using flow cytometry (n=6 for each group). The data are shown as the mean ± standard deviation of the results for each individual in each group (*p<0.01 between groups shown in the figure).

[0016] The present invention provides a tumor microenvironment improving agent containing an interleukin-26 (IL-26) inhibitor (hereinafter also referred to as "the improving agent of the present invention").

[0017] In this specification, "improvement of the tumor microenvironment" means altering the tumor microenvironment to suppress immune evasion at the site of the tumor. More specifically, it means suppressing the infiltration of neutrophils into the tumor, promoting the infiltration of tumor-attacking immune cells, including T cells, and promoting their activation. In other words, "improvement of the tumor microenvironment" in this invention means changing from an immune-evading cancer (cold tumor) state to a cancer with enhanced tumor immunity (hot tumor) state, and also includes suppressing the immune evasion mechanism by the cancer that attempts to change from a hot tumor to a cold tumor state, thereby maintaining the hot tumor state.

[0018] For ICI therapy to exert its antitumor effect, it is a prerequisite that tumor-attacking immune cells such as T cells infiltrate the tumor site (i.e., it is in a hot tumor state). Therefore, "improvement of the tumor microenvironment" in this invention can also be understood as an increase in the cancer's sensitivity to ICI. Here, "increased sensitivity to ICI" may include not only a change from ICI non-responsiveness to ICI responsiveness, and a change from low ICI sensitivity to high ICI sensitivity, but also the suppression of changes from ICI responsiveness to ICI non-responsiveness, and from high ICI sensitivity to low ICI sensitivity, that is, the suppression of the acquisition of resistance to ICI.

[0019] The active ingredient in the present invention's improving agent, "IL-26 inhibitor," refers to a substance that inhibits the function or expression of IL-26. IL-26 is a cytokine belonging to the IL-10 family, and human IL-26 consists of 150 amino acids as a mature polypeptide. The amino acid sequence of human IL-26 is registered in the NCBI database with accession number NP_060872. IL-26 binds to a heterodimer consisting of IL-20Rα and IL-10Rβ as a receptor and activates cells via phosphorylation of STAT3. The inventors previously reported that Ephrin type-A receptor 3 (EphA3), expressed in triple-negative breast cancer (TNBC), is involved in the transmission of downstream signals of IL-26, and that this downstream signal bypasses the signal mediated by EGFR tyrosine kinase, thereby contributing to the acquisition of resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in TNBC (see Patent Document 1 and Non-Patent Document 1).

[0020] In the present invention, "substances that inhibit the function of IL-26" can be any substance as long as it suppresses the function of IL-26, once functionally produced, that promotes cancer-related inflammation. Preferably, however, it is a substance that inhibits the interaction between IL-26 and molecules involved in downstream signaling (e.g., IL-26 receptor, EphA3; hereinafter, these may be collectively referred to as "receptors, etc."). For example, substances that inhibit the function of IL-26 include, but are not limited to, antagonists of IL-26 or its receptors, etc. Examples of antagonists for IL-26 or its receptors include antibodies against IL-26, fragments of soluble IL-26 receptors (e.g., the extracellular domain of IL-20Rα, the extracellular domain of IL-10Rβ, or a complex thereof, or the extracellular domain of EphA3), aptamers against IL-26, antibodies against IL-26 receptors (e.g., IL-20Rα, IL-10Rβ, or a complex thereof, or EphA3), aptamers against IL-26 receptors, and dominant-negative forms of IL-26.

[0021] In one preferred embodiment, the substance that inhibits the function of IL-26 may be an antibody against IL-26. The antibody may be either a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to known methods for producing antibodies or antisera. The isotype of the antibody is not particularly limited, but preferably IgG, IgM, or IgA, and particularly preferably IgG. Furthermore, the antibody is not particularly limited as long as it has at least a complementarity-determining region (CDR) for specifically recognizing and binding to IL-26, and may be a complete antibody molecule, or a derivative thereof modified with a protein-stabilizing molecule such as polyethylene glycol (PEG), for example, fragments such as Fab, Fab', F(ab')2, genetically engineered conjugate molecules such as scFv, scFv-Fc, minibody, diabody, etc.

[0022] In one preferred embodiment, since the antagonist antibody against IL-26 is used as a pharmaceutical drug intended for human administration, the antibody (preferably a monoclonal antibody) is an antibody with reduced risk of exhibiting antigenicity when administered to humans, specifically, a fully human antibody, a humanized antibody, a mouse-human chimeric antibody, etc., and a humanized antibody or a fully human antibody is particularly preferred. Humanized antibodies and chimeric antibodies can be produced genetically engineered according to conventional methods. Furthermore, while fully human antibodies can also be produced from human-human (or mouse) hybridomas, it is preferable to produce them using human antibody-producing mice or phage display methods.

