Intestinal tumor suppressant, PGE2 production inhibitor, and IL-22BP production promoter

Abstract

The present invention provides an intestinal tumor suppressant capable of suppressing intestinal tumors, a PGE2 production inhibitor capable of suppressing PGE2 production, and an IL-22BP production promoter capable of promoting IL-22BP production. [Solution] An intestinal tumor inhibitor containing a dectin-1 inhibitor or a β-glucan having dectin-1 inhibitory activity that suppresses intestinal tumors via dectin-1 inhibition; a PGE2 production inhibitor containing a β-glucan having dectin-1 inhibitory activity; and an IL-22BP production promoter containing a β-glucan having dectin-1 inhibitory activity.

Description

[Technical Field]

[0001] This disclosure relates to an intestinal tumor suppressant, a PGE2 production inhibitor, and an IL-22BP production promoter. [Background technology]

[0002] Colorectal cancer (CRC) is the third most common cancer worldwide, with more than one million new cases and 500,000 deaths each year (Bray, F., et al., CA Cancer J Clin 68, 394-424 (2018)). Many CRC cases occur in older adults and those with lifestyles typical of developed countries (Bosman, F. & Yan, P., Pol J Pathol 65, 257-266 (2014)), suggesting that diet may be a risk factor for CRC. However, the fundamental mechanisms controlling the development of intestinal tumors are not yet fully understood.

[0003] C-type lectin receptors (CLRs) are pattern recognition receptors that primarily play a role in host defense against pathogens by recognizing pathogen-specific sugar chain structures. Dectin-1 (also known as DECTIN-1, gene symbol: Clec7a or CLEC7A), a member of the bone marrow type II C-type lectin family, is one of the CLRs preferentially expressed in myeloid-derived cells and is a receptor for β-1,3-linked glucans (β-glucans) or glucans with a β-1,3-linked main chain and a β-1,6-linked branched chain (β-glucans) (Non-patent Literature 1: Taylor, PR, et al., J Immunol 169, 3876-3882 (2002)). Because glucans are major cell wall components of most fungi, dectin-1 is at the forefront of the host's defense against fungal infections (Taylor, PR, et al., Nat Immunol 8, 31-38 (2007), Saijo, S., et al., Nat Immunol 8, 39-46 (2007)). However, recent studies have revealed that this receptor is also involved in the development of allergic diseases, autoimmune diseases, and cancers (Tang, C., Makusheva, Y., Sun, H., Han, W. & Iwakura, Y., J Leukoc Biol 106, 903-917 (2019), Brown, GD, Willment, JA & Whitehead, L., Nat Rev Immunol 18, 374-389 (2018)).

[0004] Administration of curdlan, an agonist ligand for Dectin-1, induces arthritis, spondyloarthritis, and ileitis in SKG mice, an autoimmune mouse with a spontaneous mutation in ZAP-70 (Ruutu, M., et al., Arthritis Rheum 64, 2211-2222 (2012), Yoshitomi, H., et al., J Exp Med 201, 949-960 (2005)). Dectin-1-induced IL-22 exacerbates airway hyperresponsiveness by inducing proallergic chemokines and mucus (Lilly, LM, et al., J Immunol 189, 3653-3660 (2012)). Recently, it was reported that deficiency of Dectin-1 leads to excessive proliferation of the commensal bacterium Lactobacillus murinus, which can induce differentiation of regulatory T (Treg) cells, in the mouse colon, and as a result, dextran sulfate sodium (DSS)-induced colitis is suppressed (Tang, C., et al., Cell Host Microbe 18, 183-197 (2015)). Furthermore, Dectin-1 signaling induces IL-17F downstream and induces specific antimicrobial proteins, thereby suppressing the proliferation of a group of commensal bacteria, including Clostridium Cluster XIVa, which promotes Treg differentiation (Kamiya, T., et al., Mucosal Immunol 11, 763-773 (2018), Tang, C., et al., Nat Immunol 19, 755-765 (2018)). Therefore, while Dectin-1 essentially activates the immune system to eliminate pathogens, this activity can also trigger or exacerbate inflammation-related diseases.

[0005] Conflicting results have been reported regarding the role of Dectin-1 in tumor (hereinafter also called polyp) development (Tang, C., Makusheva, Y., Sun, H., Han, W. & Iwakura, Y., J Leukoc Biol 106, 903-917 (2019)). Dectin-1 signaling binds to N-glycan in B16 melanoma cells and induces INAM in DCs, thereby enhancing the tumor-killing activity of NK cells (Chiba, S., et al., Elife 3, e04177 (2014)). Dectin-1 activates Raf1 and NF-κB, increasing the expression of TNFSF15 and OX40L on DCs and promoting the differentiation of antitumor Th9 cells (Zhao, Y., et al., Nat Commun 7, 12368 (2016)). Furthermore, it has been reported that Dectin-1 suppresses the development of hepatocellular carcinoma caused by chemical carcinogens by inducing M-CSF and inhibiting the expression of Toll-like receptor (TLR) 4 and CD14 (Seifert, L., et al., Cell Rep 13, 1909-1921 (2015)). On the other hand, Dectin-1 recognizes the non-canonical ligand galectin-9 expressed in pancreatic ductal adenocarcinoma and suppresses the differentiation of antitumor M1 macrophages, suggesting that Dectin-1 signaling plays a facilitative role in the development of pancreatic tumors (Non-patent Literature 2: Daley, D., et al., Nat Med 23, 556-567 (2017)). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Taylor, PR, et al. The beta-glucan receptor, dectin-1, is predominantly expressed on the surface of cells of the monocyte / macrophage and neutrophil lineages. J Immunol 169, 3876-3882 (2002) [Non-Patent Document 2] Daley, D., et al. Dectin 1 activation on macrophages by galectin 9 promotes pancreatic carcinoma and peritumoral immune tolerance. Nat Med 23, 556-567 (2017) [Overview of the project] [Problems that the invention aims to solve]

[0007] However, knowledge regarding drugs and other treatments for intestinal tumors in previous reports is limited.

[0008] This disclosure is made in view of the above, and aims to provide an intestinal tumor suppressant that can suppress intestinal tumors, a PGE2 production inhibitor that can suppress PGE2 production, and an IL-22BP production promoter that can promote IL-22BP production. [Means for solving the problem]

[0009] The following embodiments are included as specific means to solve the above problems. <1> An intestinal tumor suppressant containing β-glucan with dectin-1 inhibitory activity, which suppresses intestinal tumors through dectin-1 inhibition. <2> The β-glucan is a β-glucan containing β-1,3 links or a β-glucan containing a β-1,3 linkage main chain and a β-1,6 linkage branched chain. <1> An intestinal tumor suppressant as described above. <3> The molecular weight of the aforementioned β-glucan is 0.2K to 100K. <1> or <2> An intestinal tumor suppressant as described above. <4> The aforementioned β-glucan is laminarin. <1> ~ <3> An intestinal tumor suppressant as described in any one of the items. <5> A PGE2 production inhibitor containing β-glucan with dectin-1 inhibitory activity. <6> The β-glucan is a β-glucan containing β-1,3 links or a β-glucan containing a β-1,3 linkage main chain and a β-1,6 linkage branched chain. <5> A PGE2 production inhibitor as described above. <7> The molecular weight of the aforementioned β-glucan is 0.2K to 100K. <5> or <6> A PGE2 production inhibitor as described above. <8> The aforementioned β-glucan is laminarin. <5> ~ <7> A PGE2 production inhibitor as described in any one of the items. <9> An IL-22BP production promoter containing β-glucan with dectin-1 inhibitory activity. <10> The β-glucan is a β-glucan containing β-1,3 links or a β-glucan containing a β-1,3 linkage main chain and a β-1,6 linkage branched chain. <9> An IL-22BP production promoter as described above. <11> The molecular weight of the aforementioned β-glucan is 0.2K to 100K. <9> or <10> An IL-22BP production promoter as described above. <12> The aforementioned β-glucan is laminarin. <9> ~ <11> An IL-22BP production promoter as described in any one of the items. <13> An intestinal tumor suppressant containing a dectin-1 inhibitor, which suppresses intestinal tumors through dectin-1 inhibition. <14> The aforementioned dectin-1 inhibitor is a dectin-1 antagonist. <13> An intestinal tumor suppressant as described above. <15> The Dectin-1 inhibitor is an anti-Dectin-1 inhibitory antibody or a Dectin-1 inhibitory small molecule compound. <13> An intestinal tumor suppressant as described above. <16> The aforementioned intestinal tumor is either an intestinal tumor accompanied by increased PGE2 expression or an intestinal tumor accompanied by decreased IL-22BP expression. <1> ~ <4> , <13> , and <14> An intestinal tumor suppressant as described in any one of the items. <17> A β-glucan having dectin-1 inhibitory activity for use in the prevention or treatment of intestinal tumors. <18> The aforementioned intestinal tumor is an intestinal tumor accompanied by increased expression of PGE2, <17> A β-glucan having dectin-1 inhibitory activity as described above. <19> The aforementioned intestinal tumor is an intestinal tumor accompanied by decreased expression of IL-22BP, <17> or <18> A β-glucan having dectin-1 inhibitory activity as described above. <20> Use of β-glucan having dectin-1 inhibitory activity in the manufacture of pharmaceuticals for the prevention or treatment of intestinal tumors. <21> The aforementioned intestinal tumor is an intestinal tumor accompanied by increased expression of PGE2, <20> Use as described above. <22> The aforementioned intestinal tumor is an intestinal tumor accompanied by decreased expression of IL-22BP, <20> or <21> Use as described above. <23> A method for predicting the severity of an intestinal tumor, comprising measuring at least one of the dectin-1 protein expression level and the CLEC7A gene expression level in tumor tissue. [Effects of the Invention]

