Composition comprising circadian rhythm period extending agent, and use thereof

Abstract

To provide a composition that enables improvement of a cancer microenvironment, suppression of immune exclusion, inhibition of fibroblast differentiation, promotion of T cell infiltration into tumor tissue, or enhancement of the anti-cancer effect of an immune checkpoint inhibitor.SOLUTION: A composition comprising a circadian rhythm period extending agent for use in at least one selected from the group consisting of improvement of a cancer microenvironment, suppression of immune exclusion, inhibition of fibroblast differentiation, promotion of T cell infiltration into tumor tissue, and enhancement of the anti-cancer effect of an immune checkpoint inhibitor.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a composition containing a circadian rhythm period extender and its application technology. Specifically, the present disclosure relates to a composition containing a circadian rhythm period extender for use in at least one selected from the group consisting of improving the cancer microenvironment, suppressing immune elimination, suppressing fibrocyte differentiation, promoting T cell infiltration into tumor tissue, and enhancing the anti-cancer effect of an immune checkpoint inhibitor. Furthermore, the present disclosure relates to a composition or kit for treating and / or preventing cancer, which contains a circadian rhythm period extender and an immune checkpoint inhibitor; a method for screening an active ingredient for use in at least one selected from the group consisting of improving the cancer microenvironment, suppressing immune elimination, suppressing fibrocyte differentiation, promoting T cell infiltration into tumor tissue, and enhancing the anti-cancer effect of an immune checkpoint inhibitor; and a method for screening an active ingredient for use in combination with an immune checkpoint inhibitor. [Background technology]

[0002] Immunotherapy, including immune checkpoint inhibitors, is currently at the center of advanced cancer treatment. However, one of the causes of resistance to immunotherapy is immune exclusion, where the cancer stroma, primarily composed of αSMA-positive cancer-associated fibroblasts, inhibits the infiltration of immune cells such as T cells (i.e., immune cells remain in the cancer stroma).

[0003] Fibrocytes are monocytic blood cells that possess fibroblast-like properties, such as high collagen production, and are also reported to act as precursors of some fibroblasts.

[0004] The present inventors have previously recovered fibrocytes as CD45 and CD34 positive cells from mouse tumors and confirmed that collagen1a1 (COL1A1) is highly expressed in these cells (Non-Patent Document 1). Furthermore, the tumor-derived fibrocytes were treated with CD8 +It has been confirmed that co-culturing T cells promotes T cell proliferation, and that this effect is enhanced by anti-PD-L1 antibodies (Non-Patent Document 1). On the other hand, it has also been confirmed that when the tumor-derived fibrocytes are stimulated with TGF-β, they undergo a morphological change to a myofibroblast-like state, and expression of the αSMA (α-smooth muscle actin) gene increases, leading to differentiation into cancer-associated fibroblasts (CAFs) (Non-Patent Document 1). In other words, fibrocytes act as intratumoral antigen-presenting cells and act as immune cells that promote the therapeutic effects of immune checkpoint inhibitors. On the other hand, they are also known to act as CAF precursors with tumor-promoting properties, such as by inducing the acquisition of cancer stem cell-like properties and promoting tumor angiogenesis (Non-Patent Documents 2 and 3).

[0005] Meanwhile, clock genes have been identified as a group of genes that form circadian rhythms, and circadian rhythms are formed through a feedback loop involving transcriptional repression by the key transcription factors BMAL1 and CLOCK, as well as PER and CRY. Numerous therapeutic drugs targeting these clock genes have been developed, primarily for metabolic and psychiatric disorders (Non-Patent Document 4). The importance of circadian rhythms in cancer progression has also been attracting attention in recent years, but there has been insufficient progress in elucidating their mechanisms and developing therapeutic applications. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Cell Rep.2023 Mar 28;42(3):112162. [Non-patent document 2] Cancer Letters 421(2018)17-27. [Non-patent document 3] Nat Commun 2015 Dec 4:6:8792. [Non-patent document 4] Science.2012 Aug 31;337(6098):1094-7. [Non-Patent Document 5] Nucleic Acids Res. 2016 Jun 20;44(11):5010-21. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention addresses the problem of providing a composition for use in improving the cancer microenvironment, suppressing immune elimination, suppressing fibrocyte differentiation, promoting T cell infiltration into tumor tissue, or enhancing the anti-cancer effect of immune checkpoint inhibitors. Another problem is to provide a method for screening active ingredients for use in improving the cancer microenvironment, suppressing immune elimination, suppressing fibrocyte differentiation, promoting T cell infiltration into tumor tissue, or enhancing the anti-cancer effect of immune checkpoint inhibitors, or for use in combination with immune checkpoint inhibitors. [Means for solving the problem]

[0008] The inventors discovered that treating fibrocytes with KL001, a CRY stabilizer (a circadian rhythm period extender), suppresses the expression of the Acta2 (αSMA) gene and inhibits the differentiation of fibrocytes into αSMA-positive cancer-associated fibroblasts, and have made further improvements.