[0023] In one embodiment, as antagonist antibodies against IL-26, the inventors may use clones 69-10, 20-3, 31-4, and 2-2 antibodies (Patent No. 7061750) produced from hybridomas deposited with the National Institute of Technology and Evaluation (NITE) Patent Microorganism Depository Center (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture) under accession numbers NITE P-02577, NITE P-02578, NITE P-02579, and NITE P-02580, respectively, or antibodies having complementarity-determining regions for their heavy and light chains, or antibodies having variable regions for their heavy and light chains. Similarly, variants and modifications of these antibodies are also preferably used. Such variants and modifications may, for example, be those described in Patent Document 1, and can be manufactured by conventional methods.

[0024] In another preferred embodiment, the substance that inhibits the function of IL-26 may be a fragment of a soluble IL-26 receptor. Examples of receptors that interact with IL-26 include IL-20Rα, IL-10Rβ, their complex, or EphA3. These receptors are single-pass transmembrane proteins, and their extracellular domain fragments (i.e., soluble fragments) interact with IL-26 to inhibit IL-26 from interacting with cell surface receptors and transmitting signals downstream. Since IL-20Rα or IL-10Rβ are common receptors for other IL-10 family cytokines, it may be preferable to use a complex of the extracellular domains of IL-20Rα and IL-10Rβ as the soluble fragment. The amino acid sequences of human IL-20Rα, IL-10Rβ, and EphA3 are registered in the NCBI database with accession numbers NP_055247, NP_000619, and NP_005224, respectively. Based on this sequence information, soluble IL-26 receptors and other recombinant products can be designed and manufactured.

[0025] In the present invention, "substances that inhibit IL-26 expression" may act at any stage of the IL-26 gene process, such as at the transcription level, post-transcriptional regulation level, protein translation level, or post-translational modification level. Therefore, examples of substances that inhibit IL-26 expression include substances that inhibit the transcription of the IL-26 gene (e.g., antigens), substances that inhibit the processing of early transcripts to mRNA, substances that inhibit the transport of mRNA to the cytoplasm, substances that inhibit the translation of mRNA to protein (e.g., antisense nucleic acids, miRNAs) or degrade mRNA (e.g., siRNAs, ribozymes, miRNAs), and substances that inhibit post-translational modification of early translation products. Substances that act at any stage can be used, but substances that bind complementaryally to mRNA to inhibit protein translation or degrade mRNA are preferred.

[0026] In one preferred embodiment, the substance that inhibits IL-26 expression is an siRNA or shRNA against IL-26, or an antisense nucleic acid against IL-26. These nucleic acid molecules can be easily searched for based on the sequence information of IL-26 mRNA (for example, the nucleotide sequence of human IL-26 is registered in the NCBI database as accession number NM_018402), for example, using search software that is freely available on various websites.

[0027] The "cancer" targeted by the improving agent of the present invention is not particularly limited and is applicable to all cancers. In this specification, "cancer" and "tumor" are used interchangeably and refer to cells that have undergone malignant transformation to be pathogenic to the host. Cancer may be primary cancer or metastatic cancer. Examples of cancers include, but are not limited to, colorectal cancer (e.g., colon cancer, rectal cancer), pancreatic cancer, liver cancer, stomach cancer, cervical cancer, head and neck cancer, esophageal cancer, gallbladder and bile duct cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, bone and soft tissue sarcoma, malignant lymphoma, leukemia, multiple myeloma, skin cancer, and brain tumors.

[0028] In one preferred embodiment, the improving agent of the present invention improves the tumor microenvironment in colorectal cancer. In human colorectal cancer tissue, IL-26 is highly expressed compared to normal colorectal tissue, and single-cell RNA-seq analysis revealed that it is particularly highly expressed in T cells and neutrophil-like myeloid-derived immunosuppressive cells (MDSCs). T cells that infiltrate the tumor site play a central role in antitumor immunity, and it is thought that IL-26 secreted from activated T cells migrates and recruits immunosuppressive neutrophils and MDSCs, inducing T cell elimination and apoptosis. Therefore, colorectal cancer can be a suitable therapeutic target for the improving agent of the present invention, which contains an IL-26 inhibitor as its active ingredient.