[0010] This disclosure provides an intestinal tumor suppressor capable of suppressing intestinal tumors, a PGE2 production inhibitor capable of suppressing PGE2 production, and an IL-22BP production promoter capable of promoting IL-22BP production. [Brief explanation of the drawing]

[0011] [Figure 1a] Dectin-1 deficiency suppresses colorectal tumors, and this suppression is independent of commensal microorganisms. 8-10 week old ApcMin / + and ApcMin / +Clec7a- / - mice were administered 1 mass %DSS dissolved in drinking water for 7 days and euthanized after 4 weeks. Colorectal tumors were observed macroscopically (n=5 / group). [Figure 1b]ApcMin / + and ApcMin / +Clec7a- / - mice aged 8-10 weeks were administered 1% by mass DSS dissolved in drinking water for 7 days and euthanized 4 weeks later. The number of colon polyps of indicated size was counted (n=5 / group). [Figure 1c] ApcMin / + and ApcMin / +Clec7a- / - mice were reared under SPF conditions and euthanized at 20-23 weeks of age. Colon tumors were observed macroscopically (n=14 / ApcMin / + group, n=17 / ApcMin / +Clec7a- / - group). [Figure 1d] ApcMin / + and ApcMin / +Clec7a- / - mice were reared under SPF conditions and euthanized at 20-23 weeks of age. The number of colon polyps was counted (n=14 / ApcMin / + group, n=17 / ApcMin / +Clec7a- / - group). [Figure 1e] Wild (WT) and Clec7a- / - mice were housed separately and administered AOM intraperitoneally. Seven days later, they were given 3 cycles of 1 mass% DSS as described in the "Materials and Methods" section below. Sixteen weeks after the initial AOM administration, the mice were euthanized, and colon tumors were observed macroscopically (n=5 / group). [Figure 1f] Wild-type (WT) and Clec7a- / - mice were housed separately. AOM was administered intraperitoneally, and seven days later, 1 mass %DSS was administered for three cycles as described in the "Materials and Methods" section below. Sixteen weeks after the initial AOM administration, the mice were euthanized, and the number of colon polyps of the indicated size was counted (n=5 / group). [Figure 1g] Wild (WT) and Clec7a- / - mice were cohabited after weaning at 4 weeks of age. Four weeks later, AOM was administered intraperitoneally, followed by 3 cycles of 1% by mass DSS (as described in <Materials and Methods>). Sixteen weeks after the initial AOM administration, the mice were euthanized, and macroscopic observation of colorectal tumors was measured (n=5 / group). [Figure 1h]WT and Clec7a- / - mice were cohabited after weaning at 4 weeks of age. Four weeks later, AOM was administered intraperitoneally, followed by 3 cycles of 1% by mass DSS (as described in <Materials and Methods>). Sixteen weeks after the initial AOM administration, the mice were euthanized, and the number of colon polyps of indicated size was measured (n=5 / group). [Figure 1i] Sterile (GF) wild-type (WT) and Clec7a- / - mice were administered AOM, followed by 3 cycles of 1% by mass DSS administration. These mice were euthanized 36 weeks after AOM administration. Colon tumors were observed (n=4 / GF WT group, n=5 / GF Clec7a- / - group). [Figure 1j] Sterile (GF) wild-type (WT) and Clec7a- / - mice were administered AOM, followed by three cycles of 1% by mass DSS administration. These mice were euthanized 36 weeks after AOM administration. The frequency of individuals with polyps 1 mm or larger in diameter was measured (n=4 / GF WT group, n=5 / GF Clec7a- / - group). [Figure 1k] Sterile (GF) WT and Clec7a- / - mice were administered AOM, followed by 3 cycles of 1% by mass DSS administration. These mice were euthanized 36 weeks after AOM administration. The volume of colonic polyps was calculated as (diameter) 3 (n=4 / GF WT group, n=5 / GF Clec7a- / - group). The data in Figures 1a-1j are representative of at least three independent experiments. Data in Figures 1a, 1c, 1d, 1e, 1g, 1h, 1j are expressed as mean ± SD. *p<0.05, **p<0.01.

[0012] [Figure 2a] In colorectal tumors, dectin-1 signaling promotes the differentiation of MDSCs. ApcMin / + and ApcMin / +Clec7a- / - mice were administered 1 mass% DSS for 7 days and euthanized after 4 weeks. The proportion of T cells, B cells, and DCs in the total tumor-infiltrating cells was examined by flow cytometry (n=3 / ApcMin / + group, n=5 / ApcMin / +Clec7a- / - group). [Figure 2b]ApcMin / + and ApcMin / +Clec7a- / - mice were administered 1% by mass DSS for 7 days and euthanized after 4 weeks. The proportion of MHC-II+CD11c+ cells and MHC-II+CD11c-CD11b+ cells among CD45+ cells infiltrating non-polyp tissue or colonic polyps was examined by flow cytometry (n=3 / ApcMin / + group, n=5 / ApcMin / +Clec7a- / - group). [Figure 2c] WT and Clec7a- / - mice were administered AOM, followed by 3 cycles of 1% by mass DSS administration, and euthanized 12 weeks after AOM administration. Flow cytometry analysis was performed to identify dectin-1 expressing cells in colon polyps and non-polyp tissue. [Figure 2d] WT SPF mice were administered AOM, followed by 3 cycles of DSS. After 11 weeks, these mice were euthanized, and CD11b+ and CD11c+ cells were purified from colon polyps using autoMACS. The purified cells were stimulated with the agonist β-glucan OXCA, or treated with the antagonist β-glucan laminarin for 3 hours followed by OXCA stimulation for 16 hours, and Il6 and Il1b expression was measured by qPCR. Note that the data in (Figures 2a-2d) are representative of two independent experiments, and the data in (Figures 2a, 2b, 2d) are expressed as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0013] [Figure 3a]The expression of most genes representative of the tumor-associated immune system is dependent on Dectin-1 signaling but is not affected by commensal microorganisms. WT and Clec7a- / - GF mice were administered AOM, followed by 3 cycles of 1% by mass DSS. These mice were euthanized 36 weeks after AOM administration (same as the experiment described in Figures 1i-1k), and RNA was purified from polyps and surrounding non-polyp tissue and analyzed by RNA-seq. A heatmap shows a comparison of cytokines, cytokine receptors, chemokines, cell surface markers, and T-cell transcription factor-related genes in WT and Clec7a- / - mice in polyp and non-polyp tissue. [Figure 3b] Using the KEGG database, we compared cell populations, signaling pathways, and metabolic pathways between polyps from GF Clec7a- / - mice and polyps from WT mice. [Figure 3c] A heatmap shows a comparison of WT and Clec7a- / - mice for MDSC and non-MDSC-related genes. [Figure 3d] A heatmap shows a comparison of the prostaglandin E2 (PGE2) synthase coding gene between WT and Clec7a- / - mice. [Figure 3e] Dectin-1 mRNA (Clec7a) expression in colon polyps and non-polyp tissues of GF-treated WT and Clec7a- / - mice was investigated by qPCR (WT n=3, Clec7a- / - n=4). The data in Figure 3e are representative of two independent experiments and are expressed as mean ± SD. *p<0.05.