[0009] The present disclosure encompasses, for example, the subject matter described in the following sections: Section 1. including circadian rhythm period extenders, A composition for use in at least one selected from the group consisting of improving the cancer microenvironment, suppressing immune elimination, suppressing fibrocyte differentiation, promoting T cell infiltration into tumor tissue, and enhancing the anti-cancer effect of immune checkpoint inhibitors. Section 2. Circadian rhythm period extenders a stabilizer and / or an expression promoter for at least one protein selected from the group consisting of PER and CRY, and / or Item 1. The composition according to Item 1, comprising an inhibitor of the function of at least one protein selected from the group consisting of BMAL1 and CLOCK. Section 3. Item 1 or 2. The composition according to Item 1 or 2, for use in combination with an immune checkpoint inhibitor. Section 4. A composition or kit for treating and / or preventing cancer, comprising a circadian rhythm period extender and an immune checkpoint inhibitor. Section 5. Item 5. The composition or kit according to Item 4, wherein the cancer is a solid cancer. Section 6. Expression of at least one protein selected from the group consisting of PER and CRY in fibrocytes, and / or The function of at least one protein selected from the group consisting of BMAL1 and CLOCK is used as an index. A method for screening active ingredients for use in at least one selected from the group consisting of improving the cancer microenvironment, suppressing immune elimination, suppressing fibrocyte differentiation, promoting T cell infiltration into tumor tissue, and enhancing the anti-cancer effect of immune checkpoint inhibitors. Section 7. Expression of at least one protein selected from the group consisting of PER and CRY in fibrocytes, and / or The function of at least one protein selected from the group consisting of BMAL1 and CLOCK is used as an index. A method for screening active ingredients for use in combination with immune checkpoint inhibitors. [Effects of the Invention]

[0010] The effects of improving the cancer microenvironment, suppressing immune elimination, inhibiting fibrocyte differentiation, promoting T cell infiltration into tumor tissue, or enhancing the anticancer effect of immune checkpoint inhibitors are exhibited. Also provided is a method for screening active ingredients for use in improving the cancer microenvironment, suppressing immune elimination, inhibiting fibrocyte differentiation, promoting T cell infiltration into tumor tissue, or enhancing the anticancer effect of immune checkpoint inhibitors, or for use in combination with immune checkpoint inhibitors. Furthermore, excellent anticancer effects are exhibited by using a circadian rhythm period extender in combination with an immune checkpoint inhibitor. [Brief explanation of the drawings]

[0011] [Figure 1] Identification of intratumoral fibrocytes by single-cell RNA sequencing and the results of searching for activating transcription factors are shown. [Figure 2] This shows the expression levels of Acta2 and COL1A1 genes in CD45+CD34+ fibrocytes obtained from tumor tissues of mice subcutaneously implanted with AB1-HA. [Figure 3] This shows the expression levels of the Acta2 gene in COL1A2+CD45- fibroblasts and COL1A2+CD45+ fibrocytes obtained from tumor tissues of mice subcutaneously implanted with LLC. [Figure 4] The results of measuring tumor volume and fluorescent staining in mice subcutaneously implanted with AB1-HA are shown. [Figure 5] This shows the results of measuring tumor volume in BALB / c nude mice into which AB1-HA was subcutaneously implanted. [Figure 6] 1 shows the results of measuring tumor volume in mice subcutaneously implanted with LLC. DETAILED DESCRIPTION OF THE INVENTION

[0012] Each embodiment included in the present disclosure will be described in further detail below. The compositions encompassed by the present disclosure include a circadian rhythm period extender, and may be referred to herein as "compositions of the present disclosure."