[0029] In another preferred embodiment, the improving agent of the present invention improves the tumor microenvironment in mismatch repair deficiency (MMRd) or high microsatellite instability (MSI-high) cancers, preferably MMRd / MSI-high colorectal cancer. MMRd / MSI-high cancers have many mutations and many cancer antigens (high immunogenicity), and therefore many T cells accumulate around the tumor, resulting in a high response rate to ICIs. However, when IL-26 is overexpressed locally in the tumor, neutrophil infiltration is promoted while T cell infiltration is suppressed, and the activation of infiltrated T cells is also suppressed, so the inherent high sensitivity to ICIs is thought to decrease. By inhibiting the antitumor immunosuppressive effect of IL-26 with an IL-26 inhibitor, the high sensitivity to ICIs in MMRd / MSI-high cancers can be restored. Whether the cancer targeted for treatment is MMRd / MSI-high can be determined by examining the expression of four MMR proteins (MLH1, PMS2, MSH2, MSH6) in a cancer biopsy sample using commercially available test kits (e.g., MSI test kit (FALCO) and mismatch repair deficiency detection kit (Roche Diagnostics)) by immunohistochemistry. If the expression of one or more proteins is absent (MMRd), and if two or more MSI markers (BAT-26, NR-21, BAT-25, MONO-27, NR-24) are detected by PCR, this can be determined by whether two or more MSI markers are positive (MSI-high).

[0030] In one preferred embodiment, the cancer targeted by the improving agent of the present invention may be a cancer that exhibits resistance to ICI (low sensitivity to ICI). As a mechanism for acquiring resistance to ICI, when T cells infiltrating the tumor are activated by ICI, IL-26 expression is induced. IL-26 secreted from T cells causes the production of large amounts of chemokines that attract neutrophils to cancer cells, increasing the number of neutrophils in the tumor. This suppresses the infiltration and activation of CD8-positive T cells (CTLs), thereby reducing the antitumor effect of ICI. Therefore, by blocking signal transduction from IL-26 through the action of an IL-26 inhibitor, neutrophil infiltration into the tumor site can be suppressed, and the infiltration of tumor-attacking immune cells, including CTLs, can be promoted. This activates the immune cells infiltrated by ICI, thereby increasing the sensitivity of ICI-resistant cancer to ICI.

[0031] Whether or not a target cancer is ICI-resistant can be tested, for example, by culturing a biopsy sample containing cancer cells taken from the target cancer in the presence of ICI to see if the proliferation of cancer cells can be suppressed, or by administering ICI to an immunodeficient animal to which the biopsy sample has been transplanted and seeing if the tumor size increases. Alternatively, it can be tested using other methods well known in the art. For example, in the tumor site of ICI-resistant cancer, it is thought that neutrophil infiltration is promoted while the infiltration of T cells such as CTLs is suppressed, and / or infiltrated T cells are eliminated. Therefore, for example, the sensitivity of the target cancer to ICI can be predicted by analyzing the cells contained in the cancer biopsy by flow cytometry to determine the proportion of neutrophils and / or T cells (e.g., CD8-positive T cells) in the white blood cells. Alternatively, the sensitivity of the target cancer to ICI can also be predicted by measuring the expression level of IL-26 by subjecting the cancer biopsy sample to quantitative RT-PCR or single-cell RNA-seq analysis.

[0032] In a preferred embodiment, the "cancer" targeted by the improving agent of the present invention may be inflammation-related cancer. In this specification, "inflammation-related cancer" means cancer induced by chronic inflammation or recurrent acute inflammation. Examples of inflammation-related cancers include, but are not limited to, colorectal cancer induced by inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, gastric cancer induced by chronic gastritis (e.g., due to Helicobacter pylori infection), liver cancer induced by chronic hepatitis (e.g., hepatitis B, hepatitis C, drug-induced hepatitis), pancreatic cancer induced by chronic pancreatitis, and cervical cancer and head and neck cancer induced by chronic inflammation of the cervix or head and neck due to HPV infection. In a preferred embodiment, the improving agent of the present invention improves the tumor microenvironment in enteritis-related colorectal cancer.

[0033] IL-26 inhibitors have 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.

[0034] IL-26 inhibitors are preferably prepared as pharmaceutical compositions according to conventional methods. Furthermore, the improving agent of the present invention may optionally contain pharmaceutically acceptable carriers and / or additives. For example, it may contain surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), colorants, flavorings, preservatives, stabilizers, buffers (phosphoric acid, citric acid, other organic acids, etc.), chelating agents (EDTA, etc.), suspending agents, isotonic agents, binders, disintegrants, lubricants, flow enhancers, flavoring agents, etc. However, the pharmaceutical composition is not limited to these and may appropriately contain other commonly used carriers. Specifically, examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, etc. It may also contain other low molecular weight polypeptides, proteins such as serum albumin, gelatin, and immunoglobulins, as well as amino acids. For aqueous solutions for injection, the IL-26 inhibitor is dissolved in an isotonic solution containing, for example, physiological saline, glucose, or other adjuvants. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, and sodium chloride. Furthermore, it may be used in combination with appropriate solubilizers, such as alcohol (ethanol, etc.), polyalcohols (propylene glycol, PEG, etc.), and nonionic surfactants (polysorbate 80, HCO-50).