[0014] [Figure 4a] IL-22bp, which is upexpressed in Clec7a- / - mice, suppresses the development of intestinal tumors in ApcMin mice. ApcMin / + and ApcMin / +Clec7a- / - mice were euthanized after being raised to 20 weeks of age, and the expression levels of Il-22bp and Il18 in intestinal polyps and non-polyp tissue were measured by qPCR (ApcMin / + n=3, ApcMin / +Clec7a- / - n=4). [Figure 4b]The survival rates of the mice shown are (ApcMin / + n=19, ApcMin / +Clec7a- / - n=66, ApcMin / +Clec7a- / -Il-22bp+ / - n=8, ApcMin / +Clec7a- / -Il-22bp- / - n=8). [Figure 4c] Representative images of the small intestine of 20-week-old ApcMin / + and ApcMin / +Clec7a- / - mice, and 15-week-old ApcMin / +Clec7a- / -Il-22bp- / - mice are shown. [Figure 4d] Whole cells from intestinal polyps collected from 23-week-old ApcMin / + and ApcMin / +Clec7a- / - mice were separated into epithelial cells and leukocytes using autoMACS. Il-22bp expression was then examined by qPCR. [Figure 4e] Polyp-infiltrating cells from ApcMin / + mice were analyzed by flow cytometry to examine intracellular expression of IL-22bp in three displayed myeloid cell populations. The numbers in the IL-22bp panel represent the average fluorescence intensity. [Figure 4f] Clec7a- / - mice were administered AOM, followed by 3 cycles of 1% by mass DSS administration, and euthanized 12 weeks after AOM administration. Cells of the indicated types from polyp and non-polyp tissues were purified by autoMACS, and Il-22bp expression was examined by qPCR. [Figure 4g] CD11c+ and CD11c-CD11b+ cells were purified using autoMACS, and Il-22bp expression in these subsets was examined by qPCR. [Figure 4h]C57BL / 6J mice were administered AOM, followed by 3 cycles of DSS. Eleven weeks after AOM administration, these mice were euthanized, and CD11b+CD11c+ cells were purified from colon polyps. Subsequently, the cells were stimulated with OXCA, and Il-22bp expression was measured by qPCR. In the last experimental group, laminarin treatment was performed 3 hours before OXCA treatment. Note that the data in (Figure 4a, Figure 4d~4h) are representative of two independent experiments, and the data in (Figure 4a, 4d, 4f~4h) are expressed as mean ± SD. **p<0.01, ***p<0.001, ****p<0.0001.

[0015] [Figure 5a] The gene involved in PGE2 synthesis is activated by the Dectin-1 signaling pathway. WT and Clec7a- / - mice were administered AOM, followed by 3 cycles of 1% by mass DSS administration, and euthanized 16 weeks after AOM administration. Expression of Ptgs2, which encodes Cox2, was measured by qPCR in colonic polyps and non-polyp sites (WT n=3, Clec7a- / - n=4). [Figure 5b] WT and Clec7a- / - mice were administered AOM, followed by 3 cycles of 1% by mass DSS administration, and euthanized 16 weeks after AOM administration. The concentration of PGE2 in tissue homogenates was measured by ELISA (WT n=5, Clec7a- / - n=4). [Figure 5c] C57BL / 6J mice were administered AOM, followed by 3 cycles of 1% by mass DSS administration, and euthanized 12 weeks after AOM administration. CD11c+ cells and CD11c-CD11b+ cells were purified from colon polyps and non-polyp tissues using autoMACS, and mRNA expression of the indicated genes was measured by qPCR. [Figure 5d] Ptgs2 expression was measured by qPCR in intestinal polyps and non-polyp tissues of 20-week-old ApcMin / + and ApcMin / +Clec7a- / - mice (n=3 / group). [Figure 5e]Ptgs2 expression in tissue-infiltrating CD45+ leukocytes of 20-week-old ApcMin / + and ApcMin / +Clec7a- / - mice was measured by qPCR (n=3 / group). [Figure 5f] All polyp-infiltrating cells from ApcMin / + mice were collected and stimulated with curdlan for 20 hours. Gene expression of PGE2 synthesis-related genes was then measured by qPCR. Laminarin treatment was performed 4 hours prior to curdlan treatment. [Figure 5g] Purified CD11b+ polyp-infiltrating cells were collected from ApcMin / + mice, stimulated with curdlan for 20 hours, and then the expression of genes involved in PGE2 synthesis was measured by qPCR. Laminarin treatment was performed for 4 hours prior to curdlan treatment. The data in Figures 5a-5g are representative of two independent experiments and are expressed as mean ± SD. *p<0.05, **p<0.01, ***p<0.001.

[0016] [Figure 6a] Oral administration of laminarin suppresses tumor formation in the colon, while PGE2 administration promotes tumor formation. C57BL / 6J mice were administered AOM, and from 3 days prior to the administration until the end of 1 week of DSS administration, they were given a powder food containing 5% by mass of laminarin (continued for 10 days / cycle), for a total of 3 cycles. The mice were euthanized 11 weeks after the first AOM administration, and tumor development in the colon was observed macroscopically (n=7 / group). [Figure 6b] C57BL / 6J mice were administered AOM, and from 3 days prior to the administration of AOM until the end of the 1-week DSS administration, they were given a powdered food containing 5% by mass of laminarin (continued for 10 days / cycle), for a total of 3 cycles. The mice were euthanized 11 weeks after the first AOM administration, and the number of colon polyps of the indicated size was counted (n=7 / group). [Figure 6c] C57BL / 6J mice were administered AOM, and from 3 days prior to the administration of AOM until the end of the 1-week DSS administration period, they were given a powdered food containing 5% by mass of laminarin (continued for 10 days / cycle), for a total of 3 cycles. The mice were euthanized 11 weeks after the first AOM administration, and the PGE2 concentration in tissue homogenate was measured by ELISA (n=7 / group). [Figure 6d] C57BL / 6J mice were administered AOM, and from 3 days prior to the administration until the end of the 1-week DSS administration period, they were given a powdered food containing 5% by mass of laminarin (continued for 10 days / cycle), for a total of 3 cycles. The mice were euthanized 11 weeks after the first AOM administration, and the mRNA expression of CD11b (Itgam) was examined by qPCR (n=7 / group). [Figure 6e] Eight-week-old ApcMin / +Clec7a- / - mice (n=3) were administered 2% by mass DSS for one week, followed by intraperitoneal administration of PGE2 (40g / mouse) or PBS every other day for four weeks. After euthanasia, the development of colon polyps in ApcMin / +Clec7a- / - mice was compared to that of ApcMin / + mice, regardless of whether or not they were treated with PGE2 (n=3 / group). [Figure 6f] Eight-week-old ApcMin / +Clec7a- / - mice (n=3) were administered 2% by mass DSS for one week, followed by intraperitoneal administration of PGE2 (40g / mouse) or PBS every other day for four weeks. After euthanasia, the development of colon polyps in ApcMin / +Clec7a- / - mice was compared to that of ApcMin / + mice, regardless of whether or not they were treated with PGE2 (n=3 / group). [Figure 6g] Eight-week-old ApcMin / +Clec7a- / - mice (n=3) were administered 2% by mass DSS for one week, followed by intraperitoneal administration of PGE2 (40g / mouse) or PBS every other day for four weeks. After euthanasia, the populations of MHC-II+CD11c+DCs and MHC-II-CD11b+MDSCs in polyps of ApcMin / +Clec7a- / - mice were measured by flow cytometry (n=3 / group). [Figure 6h] Eight-week-old ApcMin / +Clec7a- / - mice (n=3) were administered 2% by mass DSS for one week, followed by intraperitoneal administration of PGE2 (40g / mouse) or PBS every other day for four weeks. After euthanasia, gene expression was examined by qPCR in ApcMin / +Clec7a- / - mice (n=3 / group). [Figure 6i]Tumor-infiltrating cells (n=2, pooled at the time of collection) from ApcMin / + mice were collected and stimulated with the indicated dose of PGE2 for 20 hours. Gene expression was then measured by qPCR. Data are the mean ± SD of the 3 wells. [Figure 6j] This shows the correlation between Il-22bp and Ptgs2 expression levels in colon polyps of GF or SPF mice after AOM+DSS treatment (n=8 / GF group, n=11 / SPF group). Figures 6a-6h are representative of two independent experiments, and Figure 6i is representative of three independent experiments. Data in Figures 6b-6d, 6f-6i are expressed as mean ± SD. *p<0.05, **p<0.01.