[0013] Clock genes, such as the Per (Period) gene, Cry (Cryptochrome) gene, Bmal1 gene, and Clock gene, are known to form a group of genes that regulate circadian rhythms. The Per gene has three homologs, Per1, Per2, and Per3, and the Cry gene has two homologs, Cry1 and Cry2. The BMAL1 protein and the CLOCK protein form a heterodimer, activating the transcription of the Per and Cry genes (BMAL1 and CLOCK proteins function as positive regulators). The PER and CRY2 proteins then form a complex, which translocates from the cytoplasm to the nucleus and suppresses the transcriptional activation of the Bmal1 and Clock genes (PER and CRY2 proteins function as negative regulators). The PER protein is then degraded by CKI (casein kinase I), and the CRY protein is degraded by FBXL3 (F-box / LRR-repeat protein 3). It is believed that circadian rhythms are formed by a feedback loop mediated by transcription and translation of clock genes. For example, it has been reported that the CRY stabilizer KL001 specifically binds to the CRY protein and inhibits its degradation, thereby extending the period of the circadian rhythm (Non-Patent Document 4).

[0014] The circadian rhythm period extender is not particularly limited as long as it can extend the period of the circadian rhythm. The circadian rhythm period extender may include, for example, a stabilizer and / or an expression promoter for at least one protein selected from the group consisting of PER and CRY. Furthermore, the circadian rhythm period extender may include, for example, a function inhibitor for at least one protein selected from the group consisting of BMAL1 and CLOCK. Among these, a stabilizer for the CRY protein is preferred. In this specification, "stabilizing a protein" means inhibiting protein degradation. In other words, a protein stabilizer can be said to be an agent for inhibiting protein degradation. Specific examples of circadian rhythm period extenders include CRY stabilizers such as KL001, KL044, KL101, KL201, TH129, and TH301. Of these, KL001 is preferred. The circadian rhythm period extenders can be used singly or in combination of two or more.

[0015] As used herein, "stabilization of a protein" and "promotion of protein expression" can be evaluated by measuring the expression level of the protein. Furthermore, "inhibition of protein function" can be evaluated, for example, by measuring the expression level of downstream genes (more specifically, PER, CRY, etc.) whose expression is promoted by activation of the protein (specifically, at least one protein selected from the group consisting of BMAL1 and CLOCK); or by measuring the luminescence intensity by a reporter assay using a reporter plasmid in which a reporter gene such as luciferase is linked downstream of the protein (specifically, at least one protein selected from the group consisting of BMAL1 and CLOCK). Furthermore, as shown in the Examples below, a stabilizer for at least one protein selected from the group consisting of circadian rhythm period extenders such as KL001, PER, and CRY can improve the cancer microenvironment, suppress immune elimination, suppress fibrocyte differentiation, promote T cell infiltration into tumor tissue, or enhance the anti-cancer effect of immune checkpoint inhibitors. Furthermore, as described above, PER and CRY are normally degraded. However, when an expression promoter for at least one protein selected from the group consisting of PER and CRY, which are circadian rhythm period extenders, is used, the expression of the protein is promoted while being degraded, and it is presumed that, similar to stabilizers of at least one protein selected from the group consisting of PER and CRY, such as KL001, the effects of improving the cancer microenvironment, suppressing immune elimination, suppressing fibrocyte differentiation, promoting infiltration of T cells into tumor tissue, or enhancing the anti-cancer effect of immune checkpoint inhibitors are exhibited. Furthermore, as described above, PER and CRY have the effect of suppressing the transcriptional activation of the Bmal1 gene and the Clock gene, and therefore, it is believed that a stabilizer of at least one protein selected from the group consisting of KL001 or the like, PER, and CRY will ultimately suppress the transcriptional activation of the Bmal1 gene and the Clock gene. Therefore, although a function inhibitor of at least one protein selected from the group consisting of BMAL1 and CLOCK, which are circadian rhythm period extenders, suppresses the expression of the downstream genes PER and / or CRY, it is presumed that, as with a stabilizer of at least one protein selected from the group consisting of KL001 or the like, PER, and CRY, it will ultimately exhibit the effects of improving the cancer microenvironment, suppressing immune elimination, inhibiting fibrocyte differentiation, promoting T cell infiltration into tumor tissue, or enhancing the anti-cancer effect of immune checkpoint inhibitors.

[0016] The composition of the present disclosure can be used to improve the cancer microenvironment, suppress immune elimination, suppress fibrocyte differentiation, promote T cell infiltration into tumor tissue, or enhance the anti-cancer effect of immune checkpoint inhibitors. These may be used alone or in combination of two or more.