[0035] Furthermore, polypeptides can be encapsulated in microcapsules (such as hydroxymethylcellulose, gelatin, or poly[methylmethacrylate] microcapsules) or incorporated into colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, etc.) as needed (see Remington's Pharmaceutical Science 16th edition &, Oslo Ed. (1980), etc.).

[0036] The content of the IL-26 inhibitor in the pharmaceutical composition is, for example, about 0.01 to 100% by weight of the total pharmaceutical composition, preferably 0.1 to 99.9% by weight.

[0037] The above pharmaceutical composition can be administered orally or parenterally, but parenterally is preferred. Specifically, it is administered to patients by injection and transdermal administration. Examples of injectable formulations include, for example, intravenous injection, intramuscular injection, or subcutaneous injection, which can be administered systemically or locally. Local injection, particularly intramuscular injection, may be administered to or around the treatment site. Examples of transdermal formulations include, for example, ointments, gels, creams, patches, and transdermal patches, which can be administered systemically or locally. Furthermore, the method of administration can be appropriately selected depending on the patient's age and symptoms. As for the dosage, for example, it is possible to select a range of 0.5 mg to 10 mg of IL-26 inhibitor per kg of body weight per dose. However, the improving agent of the present invention is not limited to these dosages.

[0038] The improving agent of the present invention contains an IL-26 inhibitor as an active ingredient and is intended to improve the tumor microenvironment in the target of administration. Surprisingly, it can suppress tumor growth on its own without being used in combination with other cancer treatments (e.g., ICIs). That is, the improving agent of the present invention can be administered alone to a subject with cancer as a cancer treatment agent. In this specification, "cancer treatment" is used as a concept that encompasses all of the following: suppression of tumor growth, cancer progression, invasion and metastasis, prevention and delay of carcinogenesis, and prevention of recurrence. Accordingly, in one embodiment, the present invention provides a cancer treatment agent containing an IL-26 inhibitor, characterized in that it is not used in combination with other drugs having antitumor activity. The cancers that exhibit therapeutic effects with the monotherapy of the improving agent of the present invention are not particularly limited, but preferably cancers in which IL-26 expression is upregulated in the tumor microenvironment (e.g., colorectal cancer), or inflammation-related cancers (e.g., colitis-related colorectal cancer).

[0039] The improving agent of the present invention can improve the tumor microenvironment and enhance the antitumor effect of other drugs having antitumor activity. Therefore, in preferred embodiments, it is used in combination with at least one drug having antitumor activity. The drugs used in combination are not particularly limited as long as they have antitumor activity, and examples include well-known chemotherapeutic agents, molecular targeted drugs, ICIs, etc. However, since the improving agent of the present invention inhibits the function of IL-26 in the tumor microenvironment, suppresses the infiltration of immunosuppressive neutrophils, and promotes the infiltration and activation of immune cells such as T cells that are responsible for host antitumor immunity, it is more preferable to use it in combination with an ICI that promotes the activation of T cells that have infiltrated the tumor site.

[0040] As an ICI, there are no particular limitations 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 a 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.

[0041] 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.

[0042] 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, heavy chain antibodies, or derivatives thereof modified with molecules that have protein-stabilizing effects, such as polyethylene glycol (PEG).

[0043] In a preferred embodiment, since the antibody against an immune checkpoint molecule or its ligand is used as a pharmaceutical product for human administration, the antibody (preferably a monoclonal antibody) is an antibody with a reduced risk of showing antigenicity when administered to humans, specifically, a fully human antibody, a humanized antibody, a mouse-human chimeric antibody, etc., and particularly preferably a fully human antibody. Humanized antibodies and chimeric antibodies can be produced genetically engineered according to conventional methods. Also, although fully human antibodies can be produced from human-human (or mouse) hybridomas, in order to stably and cost-effectively provide a large amount of antibodies, it is desirable to produce them using human antibody-producing mice or phage display methods.

[0044] Some monoclonal antibodies against immune checkpoint molecules or their ligands have already been marketed as pharmaceutical products or are in clinical trials and can be used. For example, as anti-PD-1 antibodies, nivolumab (Opdivo (registered trademark)), pembrolizumab (Keytruda (registered trademark)), AMP-514 (MEDI0680), pidilizumab (CT-011), etc.; as anti-PD-L1 antibodies, atezolizumab (RG7446, MPDL3280A), durvalumab (MEDI4736), avelumab (PF-06834635, MSB0010718C), BMS-936559 (MDX1105), etc.; as anti-CTLA4 antibodies, ipilimumab (Yervoy (registered trademark)), tremelimumab, etc.; as anti-TIM-3 antibodies, MBG453, etc.; as anti-LAG-3 antibodies, BMS-986016, LAG525, etc.; as anti-KIR antibodies, lirilumab, etc.