[0017] [Figure 7a] In CRC patients, the expression of PGE2 synthase and IL22RA is altered. Samples were collected from 36 CRC patients, and postoperative survival rates are shown for patients with dectin-1 mRNA expression levels of less than 2 (n=12) or 2-20 (n=24) in tumor tissue (normalized by GAPDH). [Figure 7b] Samples were collected from 36 CRC patients, and the correlation between dectin-1 mRNA expression levels and tumor TNM stage was shown for patients with dectin-1 mRNA expression levels less than 2 (n=12) or between 2 and 20 (n=24) (normalized by GAPDH) in tumor tissue. Tumor TNM stage was calculated according to individual clinical information. [Figure 7c] Samples were collected from 36 CRC patients, and the correlation between the proportion of patients with Dectin-1 mRNA expression levels of 2 or higher at each stage was shown for patients with Dectin-1 mRNA expression levels of less than 2 (n=12) or 2-20 (n=24) (normalized by GAPDH) in tumor tissue. Tumor TNM stages were calculated according to individual clinical information. [Figure 7d] From a total of 68 CRC patients, 23 sets of tumor and non-tumor specimens were collected from the same individuals. The expression of IL-22BP and PTGS2 in tumor and non-tumor tissues was examined by qPCR (tumor group n=47, non-tumor group n=44). [Figure 7e] Twenty-three sets of tumor and non-tumor specimens were collected from the same individual, and the expression of IL-22BP and PTGS2 was examined (n=23 / group). [Figure 7f] Correlation between PGE2 synthase and dectin-1 expression levels in CRC tissue (n=47 / group). [Figure 7g] CRC samples were collected, and tumor-infiltrating cells were stimulated with curdlan for 20 hours. In the case of laminarin treatment, cells were first treated with laminarin for 4 hours, and then cultured with curdlan. PGE2 synthase expression was measured by qPCR. Data are mean ± SD of 3 wells. [Figure 7h] CRC samples were collected, and tumor-infiltrating cells and normal tissue-infiltrating cells were stimulated with curdlan for 20 hours. In the case of laminarin treatment, cells were first treated with laminarin for 4 hours, and then cultured with curdlan. CD33 expression was measured by qPCR. Data are the mean ± SD of 3 wells. [Figure 7i] Tumor tissue was collected from CRC patients, minced, and cultured for 20 hours in the presence of PGE2. Data are the mean ± SD of 3 wells. Note that the data in Figures 7g to 7i are representative of two independent experiments. *p<0.05, **p<0.01, ****p<0.0001.

[0018] [Figure 8a] Dectin-1 deficiency suppresses colorectal tumors, and this suppression is independent of commensal microorganisms. ApcMin / + and ApcMin / +Clec7a- / - mice aged 8-10 weeks were administered 1% by mass DSS for 7 days and euthanized 4 weeks later. Body weight changes were measured over time after DSS administration (n=5 / group). [Figure 8b] ApcMin / + and ApcMin / +Clec7a- / - mice aged 8-10 weeks were administered 1 mass %DSS for 7 days and euthanized 4 weeks later. Colon length was measured after dissection (n=5 / group). [Figure 8c] ApcMin / + and ApcMin / +Clec7a- / - mice were euthanized at 20-23 weeks of age. Small intestinal tumors in the mice were observed macroscopically (n=10 / ApcMin / + group, n=8 / ApcMin / +Clec7a- / - group). [Figure 8d]ApcMin / + and ApcMin / +Clec7a- / - mice were euthanized at 20-23 weeks of age. The number of polyps in the mice was counted (n=10 / ApcMin / + group, n=8 / ApcMin / +Clec7a- / - group). [Figure 8e] Wild (WT) and Clec7a- / - mice were housed separately and administered AOM intraperitoneally. Seven days later, they were given 3 cycles of 1% by mass DSS as described in the "Materials and Methods" section below. Weight loss was measured over time during the induction of colorectal tumors (n=5 / group). [Figure 8f] Wild-type (WT) and Clec7a- / - mice were housed separately and administered AOM intraperitoneally. Seven days later, they were given 3 cycles of 1% by mass DSS as described in the "Materials and Methods" section below. Sixteen weeks after the initial AOM administration, the mice were euthanized, and the length of the colon was measured. [Figure 8g] Wild (WT) and Clec7a- / - mice were administered AOM, and 7 days later, 1 mass% DSS was administered for only 7 days. They were then kept in normal care for another 10 weeks before euthanasia (WT n=8, Clec7a- / - n=9). Anal prolapse was observed in the mice. [Figure 8h] Wild (WT) and Clec7a- / - mice were administered AOM, and 7 days later, 1% by mass DSS was administered for only 7 days, followed by a further 10 weeks of normal rearing before euthanasia (WT n=8, Clec7a- / - n=9). The incidence of anal prolapse and colorectal tumors in the mice was statistically calculated. [Figure 8i] Wild (WT) and Clec7a- / - mice were administered AOM, and 7 days later, 1 mass% DSS was administered for 7 days only. They were then kept in normal care for another 10 weeks before euthanasia (WT n=8, Clec7a- / - n=9). Colon length was measured. [Figure 8j] Wild (WT) and Clec7a- / - mice were administered AOM, and 7 days later, 1 mass% DSS was administered for 7 days only. They were then kept in normal care for another 10 weeks before euthanasia (WT n=8, Clec7a- / - n=9). Colon length was measured. [Figure 8k]Wild (WT) and Clec7a- / - mice were administered AOM, and 7 days later, they were administered 1 mass %DSS for only 7 days, followed by a further 10 weeks of normal rearing before euthanasia (WT n=8, Clec7a- / - n=9). The number of rectal polyps in the mice was measured. [Figure 8l] WT and Clec7a- / - mice were cohabited after weaning at 4 weeks of age. Four weeks later, AOM was administered intraperitoneally, followed by 3 cycles of 1% by mass DSS (see Materials and Methods). Weight loss was measured over time from the start of the first DSS administration, as shown on the x-axis (n=5 / group) (n=5 / group). [Figure 8m] Wild (WT) and Clec7a- / - mice were cohabited after weaning at 4 weeks of age. Four weeks later, AOM was administered intraperitoneally, followed by 3 cycles of 1% by mass DSS (see Materials and Methods). Sixteen weeks after the initial AOM administration, the mice were euthanized, and the length of the colon was measured (n=5 / group). [Figure 8n] Sterile (GF) WT and Clec7a- / - mice were administered AOM, followed by 3 cycles of 1% by mass DSS administration. 36 weeks after AOM administration, these mice were euthanized, and colon length was measured. Data in Figures 8a, 8b, 8e, 8f, 8l, and 8m are representative of three experiments, while data in Figure 8n are representative of two independent experiments. Data in Figures 8c and 8d are pooled data from the three independent experiments. Data in Figures 8a, 8b, 8d, 8e, 8f, and 8j-8n are expressed as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0019] [Figure 9a] The majority of dectin-1 expressing cells in colorectal tumors are MHC-II-myeloid cells. WT or Clec7a- / - mice were administered AOM, followed by 3 cycles of 1 mass% DSS for 7 days each, 7 days later. Mice were euthanized 16 weeks after the initial AOM administration, and mRNA expression of the indicated genes in colon polyps or non-polyp tissue was determined by qPCR (n=3). [Figure 9b]Wild (WT) or Clec7a- / -GF mice were administered AOM, followed by 3 cycles of 1% by mass DSS for 5 days each, 7 days later. Mice were euthanized 36 weeks after the initial AOM administration, and mRNA expression of the indicated genes in colon polyps or non-polyp tissue was determined by qPCR (WT n=4, Clec7a- / - n=5). [Figure 9c] The experiment described in Figure 2a was performed, and a dot plot panel showing the ratios of T cells, B cells, and DCs in tumor-infiltrating cells measured by flow cytometry is shown. [Figure 9d] Wild-type mice were administered AOM, and 7 days later, 1% by mass DSS was administered for a total of 3 cycles of 7 days each. Thirteen weeks after the initial AOM administration, the mice were euthanized, and the dectin-1 expressing cell type infiltrating the colon polyps was measured by flow cytometry. Note that the data in Figures 9a-9d are representative of two independent experiments.