[0017] As used herein, the term "cancer microenvironment" refers to the site of interaction between cancer cells and surrounding cells or the extracellular environment. The cancer microenvironment is composed of cellular and non-cellular components, and the cellular components are classified into parenchyma (cancer cells) and stroma (non-cancer cells). The stroma is known to be composed of, for example, fibroblasts; vascular cells (e.g., vascular endothelial cells, lymphatic endothelial cells, pericytes, etc.); hematopoietic or immune cells (e.g., lymphocytes, various granulocytes, monocytes, macrophages, dendritic cells, mast cells, platelets, etc.); nervous system cells; and adipocytes. Non-cellular components include, for example, low-molecular-weight metabolites, humoral factors involved in signal transduction (e.g., growth factors, cytokines, etc.), and extracellular matrix (ECM, collagen, fibronectin, hyaluronic acid, etc.) that provides mechanical strength to tissues. In addition, fibroblasts proliferating in the cancer microenvironment are sometimes referred to as cancer-associated fibroblasts (CAFs), and the myofibroblast-like fraction that highly expresses αSMA is known to be an important cell type in cancer progression.

[0018] As shown in the Examples below, circadian rhythm extenders such as the CRY stabilizer KL001 have been shown to suppress the differentiation of fibrocytes into αSMA-positive cancer-associated fibroblasts. αSMA-positive cancer-associated fibroblasts are important cells in the cancer microenvironment and are known to promote angiogenesis, cancer cell proliferation, and infiltration. It is also known that the cancer stroma, primarily composed of αSMA-positive cancer-associated fibroblasts, inhibits the infiltration of immune cells such as T cells (immune exclusion), leading to treatment resistance. Fibrosis in the cancer microenvironment significantly contributes to the formation of a stromal barrier. Therefore, the composition of the present disclosure can be used to improve the cancer microenvironment (more specifically, to suppress fibrosis in the cancer microenvironment).

[0019] As shown in the Examples below, circadian rhythm extenders such as the CRY stabilizer KL001 have been shown to reduce αSMA-positive cancer-associated fibroblasts at the tumor margin and promote T cell infiltration into tumor tissue. It is known that the cancer stroma, primarily composed of αSMA-positive cancer-associated fibroblasts, inhibits the infiltration of immune cells such as T cells (cancer immune exclusion), leading to treatment resistance. Therefore, the composition of the present disclosure can be used to suppress immune exclusion. It is predicted that suppressing immune exclusion will promote the infiltration of immune cells such as T cells, thereby suppressing tumor progression. Furthermore, since immune exclusion leads to resistance to immune checkpoint inhibitors, the compositions of the present disclosure can also be used to suppress resistance to immune checkpoint inhibitors.

[0020] As shown in the examples below, circadian rhythm period extenders such as the CRY stabilizer KL001 have been confirmed to suppress the differentiation of fibrocytes into αSMA-positive cancer-associated fibroblasts. Therefore, the composition of the present disclosure can be used to suppress the differentiation of fibrocytes, more specifically, to suppress the differentiation of fibrocytes in cancer tissue (tumor) into αSMA-positive cancer-associated fibroblasts. As used herein, the term "fibrocyte" refers to a bone marrow-derived monocytic cell capable of producing collagen, and is a CD45 and CD34 double-positive cell.

[0021] As shown in the Examples below, circadian rhythm extenders such as the CRY stabilizer KL001 have been shown to promote T cell infiltration into tumor tissues. Therefore, the composition of the present disclosure can be used to promote T cell infiltration into tumor tissues.

[0022] As will be described in the Examples below, it has been confirmed that circadian rhythm extenders, such as the CRY stabilizer KL001, have an enhanced effect in suppressing tumor progression when combined with immune checkpoint inhibitors, such as anti-PD-L1 antibodies. Therefore, the compositions of the present disclosure can be used to enhance the anti-cancer effect of immune checkpoint inhibitors.

[0023] Furthermore, as described above, it has been confirmed that the effect of circadian rhythm extenders, such as the CRY stabilizer KL001, in suppressing tumor progression is enhanced when combined with immune checkpoint inhibitors, such as anti-PD-L1 antibodies. Therefore, the composition of the present disclosure can be used in combination with immune checkpoint inhibitors.

[0024] The immune checkpoint inhibitor is not particularly limited, and examples thereof include known ones, such as anti-PD-L1 antibodies, anti-PD-1 antibodies, and anti-CTLA-4. Of these, anti-PD-L1 antibodies are preferred.

[0025] Examples of cancers (tumors) targeted by the composition of the present disclosure include solid cancers. Examples of solid cancers include mesothelioma, lung cancer, colon cancer, gastric cancer, breast cancer, prostate cancer, pancreatic cancer, and hepatocellular carcinoma. Of these, mesothelioma and lung cancer are preferred.