[0045] Alternatively, as a substance that binds to an immune checkpoint molecule and inhibits the binding of the immune checkpoint molecule to its ligand expressed on antigen-presenting cells such as cancer cells and tumor-infiltrating macrophages, a fragment containing a portion (e.g., extracellular domain) necessary for the binding of the ligand to the immune checkpoint molecule and having no ability to transmit an immunosuppressive signal can be used. Furthermore, a substance obtained by conjugating a molecule capable of removing immune-exhausted effector cells to the fragment can be used. As an example of the latter, a fusion protein of the extracellular domain of PD-L1 or PD-L2 and the Fc region of an IgG1 or IgG2 antibody (e.g., AMP-224, etc.) can be mentioned. <​​​​​​​​​​​​

[0050] Both IL-26 inhibitors and ICIs have 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.

[0051] The combination agent of the present invention may be formulated by separately developing each active ingredient, or by formulating two or more of them as a combination agent.

[0052] When IL-26 inhibitors and ICIs are formulated separately, IL-26 can be formulated with pharmaceutically acceptable carriers or additives, as exemplified in the improved agent of the present invention. ICIs can also be formulated in the same manner as IL-26 inhibitors. When IL-26 inhibitors and ICIs are manufactured as a combination product, these active ingredients can be formulated by appropriately incorporating pharmaceutically acceptable carriers or additives, as exemplified in the improved agent of the present invention.

[0053] The dosage of each active ingredient varies depending on the target patient, the type of cancer, the symptoms, and the route of administration. For example, when used to treat colorectal cancer in adults, it is possible to appropriately select an IL-26 inhibitor and an ICI in an amount ranging from 0.5 mg to 10 mg per kg of body weight per dose. However, the combination agent of the present invention is not limited to these dosages.

[0054] If the combination agent of the present invention is provided in the form of separate compositions for each active ingredient, or in the form of separate compositions for at least one active ingredient and at least one other active ingredient, each composition can be administered to a subject simultaneously or with a time difference, via the same or separate routes.

[0055] The present invention will be described in more detail below with reference to examples, but these are illustrative examples and the present invention is not limited to these examples.

[0056] 1. Materials and Methods (1) Cells The mouse colon cancer cell line MC38 was purchased from Sigma (St. Louis, MO, USA). MC38 cells were cultured in DMEM medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS). All cell cultures were performed under conditions of 5% CO2 and 37°C.

[0057] (2) Antibodies and reagents: For the humanized anti-IL-26 monoclonal antibody, clone h69-10A described in Hatano R et al. Am J Transplant (2022) and WO2021 / 251340 was used. The purified human IgG1 isotype control antibody (clone QA16A12), GoInVivo rat anti-mouse PD-1 antibody (clone 29F.1A12), and purified rat IgG2a, κ isotype control antibody (clone RTK2758) used as comparative controls were purchased from BioLegend (San Diego, CA, USA).

[0058] (3) C57BL / 6J mice (female) were purchased from Sankyo Labo-Service (Tokyo, Japan). Bacterial artificial chromosome transgenic (hIL-26BACTg) mice containing the human IL-26 gene region were provided by Dr. Thomas Aune (Vanderbilt University, Nashville, TN, USA) and mated with C57BL / 6J mice at the Juntendo University Animal Facility. Transgenic mice aged 8-14 weeks and littermates were used. All mice used in this experiment were housed in specific sterile facilities. Animal experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee.