[0020] [Figure 10a] The expression of most genes representative of the tumor-associated immune system is dependent on Dectin-1 signaling but is not affected by commensal microorganisms. Experiments described in Figures 3a-3d were performed, and the expression levels of selected genes encoding immune cell markers and tumor-associated factors in colon polyps or non-polyp tissue were determined by RNA-seq analysis. [Figure 10b] The experiments described in Figures 3a-3d were performed, and the expression levels of Th1 or Th17-related genes in colon polyps or non-polyp tissue were determined by RNA-seq analysis. [Figure 10c] WT and Rag2- / - mice were administered AOM intraperitoneally, and 7 days later, 1 mass %DSS was administered for 3 cycles as described in the "Materials and Methods" section below. Twelve weeks after colon tumor induction, these mice were euthanized, and the colon tumors were observed macroscopically (WT n=8, Rag2- / - n=6). [Figure 10d]WT and Rag2- / - mice were administered AOM intraperitoneally, and 7 days later, 1 mass %DSS was administered for 3 cycles as described in "Materials and Methods" below. Twelve weeks after colon tumor induction, these mice were euthanized and the number of polyps was counted (WT n=8, Rag2- / - n=6). Note that the data in Figures 10c and 10d are representative of two independent experiments and are expressed as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0021] [Figure 11a] Retinoic acid (RA) synthesis is not directly regulated by the Dectin-1 signaling pathway. RNA was prepared as shown in Figures 2e-2j, and the expression of IL-22bp (Il22ra2) and IL-18 (Il18) was measured by qPCR (WT n=4, Clec7a- / - n=5). [Figure 11b] Wild (WT) or Clec7a- / -GF mice were administered AOM, followed by 1 mass% DSS for 5 days for a total of 3 cycles 7 days later. Mice were euthanized 36 weeks after the initial AOM administration, and the expression of genes encoding RA-related synthases was measured by RNA-seq. [Figure 11c] Total RNA was collected from polyp or non-polyp intestinal tissue of WT and Clec7a- / - mice that had been administered AOM followed by three cycles of DSS totaling 16 weeks. The expression of genes shown to be involved in RA synthesis was examined by RTqPCR (WT n=3, Clec7a- / - n=5). [Figure 11d] WT mice were euthanized after being administered AOM followed by 3 cycles of DSS for 12 weeks. CD11b+ and CD11c+ bone marrow-derived cells from the colon were purified by autoMACS. After pre-culturing with laminarin for 3 hours, these cells were stimulated with OXCA for 16 hours, and mRNA expression of genes involved in RA synthesis was examined by RTqPCR. Data in Figures 11a, 11c, and 11d are expressed as mean ± SD. *p<0.05, **p<0.01, ***p<0.001.

[0022] [Figure 12a] Levels of potential dectin-1 endogenous ligand do not change in colorectal polyps. Figure 6i shows a dot panel of flow cytometry analysis performed. [Figure 12b] WT or Clec7a- / -GF mice were administered AOM, followed by 3 cycles of 1 mass% DSS for 5 days each, 7 days later. Mice were euthanized 36 weeks after the initial AOM administration, and the expression of the identified gene encoding a potential dectin-1 endogenous ligand was determined by RNA-seq. [Figure 12c] Total RNA was collected from polyp or non-polyp intestinal tissue of WT and Clec7a- / - mice that had been administered AOM followed by three cycles of DSS totaling 16 weeks. The expression of the indicated gene encoding a potential dectin-1 endogenous ligand was examined by qPCR (WT n=3, Clec7a- / - n=5). Data in Figures 12b and 12c are expressed as mean ± SD. *p<0.05.

[0023] [Figure 13a] PGE2 synthase expression is induced by Dectin-1 signaling in CRC patients. Disease-free survival (left panel) and progression-free survival (right panel) in patients with the CLEC7A gene mutation and control CRC patients without the mutation were compared by analyzing shared data from the TCGA database. [Figure 13b] Non-tumor specimens from a total of 44 CRC patients were collected to show the correlation between PGE2 synthase and dectin-1 expression. [Figure 13c] Fresh CRC specimens were collected postoperatively, and tumor tissue fragments were stimulated with curdlan for 20 hours. In the case of laminarin treatment, the tissue was first treated with laminarin for 4 hours, and then curdlan was added to the culture. PGE2 synthase expression was determined by qPCR. Data are mean ± SD of 3 wells. *p<0.05, **p<0.01, ****p<0.0001. [Modes for carrying out the invention]

[0024] One embodiment of this disclosure is described in detail below. However, this disclosure is not limited to the embodiment described below. In the following disclosure, the components (including elemental steps, etc.) are not essential unless otherwise explicitly stated. The same applies to numerical values ​​and their ranges, and they do not limit this disclosure. In this disclosure, the term "process" includes not only processes that are independent of other processes, but also processes that cannot be clearly distinguished from other processes, provided that the purpose of such process is achieved. In this disclosure, the numerical range indicated using "~" includes the numbers before and after "~" as the lower and upper limits, respectively. In numerical ranges described in stages within this disclosure, the upper or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Furthermore, in numerical ranges described within the text, the upper or lower limit of that range may be replaced with the values ​​shown in the examples. In this disclosure, the content of each component in a composition means the total content of multiple substances present in the composition, unless otherwise specified, if multiple substances corresponding to each component are present in the composition.

[0025] <Intestinal tumor suppressants, PGE2 production inhibitors, and IL-22BP production promoters> In one embodiment, the intestinal tumor suppressant of the present disclosure contains a dectin-1 inhibitor and suppresses intestinal tumors via dectin-1 inhibition. In one embodiment, the intestinal tumor suppressant of the present disclosure contains a β-glucan having dectin-1 inhibitory activity and suppresses intestinal tumors via dectin-1 inhibition. In one embodiment, the PGE2 production inhibitor and IL-22BP production promoter of the present disclosure contain a β-glucan having dectin-1 inhibitory activity.

[0026] The inventors found that dectin-1 deficiency suppresses the development of colorectal tumors, and that this is achieved through the suppression of PGE2 production and the promotion of IL-22BP production, independently of commensal microorganisms. Based on these findings, inhibition of dectin-1, suppression of PGE2 production, and / or promotion of IL-22BP production are considered to be useful strategies for suppressing intestinal tumors.

[0027] In this disclosure, the term "dectin-1 inhibitor" refers to a substance having inhibitory activity on the function of dectin-1. The inhibitory activity on the function of dectin-1 may be, for example, an activity that inhibits the active site of dectin-1, or an activity that inhibits a site other than the active site of dectin-1 (i.e., non-competitive inhibition). In one embodiment, the dectin-1 inhibitor of this disclosure inhibits the function of dectin-1, suppresses the production of PGE2, and promotes the production of IL-22BP.

[0028] Whether a target substance is a Dectin-1 inhibitor, that is, whether the target substance inhibits Dectin-1 function, is confirmed as follows: All cells from the spleen and regional lymph nodes are collected from male C57BL / 6 mice and tested for CD11b by MACS. + &CD11c + After purifying the cells, 4x10 5 Cells are placed in a 96-well culture plate at a cell / well count, and the target substance is added at various concentrations. The cells are then cultured for 3 hours. Subsequently, depleted-Zymosan (InvivoGen, Catalog #tlrl-zyd, USA), which has been treated with polymyxin B for 1 hour, is added to the culture wells at a concentration of 100 μg / ml. After a further 48 hours of culture, the supernatant is collected, and the amount of TNF produced is examined by ELISA. The amount of TNF produced is compared when the target substance is added and when an equal amount of solvent is added instead of the control substance. If TNF production is suppressed, it can be concluded that the function of dectin-1 has been inhibited.

[0029] Dectin-1 inhibitors include binding inhibitors that inhibit ligand binding to dectin-1, dectin-1 activity inhibitors, and signal transduction inhibitors that inhibit signal transduction from dectin-1.

[0030] In one embodiment, a dectin-1 inhibitor is a dectin-1 antagonist. Whether or not a target substance is a dectin-1 antagonist can be determined by using curdlan, which is a dectin-1 agonist ligand. 3 The activity can be confirmed by radiolabeling with [H] and evaluating the inhibitory activity of this radioactivity on the binding of dectin-1. If the binding of curdlan to dectin-1 is significantly inhibited compared to the non-inhibitory state (p=0.05), the β-glucan is evaluated as having dectin-1 inhibitory activity. Dectin-1 inhibitory activity may be inhibited by 10% or more, 20% or more, or 30% or more. Alternatively, agonist activity can also be evaluated by assessing how much cytokine production, such as IL-1β and TNFα, which is induced when dectin-1 expressing cells are stimulated with long-chain β-glucans such as curdlan, is inhibited.