[0026] The composition of the present disclosure contains a circadian rhythm period extender and may further contain other ingredients. Examples of such other ingredients include pharmacologically acceptable bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, surfactants, antioxidants, preservatives, coating agents, colorants, flavorings, pH adjusters, and other drugs such as gastric mucosa protectants. These ingredients may be used alone or in combination of two or more.

[0027] The composition of the present disclosure can be prepared by a conventional method by combining a circadian rhythm period extender and, if necessary, other ingredients.

[0028] The amount of the composition of the present disclosure to be administered (ingested) is not particularly limited and is determined depending on the age, weight, sex, severity of symptoms, administration method, etc. of the subject to be administered.

[0029] The composition of the present disclosure may be administered once a day, or may be administered in divided doses multiple times a day (for example, about 2 to 5 times).

[0030] Examples of administration methods include oral administration and parenteral (eg, intravenous, intraarterial, intramuscular, subcutaneous, peritoneal, rectal, transdermal, topical, etc.) administration.

[0031] The subject to which the composition of the present disclosure is administered is preferably a mammal. It may be not only a human but also a non-human mammal. Examples of the human subject include cancer patients or suspected cancer patients; humans in a (cancer) immunoexclusion state or suspected cancer patients; humans who are administered or have been administered an immune checkpoint inhibitor; humans with or suspected immune checkpoint inhibitor resistance; or humans with two or more of these characteristics.

[0032] The present disclosure also encompasses a composition for treating and / or preventing cancer, comprising a circadian rhythm period extender and an immune checkpoint inhibitor. In this specification, such a composition may be referred to as the "composition for treating and / or preventing cancer of the present disclosure."

[0033] The mass ratio of the circadian rhythm period extender to the immune checkpoint inhibitor in the cancer treatment and / or prevention composition of the present disclosure (circadian rhythm period extender / immune checkpoint inhibitor) is not particularly limited and can be, for example, about 0.01 to 50, preferably about 0.1 to 10.

[0034] The content of the circadian rhythm period extender in the cancer treatment and / or prevention composition of the present disclosure is not particularly limited, and can be about 0.1 to 99% by mass.

[0035] The content of the immune checkpoint inhibitor in the composition for treating and / or preventing cancer of the present disclosure is not particularly limited, and can be about 0.1 to 99% by mass.

[0036] The cancer treatment and / or prevention composition of the present disclosure includes a circadian rhythm period extender and an immune checkpoint inhibitor, and may further include other ingredients. Examples of such other ingredients include pharmacologically acceptable bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, surfactants, antioxidants, preservatives, coating agents, colorants, flavorings, pH adjusters, and other drugs such as gastric mucosa protectants. These ingredients may be used alone or in combination of two or more.

[0037] The composition for treating and / or preventing cancer of the present disclosure can be prepared by a conventional method by combining a circadian rhythm period extender and an immune checkpoint inhibitor, and, if necessary, other ingredients.

[0038] The above description can be applied to the cancers (tumors) targeted by the cancer treatment and / or prevention composition of the present disclosure. The above descriptions can be used to refer to the dosage, administration method, and administration target of the composition for treating and / or preventing cancer of the present disclosure.

[0039] The present disclosure also encompasses a kit for treating and / or preventing cancer, comprising a circadian rhythm period extender and an immune checkpoint inhibitor. Herein, such a composition may be referred to as a "kit for treating and / or preventing cancer of the present disclosure."

[0040] The kit for treating and / or preventing cancer of the present disclosure includes a circadian rhythm period extender and an immune checkpoint inhibitor, and may further include, for example, a further preferred pharmaceutical agent for treating or preventing cancer, an administration device (e.g., a syringe), or instructions (package insert).

[0041] The above description can be applied to the cancers (tumors) targeted by the cancer treatment and / or prevention kit of the present disclosure. The dosage and administration method of the circadian rhythm period extender can be referenced above. Similarly, the dosage and administration method of the immune checkpoint inhibitor can be referenced above. The administration times of the circadian rhythm period extender and the immune checkpoint inhibitor can be simultaneous or different. Furthermore, the above description of the circadian rhythm period extender can be cited for the subject of application of the cancer treatment and / or prevention kit of the present disclosure.