[0059] (4) Enteritis-related (AOM / DSS-induced) colorectal cancer model: Azoximethane (AOM, Wako) was adjusted to a concentration of 1 mg / mL in sterile PBS and administered intraperitoneally to C57BL / 6J mice and hIL-26BACTg mice (8-10 weeks old, male, each) at a dose of 200 μL (200 μg). Dextran sulfate sodium (DSS, 36-50 kDa, MP Biomedicals, Solon, OH, USA) was dissolved in sterile water to prepare 2.0% DSS-containing water, which was allowed to be freely consumed for 5 days starting 7 days after AOM administration. After 14 days of switching to normal drinking water, the cycle of consuming 2.0% DSS water for 5 days was repeated for 3 cycles. Diarrhea and bloody stools were observed, and body weight was measured 3 times a week. In antibody-based therapy experiments, control human IgG or humanized anti-IL-26 monoclonal antibody was administered continuously from the end of DSS water intake in the first cycle (day 12). The antibody solution was adjusted to a concentration of 1 mg / mL in sterile PBS and administered intraperitoneally at a dose of 100 μL (100 μg) per mouse. Mice were euthanized on the end of normal water intake in the third cycle (day 64), and the entire large intestine from just below the cecum to the rectum was collected. After counting the number of polyps throughout the entire length of the colon, the polyps were collected, finely chopped with scissors, and enzymatically treated in RPMI-1640 medium (Wako) containing 0.2 mg / mL collagenase type IV (GIBCO, Grand Island, NY, USA), 2 mg / mL dispase (GIBCO), 0.1 mg / mL DNase I, and 5% FBS at 37°C for 30 minutes. The tissue was then disrupted and filtered, and washed with PBS containing 0.5% FBS and 5 mM EDTA. The obtained cells were analyzed by flow cytometry.

[0060] (5) Mouse colorectal cancer cell line: MC38 cells of the mouse colorectal cancer cell line, a cancer-bearing model, are placed in sterile PBS in 2 x 10⁶ cells. 6 Suspend to a concentration of cells / mL and administer 100 μL per mouse subcutaneously to the flanks of C57BL / 6J mice and hIL-26BACTg mice (8-14 weeks old, respectively). 5MC38 cells were seeded one by one. The size of the tumors that formed on the flanks was measured twice a week. In antibody therapy experiments, anti-mouse PD-1 antibody or control rat IgG was administered intraperitoneally to each mouse at a dose of 100 μg 7, 10, and 14 days after seeding of MC38 cells. In combination therapy experiments with anti-mouse PD-1 antibody and anti-human IL-26 antibody, anti-mouse PD-1 antibody and humanized anti-IL-26 monoclonal antibody or control human IgG were administered intraperitoneally at a dose of 100 μg each 7, 10, and 14 days after seeding of MC38 cells subcutaneously on the flanks of hIL-26BACTg mice. The antibody solution was adjusted to a concentration of 1 mg / mL in sterile PBS and administered intraperitoneally at a dose of 100 μL (100 μg) per mouse. MC38 cells were seeded subcutaneously, and mice were euthanized on day 17 or 21. Tumors were collected from the flanks. The tumors collected from the flanks were finely chopped with scissors and enzymatically treated in RPMI-1640 medium containing 0.2 mg / mL collagenase type IV, 2 mg / mL dispase, 0.1 mg / mL DNase I, and 5% FBS at 37°C for 30 minutes. After tissue disruption and filtration, the tissue was washed with PBS containing 0.5% FBS and 5 mM EDTA. The obtained cells were analyzed by flow cytometry.

[0061] (6) Cells were prepared from polyps in the large intestine or tumors taken from the flank of flow cytometry mice. After washing with PBS containing 1% FBS and 0.1% sodium azide, unlabeled anti-mouse CD16 / 32 antibody was added to inhibit nonspecific binding of the fluorescently labeled antibody. Subsequently, the cells were stained with a fluorescently labeled antibody at 4°C for 30 minutes. Flow cytometry was performed using LSRFortessa (BD Biosciences), and the data were analyzed using FlowJo software (BD Biosciences). The flow cytometry antibodies used in this experiment are shown in Table 1. All antibodies listed in Table 1 were purchased from BioLegend.

[0062]

[0063] (7) Statistical data were analyzed using a two-tailed Student's t-test in the case of two-group comparisons. The data show the mean ± standard deviation for each individual in each group in the in vivo experiment. Significance was analyzed using GraphPad Prism 9 (GraphPad Software, San Diego, CA), and values ​​of p<0.01 were considered significant and are shown in the figures (*).

[0064] 2. Results (Example 1) Antitumor effect of humanized anti-IL-26 antibody in a colitis-related (AOM / DSS-induced) colorectal cancer model. In this example, we investigated the possibility that IL-26 promotes the malignancy of inflammation-related cancers in an environment where IL-26 is expressed and inflammation is exacerbated. As a representative model of inflammation-related cancer, we used a colitis-related colorectal cancer model induced by azoxymethane (AOM) / dextran sulfate sodium (DSS). AOM is a carcinogen that induces O6-methylguanine adducts in DNA, causing the conversion of guanine to adenine, and is particularly effective in inducing colon cancer. DSS is a sulfated polysaccharide that contains many anions, and oral ingestion of DSS-containing water causes damage to the mucosal epithelium of the large intestine. Damage to the mucosal barrier of the large intestine leads to stimulation by the gut microbiota, activation of immune cells in the large intestine, and production of inflammatory cytokines, resulting in the development of colitis. Inflammation rarely occurs in the small intestine, and is concentrated in the large intestine, making it a model that closely resembles the pathology of ulcerative colitis. Using this enteritis-associated colorectal cancer model, we investigated the differences in the development and progression of colorectal cancer between wild-type mice and hIL-26BACTg mice.