[0031] In one embodiment, β-glucans having Dectin-1 inhibitory activity may be useful as intestinal tumor suppressants. Generally, β-glucans with small molecular weights are known to inhibit Dectin-1. On the other hand, even in the case of β-glucans with large molecular weights, if they are taken up by macrophages via Dectin-1, the large molecular weight β-glucans are broken down into smaller molecular weight, soluble β-glucan fragments. That is, for example, if insoluble β-glucans with large molecular weights are taken orally, some of the β-glucans are broken down into smaller molecular weight soluble β-glucans. It is thought that these soluble β-glucans then inhibit Dectin-1 signaling in the intestinal tract, thereby suppressing PGE2 production, promoting IL-22BP production, and consequently suppressing tumor formation in the intestinal tract. This disclosure is not limited in any way to the estimation mechanism described above.

[0032] Beta-glucan is a polysaccharide formed by the polymerization of glucose via β-1,3 bonds, etc. The materials used to produce beta-glucan are not particularly limited as long as they contain beta-glucan. Beta-glucan is abundant in the cell walls of mushrooms, fungi, yeasts, seaweed, etc., and can be produced using these as materials. Beta-glucan can be produced by known methods. For example, beta-glucan can be extracted by immersing mushrooms, fungi, yeasts, seaweed, etc. in hot water. Among the beta-glucan materials used in this disclosure, seaweed is preferred as a material because it contains a large fraction of beta-glucan with a molecular weight of 0.2K to 100K. Beta-glucan contained in seaweed is called laminarin. In addition, beta-glucan extracted from mushrooms, fungi, yeasts, etc. has a large molecular weight, so it may be reduced in molecular weight by heat treatment or acid treatment, etc.β-glucan extracted from mushrooms, fungi, yeast, etc., is used, for example, in Ishimoto, Y., Ishibashi, KI, Yamanaka, D., Adachi, Y., Kanzaki, K., Okita, K., Iwakura, Y., and Ohno, N. Modulation of an innate immune response by soluble yeast b-glucan prepared by a heat degradation method. Int. J. Biol. Macromol., 104, 367-376 (2017 Nov). doi: 10.1016 / j.ijbiomac.2017.06.036. [Epub 2017 Jun 8]. Or Ishimoto, Y., Ishibashi, KI, Yamanaka, D., Adachi, Y., Kanzaki, K., Iwakura, Y., and Ohno, N. Production of low-molecular weight soluble yeast b-glucans by an acid degradation method. Int. J. It can be reduced to small molecules according to the method described in Biol. Macromol., 107, 2269-2278 (2018 Feb). doi: 10.1016 / j.ijbiomac.2017.10.094. [Epub 2017 Oct 16]. etc.

[0033] Laminarin (CAS registry number 9008-22-4) refers to a β-glucan derived from seaweed such as Eisenia Bicyclis, and is also called laminaran. Laminarin is a type of soluble β-glucan. Laminarin may be a β-glucan containing β-1,3 links, or a β-glucan containing a β-1,6 linked branch chain on a β-1,3 linked main chain. That is, laminarin may have β-1,6 linked side chains on a long chain of β-1,3 linked sugar chains. The structure of laminarin can be represented, for example, by the following general formula (H is present at the non-reducing end, and OH is present at the reducing end). In the following general formula, n1, n2, and m each represent the number of each structural unit. The sum of n1 and n2 represents the length of the 1-3 linked main chain, and m represents the length of the 1-6 linked branch chain. n1, n2, and m all represent integers of 1 or more, preferably integers between 2 and 500. Furthermore, in the general formula below, it is even more preferable that n1+n2 represent an integer between 2 and 25, from the viewpoint of superior antagonist activity against dectin-1. Also, m < (n1+n2).

[0034] [ka]

[0035] Laminarin is abundant in the cell walls of brown algae, such as those in the orders Laminariales and Fucales, and can be extracted from these brown algae. Extraction can be carried out by various methods. For example, laminarin can be extracted by immersing the material in hot water, or by the method described by Matsuda et al. in the Bulletin of Fisheries Sciences, Hokkaido University, 56(3):75-86, December 2005.

[0036] For more details on β-glucans, please refer to International Publication No. 2014 / 136982.

[0037] From the viewpoint of suppressing intestinal tumors, inhibiting PGE2 production, or promoting IL-22BP production, the β-glucan of this disclosure is preferably a soluble β-glucan, preferably a β-glucan containing β-1,3 linkages or a β-glucan containing a β-1,6 linkage branched chain in a β-1,3 linkage main chain, and preferably a β-glucan derived from seaweed. The β-glucan of this disclosure is particularly preferably laminarin.

[0038] In this disclosure, soluble β-glucan is also referred to as water-soluble β-glucan. In this disclosure, soluble β-glucan means β-glucan that can be dissolved in 1 liter of water at 25°C in a quantity of 20 g or more.

[0039] The molecular weight of the β-glucan having Dectin-1 inhibitory activity according to this disclosure (hereinafter also referred to as the β-glucan of this disclosure) is preferably 100K or less, and more preferably 0.2K to 100K, from the viewpoint of inhibiting the activation of Dectin-1. The molecular weight of the β-glucan of this disclosure may be 0.2K to 50K, 1K to 10K, 5K or less, or 0.2K to 5K. The molecular weight of the β-glucan of this disclosure is preferably 5K or less because it exhibits superior inhibitory activity against Dectin-1. The molecular weight distribution of the β-glucan of this disclosure may be unimodal, containing one type of β-glucan with a single molecular weight, or may be bimodal, trimodal or more, or exhibit a smear molecular weight distribution containing two types of β-glucans with different molecular weights.

[0040] In this specification, "molecular weight" refers to the weight-average molecular weight. The molecular weight of β-glucan can be determined by known methods. The weight-average molecular weight can be measured by gel permeation chromatography (GPC). A molecular weight of 1K indicates a molecular weight of 1000.

[0041] More specifically, the weight-average molecular weight analysis of β-glucan is performed using gel filtration chromatography (GPC) under the following conditions. Columns: G6000PWXL and SB802 Column temperature: Pump: 40℃, Column: 60℃ Mobile phase: water Flow rate: 0.5ml / min Detector: RI Sample concentration: 0.1% (w / w) Sample injection volume: 50 μl

[0042] Known methods can be used to obtain β-glucans. For example, a β-glucan mixture containing β-glucans of various molecular weights can be dissolved in distilled water (100 mg / ml), dialyzed against the distilled water at 4°C for 2 days using a dialysis membrane with an arbitrary molecular weight cutoff value, and after dialyzing, the dialyzed internal solution can be collected and freeze-dried to obtain β-glucans of any molecular weight.

[0043] Beta-glucans of a specific molecular weight can be obtained from a mixture containing beta-glucans of various molecular weights by known methods such as ultrafiltration, dialysis, and gel filtration. For example, when preparing beta-glucans with a molecular weight of 100K or less using beta-glucans with a molecular weight of over 100K as a material, the material can also be used by chemical or enzymatic hydrolysis. For chemical hydrolysis, for example, an acid can be used. For enzymatic hydrolysis, for example, an endo-β-glucanase type enzyme can be used. Alternatively, dried powder of brown algae containing a high proportion of beta-glucans of a specific molecular weight can also be used, as long as it has the effects of this disclosure.

[0044] More specifically, a method for obtaining lower molecular weight β-glucan from high molecular weight β-glucan is as follows: 1,000 mg of high molecular weight β-glucan (e.g., β-glucan derived from mold) and 50 ml of 58% (w / w) concentrated sulfuric acid are added to a 200 ml flask while cooling, and the mixture is stirred at 20°C for 3 hours. Next, 350 ml of ice water is added to the flask to dilute it eightfold, and calcium hydroxide is added to neutralize it until the pH reaches 7.0. After filtering the neutralized substance to remove calcium sulfate, the filtered substance is concentrated using an evaporator, centrifuged, and freeze-dried to obtain low molecular weight β-glucan (e.g., β-glucan with a molecular weight of 100K or less).