[0042] The present disclosure also encompasses a method for screening for an active ingredient for at least one selected from the group consisting of improving the cancer microenvironment, suppressing immune elimination, suppressing fibrocyte differentiation, promoting T cell infiltration into tumor tissue, and enhancing the anti-cancer effect of immune checkpoint inhibitors, using the expression of at least one protein selected from the group consisting of PER and CRY in fibrocytes and / or the function of at least one protein selected from the group consisting of BMAL1 and CLOCK as an indicator. In this specification, this method may be referred to as the "screening method of the present disclosure."

[0043] The screening method of the present disclosure also includes a screening method for an active ingredient to be used in combination with an immune checkpoint inhibitor, using the expression of at least one protein selected from the group consisting of PER and CRY in fibrocytes and / or the function of at least one protein selected from the group consisting of BMAL1 and CLOCK as an indicator.

[0044] Protein expression can be measured by adding a test substance to fibrocytes and measuring, for example, Western blotting. The screening method of the present disclosure preferably includes the steps of adding a test substance to fibrocytes and measuring the expression of at least one protein selected from the group consisting of PER and CRY in the cells. The function of a protein can be evaluated by, for example, adding a test substance to fibrocytes and measuring the expression level of a downstream gene (more specifically, PER, CRY, etc.) whose expression is promoted by activation of the protein (specifically, at least one protein selected from the group consisting of BMAL1 and CLOCK); or by measuring the luminescence intensity by a reporter assay using a reporter plasmid in which a reporter gene such as luciferase is linked downstream of the protein (specifically, at least one protein selected from the group consisting of BMAL1 and CLOCK). The screening method of the present disclosure preferably includes the steps of adding a test substance to fibrocytes and measuring the function of at least one protein selected from the group consisting of BMAL1 and CLOCK in the cells.

[0045] In the screening method of the present disclosure, a substance that suppresses the degradation of at least one protein selected from the group consisting of PER and CRY 24 or 48 hours after addition of the test substance when the test substance is added compared to when the test substance is not added (in other words, a substance that suppresses the decrease in the expression level of at least one protein selected from the group consisting of PER and CRY) can be selected as an active ingredient for at least one selected from the group consisting of improving the cancer microenvironment, suppressing immune exclusion, suppressing fibrocyte differentiation, promoting T cell infiltration into tumor tissue, and enhancing the anti-cancer effect of an immune checkpoint inhibitor. Similarly, the substance can be selected as an active ingredient for use in combination with an immune checkpoint inhibitor. Furthermore, in the screening method of the present disclosure, a substance that increases the expression level of at least one protein selected from the group consisting of PER and CRY when the test substance is added compared to when the test substance is not added can be selected as an active ingredient for at least one selected from the group consisting of improving the cancer microenvironment, suppressing immune exclusion, suppressing fibrocyte differentiation, promoting T cell infiltration into tumor tissue, and enhancing the anti-cancer effect of an immune checkpoint inhibitor. Similarly, the substance can be selected as an active ingredient for use in combination with an immune checkpoint inhibitor. Furthermore, in the screening method of the present disclosure, a substance that suppresses the function of at least one protein selected from the group consisting of BMAL1 and CLOCK when the test substance is added compared to when the test substance is not added can be selected as an active ingredient for at least one selected from the group consisting of improving the cancer microenvironment, suppressing immune exclusion, suppressing fibrocyte differentiation, promoting T cell infiltration into tumor tissue, and enhancing the anti-cancer effect of an immune checkpoint inhibitor. Similarly, the substance can be selected as an active ingredient for use in combination with an immune checkpoint inhibitor.

[0046] In the screening method of the present disclosure, in the step of adding a test substance to fibrocytes, TGF-β, which is known to induce differentiation of fibrocytes into fibroblasts, can be added. The concentration of TGF-β when added is not particularly limited, but can be, for example, about 1 to 50 ng / mL.

[0047] The test substance is not particularly limited and may be, for example, a compound or a composition. The compound may be, for example, a low molecular weight compound, a nucleic acid (e.g., DNA, RNA, etc.), a protein (e.g., an antibody or a portion thereof, etc.), or a high molecular weight compound such as a polymer. The composition may also be an extract obtained from an organism (e.g., an animal, a plant, a microorganism, etc.), or may be a combination of two or more compounds.

[0048] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes any and all combinations of the constituent elements described in this specification.

[0049] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0050] The contents of the present disclosure will be specifically explained using the following test examples. However, the present disclosure is not limited to these in any way. In the following, unless otherwise specified, experiments were performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "% by mass."

[0051] Six-week-old male BALB / c mice, C57BL / 6 mice, and athymic BALB / c nude mice were purchased from Charles River Japan (Japan).