[0065] Figure 1A shows the schedule for creating the AOM / DSS-induced colorectal cancer model. AOM was administered intraperitoneally to hIL-26BACTg mice and wild-type mice (day 0). After one week (day 7), ad libitum access to 2.0% DSS water was started. After 5 days of DSS water intake, the mice were switched to normal drinking water for 14 days, and then given 2.0% DSS water again for 5 days. This cycle was repeated for 3 cycles. On the last day of normal water intake in the 3rd cycle (day 64), the mice were euthanized, and the entire colon from just below the cecum to the rectum was collected. This model, which alternates between DSS water and normal water intake, is used as a model of chronic colitis that mimics the pathology of ulcerative colitis patients, who experience repeated remissions and relapses of colitis. In this model, hIL-26BACTg mice showed more severe damage to the mucosal epithelium, neutrophil and lymphocyte infiltration in the lamina propria, and a significantly higher colitis score compared to wild-type mice. After administering AOM and repeating DSS water / normal water intake for 3 cycles, the entire colon was collected on day 64, and the number of formed polyps was counted. The results showed that the number of polyps in hIL-26BACTg mice was significantly higher than in wild-type mice (Figure 1B). In addition, after counting the number of polyps on day 64, the polyps were collected, and the proportion of neutrophils (CD11b-positive, Ly6G-positive) among CD45-positive leukocytes in the polyps was analyzed using flow cytometry. Compared to wild-type mice, polyps in hIL-26BACTg mice showed significantly more neutrophil infiltration, indicating that cancer-related inflammation was strongly induced (Figure 1C).

[0066] Therefore, using the same model, we administered the humanized anti-IL-26 antibody established by the inventors and investigated its antitumor effect against colitis-associated colorectal cancer. Similar to Figure 1A, after administering AOM, intake of 2.0% DSS water was started from day 7, and from the day the DSS water intake of the first cycle ended (day 12), the humanized anti-IL-26 antibody or control human IgG was administered on the days indicated by the arrows in the figure (Figure 1D). As a result, hIL-26BACTg mice administered with control human IgG developed severe colitis and a very large number of polyps were observed, whereas hIL-26BACTg mice administered with humanized anti-IL-26 antibody showed a decrease in colitis score and a significantly smaller number of polyps (Figure 1E). Regarding the proportion of neutrophils in polyps, neutrophil infiltration was significantly suppressed in polyps of hIL-26BACTg mice treated with humanized anti-IL-26 antibody compared to hIL-26BACTg mice treated with control human IgG, indicating suppression of cancer-related inflammation (Figure 1F).

[0067] These results indicate that humanized anti-IL-26 antibodies suppress colitis in enteritis-associated colorectal cancer, thereby inhibiting the malignant progression of the cancer. Furthermore, they significantly suppress the number of tumor-immunosuppressive neutrophils infiltrating the tumor and cancer-associated inflammation. Therefore, humanized anti-IL-26 antibodies are shown to be a useful therapeutic approach for enteritis-associated colorectal cancer.

[0068] (Example 2) Combination effect of anti-PD-1 antibody and humanized anti-IL-26 antibody in a mouse colorectal cancer cell line tumor-bearing model In mismatch repair deficiency (MMRd) / high microsatellite instability (MSI-High) colorectal cancers, which have many mutations and many cancer antigens (high immunogenicity), it is known that due to high immunogenicity, many T cells accumulate around the tumor, and immune checkpoint inhibitors (ICIs) are highly effective. On the other hand, in mismatch repair function normal (MMRp) / microsatellite stable colorectal cancers, immunogenicity is low and there are few T cells around the tumor, but neutrophil infiltration is high, and it is known that the response rate to ICIs is low.Therefore, a subcutaneous tumor-bearing model of the mouse colorectal cancer cell line MC38, which has properties similar to human MMRd / MSI-High colorectal cancer, was used.