[0045] In this disclosure, the Dectin-1 inhibitory activity of β-glucan refers to the activity that can inhibit the activation of Dectin-1 by acting as an antagonist of Dectin-1. More specifically, Dectin-1 inhibitory activity may be an activity that directly inhibits the active site of Dectin-1 (i.e., competitive inhibition), or it may be an activity that inhibits a site other than the active site of Dectin-1 (i.e., non-competitive inhibition).

[0046] In this disclosure, the fact that β-glucan has dectin-1 inhibitory activity is demonstrated by curdlan, which is an agonist ligand of dectin-1 [ 3 The activity can be confirmed by radiolabeling with [H] and evaluating the inhibitory activity of this radioactivity on the binding of dectin-1. If the binding of curdlan to dectin-1 is significantly inhibited compared to the non-inhibitory state (p=0.05), the β-glucan is evaluated as having dectin-1 inhibitory activity. Dectin-1 inhibitory activity may be inhibited by 10% or more, 20% or more, or 30% or more. Alternatively, dectin-1 inhibitory activity can also be evaluated by assessing how much cytokine production, such as IL-1β and TNFα, which are induced when dectin-1 expressing cells are stimulated with long-chain β-glucans such as curdlan, is inhibited.

[0047] The suppression of intestinal tumors via dectin-1 inhibition can be confirmed by measuring the expression levels of cytokines regulated by dectin-1 and associated with intestinal tumors, such as GM-CSF, IL-6, IL-1, and TNF; as well as PGE2 and IL-22 binding proteins, using ELISA.

[0048] The method of administering a dectin-1 inhibitor, β-glucan, or a preparation containing the same to the target is not particularly limited and may be oral or parenteral (e.g., subcutaneous, intraperitoneal, intramuscular, eye drops, ear drops, nasal, inhalation, transdermal, rectal, intrathecal, or intravenous), but oral or enteral administration is preferred. Suitable dosage forms for oral administration include tablets, capsules, powders, granules, liquids, elixirs, etc.

[0049] There are no particular restrictions on the subjects to whom dectin-1 inhibitors, β-glucans, or agents containing them are administered, but mammals such as humans, monkeys, cattle, pigs, horses, donkeys, sheep, goats, deer, dogs, cats, rabbits, mice, rats, guinea pigs, hamsters, and squirrels, and birds such as chickens, ducks, geese, pheasants, pigeons, quail, guinea fowl, turkeys, parakeets, and parrots are preferred. Mammals are particularly preferred subjects.

[0050] The dectin-1 inhibitors or β-glucans used in this disclosure are preferably administered to subjects as pharmaceuticals, foods, or feed. The intestinal tumor inhibitors, PGE2 production inhibitors, and IL-22BP production promoters of this disclosure may contain other components, such as sugar chains other than β-glucans, as long as they have the effects of this disclosure.

[0051] Other components include, for example, vitamins, amino acids, excipients, thickeners, isotonic agents (e.g., solutes such as sodium chloride and glucose), and other pharmaceutical additives. Examples of pharmaceutical additives include liquid media such as water, physiological saline, dextrose, or similar sugar solutions; glycols such as ethylene glycol, propylene glycol, polyethylene glycol, and polypropylene glycol; antioxidants such as sulfites; pH adjusters and buffers such as sodium citrate, sodium acetate, and sodium phosphate; stabilizers such as sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic acid; isotonic agents such as sodium chloride and glucose; local anesthetics such as procaine hydrochloride and lidocaine hydrochloride; and surfactants such as dimethyl sulfoxide (DMSO). In addition, other known additives may be included depending on the dosage form.

[0052] The dosage of the intestinal tumor suppressor, PGE2 production inhibitor, and IL-22BP production promoter described herein should be an amount effective in suppressing intestinal tumors, inhibiting PGE2 production, or promoting IL-22BP production. The dosage should preferably be increased or decreased as appropriate depending on age, disease state, symptoms, family history, etc. Multiple doses may also be administered. When multiple doses are administered, the administration interval should preferably be increased or decreased as appropriate depending on the disease state, symptoms, etc.

[0053] In the intestinal tumor suppressant of this disclosure, the intestine may be either the large intestine (more specifically, the cecum, colon, rectum, etc.) or the small intestine (more specifically, the duodenum, jejunum, ileum, etc.), or it may be the large intestine and the small intestine.

[0054] The intestinal tumor suppressant of this disclosure may be an intestinal tumor reduction agent, administered to subjects with intestinal tumors and used therapeutically to reduce the number of tumors already formed. The intestinal tumor suppressant of this disclosure may be an intestinal tumor reduction prophylactic agent, administered to subjects without intestinal tumors and used prophylactically to reduce the number of tumors that may be formed in the future. Alternatively, the intestinal tumor suppressant of this disclosure may be an intestinal tumor reduction agent, administered to subjects with intestinal tumors and used therapeutically to reduce the size of tumors that have already been formed. The intestinal tumor suppressant of this disclosure may be an intestinal tumor reduction prophylactic agent, administered to subjects without intestinal tumors and used prophylactically to reduce the size of tumors that may be formed in the future.

[0055] By administering the intestinal tumor suppressant described herein to the target, PGE2 production in the target can be suppressed. In other words, the intestinal tumor suppressant described herein may be used as a PGE2 production inhibitor.

[0056] By administering the intestinal tumor suppressant of this disclosure to the target, IL-22BP production in the target can be promoted. In other words, the intestinal tumor suppressant of this disclosure may also be used as an IL-22BP production promoter.

[0057] PGE2 (prostaglandin E2) is a type of prostaglandin, which is a physiologically active lipid. IL-22BP (IL-22 binding protein) is a potent regulator of tumorigenesis. The intestinal tumor suppressor disclosed herein is thought to inhibit the Dectin-1 signaling pathway, thereby suppressing the synthesis (production) of PGE2 from MDSCs (myeloid-derived suppressor cells), further inhibiting the proliferation of MDSCs, promoting the production of IL-22BP, and ultimately suppressing intestinal tumorigenesis.

[0058] In this disclosure, intestinal tumor suppression can be evaluated by visual inspection, endoscopic examination, histological examination (e.g., cytology, histopathological examination, etc.), marker tests (e.g., blood tests, urine tests, etc.), or imaging diagnostics (e.g., X-ray examination, CT scan, MRI scan, PET scan, ultrasound examination, etc.) of the intestinal tract. Alternatively, intestinal tumor suppression can be evaluated by a decrease in the protein expression level or mRNA expression level of PGE2 in the intestinal tumor, or by a decrease in the mRNA expression level of enzymes or genes involved in PGE2 synthesis (e.g., Pla2g2a or Pla2g2e (Phospholipase A2 group IIA / IIE), Ptgs1 or Ptgs2 (Cox1 / Cox2), or Ptges2 or Ptges3 (PGE2synthase 2 / 3), etc.). Alternatively, as an indicator of intestinal tumor suppression, it can be evaluated by observing an increase in the protein expression level of IL-22BP or the mRNA expression level of the IL-22BP gene (Il-22bp) in intestinal tumors.

[0059] In this disclosure, suppression of PGE2 production can be determined by a decrease in the protein expression level or mRNA expression level of PGE2 in the sample. The protein expression level of PGE2 can be measured by known methods such as ELISA. The mRNA expression level of PGE2 can be measured by known methods such as real-time PCR.

[0060] In this disclosure, the promotion of IL-22BP production can be determined by an increase in the protein expression level or mRNA expression level of IL-22BP in the sample. The protein expression level of IL-22BP can be measured by known methods such as ELISA. The mRNA expression level of IL-22BP can be measured by known methods such as real-time PCR.

[0061] <<Other Embodiments>> This disclosure also provides a method for preventing or treating intestinal tumors, comprising administering an effective amount of a dectin-1 inhibitor to a target. Furthermore, this disclosure also provides a method for preventing or treating intestinal tumors, comprising administering an effective amount of a β-glucan having dectin-1 inhibitory activity to a target. The intestinal tumor may be an intestinal tumor accompanied by increased PGE2 expression, or an intestinal tumor accompanied by decreased IL-22BP expression.

[0062] Furthermore, this disclosure also provides a dectin-1 inhibitor for use in the prevention or treatment of intestinal tumors. Furthermore, this disclosure also provides a β-glucan having dectin-1 inhibitory activity for use in the prevention or treatment of intestinal tumors. The intestinal tumor may be an intestinal tumor accompanied by increased PGE2 expression, or an intestinal tumor accompanied by decreased IL-22BP expression.