[0052] <Test Example 1> Identification of factors contributing to the differentiation of tumor-derived fibrocytes Based on previous studies (Non-Patent Document 1), we compared the genes expressed in intratumoral fibrocytes and macrophages using single-cell RNA sequencing data from CD45-positive blood cells recovered from AB1-HA tumor tissue. Using the top 100 genes that were specifically expressed in each fraction, we identified a group of transcriptional genes specifically activated in fibrocytes using the wPGSA method (Non-Patent Document 5).

[0053] As shown in Figure 1, it was confirmed that genes under the control of clock genes, such as BMAL1, CLOCK, PER, and CRY, were characteristically expressed in fibrocytes. <Test Example 2> Fibrocyte isolation from mouse tumor tissue The mouse tumor cell line AB1-HA (mesothelioma, BALB / c mouse background) was subcutaneously transplanted into 6-week-old male BALB / c mice (1 × 10 6 Subcutaneous tumor tissue was harvested 21 days after implantation. The tumor tissue was treated with DMEM containing 1 mg / ml BSA (Sigma, USA), 1 mg / ml collagenase IV (Roche, Switzerland), and 100 μg / ml DNase 1 (Sigma) at 37°C for 1 hour, washed, and then physically dispersed using a cell strainer to prepare a cell suspension. The obtained tumor tissue-derived cells were incubated with PE-Cy7-labeled anti-mouse CD45 antibody (1:100 dilution, 30-F11, eBioscience, USA), FITC-labeled anti-mouse CD34 antibody (1:100 dilution, RAM34, BD Pharmingen, USA), and APC-labeled anti-mouse F4 / 80 antibody (1:100 dilution, BM8, BioLegend, USA) on ice for 30 minutes, and tumor-infiltrating fibrocytes were isolated as CD45 and CD34 double-positive cells using a LE-SH800BC cell sorter (Sony, Japan).

[0054] Differentiation induction of tumor tissue-derived fibrocytes and evaluation of the effect of KL001, a CRY stabilizer Mouse tumor tissue-derived fibrocytes collected by the above method were seeded onto a 48-well plate (1.0 × 10 4The cells were cultured for 4 days under conditions containing 1000 cells / well, the CRY stabilizer KL001 (Sigma, 8 μM, Non-Patent Document 4), and TGF-β (10 ng / mL), which is known to induce differentiation into myofibroblasts. RNA was then collected from each group of cells, reverse transcribed to cDNA, and the gene expression of Acta2 (αSMA), collagen1a1 (COL1A1), and RPS29 (a housekeeping gene) was quantified by real-time PCR using the SYBR green method. The expression was compared using the ΔΔCt method.

[0055] The expression levels of the Acta2 and COL1A1 genes are shown in Figure 2. KL001 suppressed the expression of the Acta2 gene regardless of the presence or absence of TGF-β stimulation, confirming that it inhibited the differentiation of fibrocytes into αSMA-positive cancer-associated fibroblasts.

[0056] <Test Example 3> Isolation of fibrocytes and fibroblasts from mouse tumor tissue Col1a2-GFP mice (mice expressing GFP under the collagen promoter) were kindly provided by Professor Yutaka Inagaki of Tokai University. A mouse tumor cell line, LLC (lung cancer, C57BL / 6 mouse background), was subcutaneously transplanted into the Col1a2-GFP mice (1 × 10 6 Thereafter, the tumor tissue was collected on day 21 after transplantation and a cell suspension was prepared in the same manner as in Test Example 2. The obtained tumor tissue-derived cells were reacted with a PE-Cy7-labeled anti-mouse CD45 antibody and detected by flow cytometry as GFP. + CD45 + The cells were identified as fibrocytes, and GFP was detected. + CD45 - The cells were separated as fibroblasts using a cell sorter.

[0057] The fibrocytes and fibroblasts collected by the above method were cultured in the same manner as in Test Example 2 under conditions in which the CRY stabilizer KL001 and TGF-β were added, and the expression level of the Acta2 gene was evaluated.

[0058] The expression level of the Acta2 gene is shown in Figure 3. In fibrocytes, KL001 suppressed the expression of the Acta2 gene, and it was confirmed that differentiation into αSMA-positive cancer-associated fibroblasts was suppressed. - In fibroblasts, KL001 did not affect Acta2 gene expression.