[0069] MC38 cells were seeded subcutaneously in the flanks of both hIL-26BACTg mice and wild-type mice, and the size of the tumors that formed in the flanks was measured. As a result, the rate of tumor size increase when MC38 cells were seeded subcutaneously in hIL-26BACTg mice was similar to that when seeded in wild-type mice (Figure 2A). On the other hand, when anti-mouse PD-1 antibody was administered 7 days after MC38 cell seeding, the antitumor effect was significant in wild-type mice, while the tumor size in hIL-26BACTg mice increased significantly more than in wild-type mice (Figure 2B). Subcutaneous tumors were collected, and the proportion of neutrophils (CD11b-positive, Ly6G-positive) and CD8 T cells (TCRβ-positive, CD4-negative, CD8a-positive) among the CD45-positive leukocytes infiltrating the tumors was analyzed using flow cytometry. As a result, compared to wild-type mice, hIL-26BACTg mice showed significantly more neutrophil infiltration within tumors and stronger induction of cancer-associated inflammation, while CD8 T cell infiltration was very low (Figure 2C and 2D).

[0070] Therefore, using the same model, we investigated the combined effect of administering anti-mouse PD-1 antibody in addition to the humanized anti-IL-26 antibody established by the inventors. As a result, it was shown that the tumor size of hIL-26BACTg mice administered with both anti-PD-1 antibody and humanized anti-IL-26 antibody was clearly smaller compared to the tumor size of hIL-26BACTg mice administered with anti-PD-1 antibody and control human IgG (Figure 2E). Regarding immune cells infiltrating the tumor, compared to hIL-26BACTg mice administered with control human IgG, neutrophil infiltration was significantly suppressed in the tumors of hIL-26BACTg mice administered with humanized anti-IL-26 antibody, indicating suppression of cancer-related inflammation, while CD8 T cell infiltration was greatly increased (Figures 2F and 2G).

[0071] Administration of an anti-mouse PD-1 antibody induces the activation of T cells infiltrating the tumor. In hIL-26BACTg mice, T cell activation also induces IL-26 expression. IL-26 produced by T cells is thought to cause cancer cells to produce large amounts of neutrophil migration chemokines, increasing the number of neutrophils in the tumor. This suppresses CD8 T cell activation and weakens the antitumor effect of the anti-PD-1 antibody. These results suggest that even when ICI therapy is attempted in MMRd / MSI-High colorectal cancer with high T cell infiltration, high IL-26 expression can lead to resistance by increasing tumor immunosuppressive neutrophils and decreasing T cells within the tumor. In this example, we demonstrated that combining a humanized anti-IL-26 antibody with ICI significantly suppresses the number of neutrophils infiltrating the tumor and increases the number of T cells, resulting in a remarkable antitumor effect. In summary, the combination therapy of humanized anti-IL-26 antibodies and ICIs is shown to be a useful treatment approach for ICI-resistant colorectal cancer.

[0072] The improving agent of the present invention can transform the tumor microenvironment from a cold tumor state to a hot tumor state by inhibiting the action of IL-26. Therefore, it can exert an antitumor effect even in cancers with reduced sensitivity to ICIs, either alone or in combination with other antitumor drugs, preferably ICIs. Furthermore, it can suppress the occurrence of immune-related adverse events, which are serious side effects of ICIs, and can contribute to improving the safety of ICI treatment. Thus, the improving agent and the combination agent of the present invention are extremely useful as novel and groundbreaking cancer treatment means.

[0073] This application is based on Japanese Patent Application No. 2025-007248, filed in Japan on 17 January 2025, the contents of which are incorporated herein by reference.

Claims

1. A tumor microenvironment improving agent containing an interleukin-26 (IL-26) inhibitor.

2. The agent according to claim 1, wherein the improvement of the tumor microenvironment is an increase in the cancer's sensitivity to immune checkpoint inhibitors (ICIs).

3. The agent according to claim 1 or 2, wherein the cancer is ICI-resistant cancer.

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 any one of claims 1 to 4, wherein the cancer is colorectal cancer.

6. The agent according to any one of claims 1 to 5, wherein the IL-26 inhibitor is a substance that inhibits the interaction between IL-26 and molecules involved in downstream signal transduction.

7. The agent according to claim 6, wherein the IL-26 inhibitor is an anti-IL-26 antibody.

8. The agent according to any one of claims 1 to 7, which is administered alone as a cancer treatment agent.

9. The agent according to any one of claims 1 to 7, which is used in combination with at least one drug having antitumor activity.

10. The agent according to claim 9, wherein the drug is an ICI.

11. A cancer treatment agent comprising an interleukin-26 (IL-26) inhibitor and at least one drug having antitumor activity.

12. The agent according to claim 11, wherein the IL-26 inhibitor is an anti-IL-26 antibody.

13. The agent according to claim 11 or 12, wherein the drug is an ICI.

14. The agent according to claim 13, wherein ICI is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody.

15. The agent according to any one of claims 11 to 14, wherein an IL-26 inhibitor and at least one drug having antitumor activity are formulated separately.

16. The agent according to any one of claims 11 to 14, wherein the IL-26 inhibitor and the drug having antitumor activity are in the form of a combination agent.

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