[0059] <Test Example 4> Examination of the effects of KL001, a CRY stabilizer, on subcutaneous tumor-implanted mouse models AB1-HA mesothelioma cell line was subcutaneously implanted into 6-week-old male BALB / c mice, and the CRY stabilizer KL001 was administered intraperitoneally (10 mg / kg, 6 days on, 1 day off) every day from 5 days later. The tumor volume was evaluated twice a week, and tumor tissue was collected 16 days after implantation. The tumor volume was determined by measuring the long and short diameters of the tumor. 2 2 -1 (a, major axis; b, minor axis)

[0060] The recovered tumor tissue was embedded in OCT compound (Sakura Fine Tech, Japan), frozen, and then sliced ​​at 8 μm. The sections were fixed in 4% paraformaldehyde in PBS and immunohistochemically evaluated by fluorescent staining for αSMA (rabbit anti-mouse αSMA antibody, 1:150 dilution, ERR5368, Abcam, USA) as a myofibroblast marker and CD8 (rat anti-mouse CD8a antibody, 1:150 dilution, 53-6.7, BD Pharmingen) as a cytotoxic T cell marker. Stained tissues were imaged using a Keyence BZ-9000 fluorescence microscope (Keyence, Japan). αSMA-positive myofibroblasts in the tumor tissue were quantified by measuring the area of ​​fluorescent dye-positive regions using Image J software (National Institutes of Health, USA).

[0061] The results of tumor volume measurements and fluorescent staining are shown in Figure 4. It was confirmed that KL001 inhibited tumor progression. KL001 also reduced αSMA-positive cancer-associated fibroblasts at the tumor margin and increased CD8 +It was confirmed that this promoted the infiltration of T cells into tumor tissue. <Test Example 5> Examination of the effects of KL001, a CRY stabilizer, on subcutaneous tumor-implanted mouse models Tumor volume was calculated in the same manner as in Test Example 4, except that BALB / c nude mice (deficient in mature T cells) were used instead of 6-week-old male BALB / c mice.

[0062] The results of measuring tumor volume are shown in Figure 5. In BALB / c nude mice with deficient T cell function, KL001 did not have an inhibitory effect on tumor progression.

[0063] <Test Example 6> Examination of the combined effects with immune checkpoint inhibitors The LLC lung cancer cell line was subcutaneously transplanted into 6-week-old male C57BL / 6 mice. Starting 5 days after transplantation, KL001 was administered in the same manner as in Test Example 4, and starting 7 days later, an anti-mouse PD-L1 antibody (5 mg / kg, clone 10F.9G2, BioXCell, USA) was administered. Tumor volume was assessed twice weekly, and tumor tissue was collected 21 days after transplantation. Tumor volume was determined by measuring the long and short diameters of the tumor and then dividing by ab. 2 2 -1 (a, major axis; b, minor axis)

[0064] The results of measuring tumor volume are shown in Figure 6. The combined use of KL001 and anti-PD-L1 antibody demonstrated a significant inhibitory effect on tumor progression compared to the administration of KL001 or anti-PD-L1 antibody alone.

Claims

1. including circadian rhythm period extenders, A composition for use in at least one selected from the group consisting of improving the cancer microenvironment, suppressing immune exclusion, suppressing fibrocyte differentiation, promoting T cell infiltration into tumor tissue, and enhancing the anti-cancer effect of immune checkpoint inhibitors.

2. Circadian rhythm period extenders a stabilizer and / or an expression promoter for at least one protein selected from the group consisting of PER and CRY, and / or The composition according to claim 1, comprising an inhibitor of the function of at least one protein selected from the group consisting of BMAL1 and CLOCK.

3. 3. The composition of claim 1 or 2 for use in combination with an immune checkpoint inhibitor.

4. A composition or kit for treating and / or preventing cancer, comprising a circadian rhythm period extender and an immune checkpoint inhibitor.

5. The composition or kit of claim 4 , wherein the cancer is a solid cancer.

6. Expression of at least one protein selected from the group consisting of PER and CRY in fibrocytes, and / or The function of at least one protein selected from the group consisting of BMAL1 and CLOCK is used as an indicator. A method for screening an active ingredient for use in at least one selected from the group consisting of improving the cancer microenvironment, suppressing immune elimination, suppressing fibrocyte differentiation, promoting T cell infiltration into tumor tissue, and enhancing the anti-cancer effect of immune checkpoint inhibitors.

7. Expression of at least one protein selected from the group consisting of PER and CRY in fibrocytes, and / or The function of at least one protein selected from the group consisting of BMAL1 and CLOCK is used as an indicator. A method for screening active ingredients for use in combination with immune checkpoint inhibitors.

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