[0063] Furthermore, this disclosure also provides the use of dectin-1 inhibitors in the manufacture of pharmaceuticals for the prevention or treatment of intestinal tumors. Furthermore, this disclosure also provides the use of β-glucans having dectin-1 inhibitory activity in the manufacture of pharmaceuticals for the prevention or treatment of intestinal tumors. The intestinal tumors may be intestinal tumors accompanied by increased PGE2 expression, or intestinal tumors accompanied by decreased IL-22BP expression.

[0064] In the above embodiments, "increased expression" and "decreased expression" can be evaluated by measuring the amount of the target molecule in tumor tissue and non-tumor tissue using ELISA or real-time PCR. Alternatively, the amount of the target molecule in tumor tissue can be measured and compared with a known standard amount.

[0065] Furthermore, this disclosure also provides a method for predicting the severity of intestinal tumors, which includes measuring at least one of the dectin-1 protein expression level and the CLEC7A gene expression level in tumor tissue. The measurement may be performed in vivo or in vitro.

[0066] The mechanism for predicting the severity of intestinal tumors is not clear, but it is presumed to be as follows. Intestinal tumors are promoted by activation of the Dectin-1 signaling pathway. Conversely, intestinal tumors are suppressed by inhibition of the Dectin-1 signaling pathway. Furthermore, Dectin-1 protein expression levels, or the expression levels of the CLEC7A (C-Type Lectin Domain Containing 7A) gene encoding Dectin-1 protein, are positively correlated with the severity of intestinal tumors. Therefore, by measuring Dectin-1 protein expression levels or the CLEC7A gene expression levels, it is thought that higher expression levels are associated with higher intestinal tumor severity, and lower expression levels are associated with lower intestinal tumor severity. This disclosure is not limited in any way to the estimation mechanism described above.

[0067] The method for measuring the expression levels of Dectin-1 protein and CLEC7A gene in tumor tissue is not particularly limited, and commonly performed protein quantification methods (e.g., ELISA) or gene quantification methods (e.g., mRNA) (e.g., real-time PCR) may be used.

[0068] In each of the above embodiments, the details of the requirements such as the dectin-1 inhibitor, β-glucan having dectin-1 inhibitory activity, target, intestinal tumor, PGE2, IL-22BP, etc., can be determined by applying the matters described above. [Examples]

[0069] The present disclosure will be further described below with reference to examples, but the present disclosure is not limited to the following examples unless it exceeds the spirit of the disclosure. Unless otherwise specified, "%" is based on mass.

[0070] 1. Dectin-1 deficiency suppresses the development of colorectal tumors in a manner independent of commensal microorganisms. To investigate the potential role of Dectin-1 in intestinal tumorigenesis, we first examined Apcs with mutations in the β-catenin signaling pathway. Min (Apc Min -DSS) The effect of Dectin-1 signaling on colorectal tumor formation in DSS-treated mice was investigated (Reference 18). ApcMin / + Dectin-1 deficient (Clec7a - / - ) mice showed significantly milder weight loss after DSS administration than Apc Min / + mice (Figure 8a). On day 28 after DSS administration, shortening of the colon length, one of the symptoms of colitis, was as mild as that in Clec7a - / - mice treated with only DSS (Reference 11), and was also mild in Apc Min / + Clec7a - / - mice (Figure 1a, 8b). The number of polyps in the colon was significantly decreased in Apc Min / + Clec7a - / - mice (Figure 1a, 1b).

[0071] Furthermore, the effect of Dectin-1 deficiency on tumor development in Apc Min mice without DSS administration was examined. After euthanizing mice at 20 - 23 weeks of age, it was found that polyp development in both the colon and small intestine was significantly suppressed in Apc Min / + Clec7a - / - mice compared to Apc Min / + mice (Figure 1c, 1d, 8c, 8d). In the small intestine of Apc Min / + Clec7a - / - mice, the number of polyps less than 3 mm was significantly decreased (Figure 8d).

[0072] Next, using another chemically induced colorectal tumor model, wild-type (WT) and Clec7a - / - mice were administered azoxymethane (AOM) and DSS for 3 cycles (AOM-3DSS). Clec7a - / - mice showed milder weight loss than WT mice during 3 cycles of DSS administration (Figure 8e). In Clec7a - / - mice, shortening of the colon length was significantly mild (Figure 1e, 8f). The number of polyps in Clec7a - / - mice induced by AOM-3DSS was significantly less than that in WT mice (Figure 1f). Clec7a - / -In mice, when only AOM and one cycle of DSS were performed, anal prolapse (Figure 8g, 8h) and colonic shortening (Figure 8i, 8j) were significantly milder. Colon polyps developed in all WT mice, but Clec7a - / - In mice, small polyps developed in half of the mice (Figure 8h, 8k). These results indicate that blocking the Dectin-1 signaling pathway suppresses intestinal tumor formation.

[0073] We previously found that colony formation of Lactobacillus murinus, a commensal bacterium that induces regulatory T cells (Tregs), is linked to Clec7a - / - It was reported that Foxp3 is elevated in the mouse gut and suppresses intestinal inflammation (Reference 11). In fact, real-time RT-PCR revealed that Foxp3 (Treg marker) expression in polyp and non-polyp tissue was significantly increased in Dectin-1-deficient mice (Figure 9a). Clec7a - / - To investigate the influence of the gut microbiota on the control of tumor formation in mice, we studied Clec7a after weaning. - / - We continuously housed mice and wild-type mice together and administered AOM-3DSS to these cohabiting mice to induce colorectal tumors. However, Clec7a - / - The number of polyps in the colon of mice was significantly lower than that of wild-type mice, and particularly large polyps with a diameter of 3 mm or more were observed in wild-type mice (Figure 1g, 1h).

[0074] To further analyze the influence of the commensal microbiome on the control of tumorigenesis by Dectin-1, sterile (GF) WT and Clec7a - / - Mice were administered AOM-3DSS. After 36 weeks following AOM administration, the mice were euthanized. The length of the colon was nearly identical between the two groups of mice, but colon polyps developed only in the wild-type mice. - / - This did not occur in mice (Figure 1i-1k). In these GF mice, intestinal Foxp3 expression was different from that of WT mice and Clec7a - / - The results were similar in mice (Figure 9b). From these results, Clec7a - / -The suppression of tumor development in mice was shown to be independent of commensal microorganisms.

[0075] 2.Clec7a - / - In mouse polyps, the infiltration of myelo-derived suppressor cells (MDSCs) is reduced. Next, Apc Min / + Clec7a in the background - / - Mice were administered 1% by mass DSS, and changes in the tumor-infiltrating cell population in the colon were analyzed. Flow cytometry analysis revealed that Apc Min / + Clec7a - / - In mouse polyps, T cells (including both αβ-type T cells and γδ-type T cells), B cells (IgG + (including B cells), DCs (MHC-II + CD11c + Cells) are Apc Min / + Compared to mice, MHC-II levels were significantly increased (Figure 2a, 9c), - CD11c - CD11b + A significant decrease in myeloid-derived suppressor cells (MDSCs) was observed (Figure 3b).

[0076] Dectin-1 is widely known to be expressed in DCs, but in mouse colon polyps, flow cytometry analysis revealed that Dectin-1 expression is mainly in CD103 - CD11c - CD11b + It was observed in cells (Figure 2c, 92.4%). Furthermore, when the population expressing Dectin-1 was analyzed, Dectin-1 + Nearly 75% of them are MHC-II - And nearly 50% of them are Ly6C int Ly6G + CD11b + 5% is Ly6C hi CD11b + 19% have CD11c - MHC-II - Gr1 - CD11b +These were cells (Figure 9d). These tumor-infiltrating myeloid cells are generally known as granulocytic polymorphonuclear cells and monocytic MDSCs (Reference 22). IL-6 and IL-1β have been reported to promote the proliferation of MDSCs (References 23, 24), and CD11b cells infiltrating intestinal polyps were observed. + Cells and CD11c + Stimulation of cells with Candida albicans-derived β-glucan OXCA induced the expression of Il6 and Il1b, and this induction was inhibited by the dectin-1 antagonist laminarin (Figure 2d), suggesting that MDSC-promoting factors are directly induced by the dectin-1 signaling pathway.

[0077] 3. Several tumor control pathways are dependent on dectin-1 signaling. To elucidate the mechanism by which Dectin-1 signaling controls tumorigenesis, gene expression in tumor and non-tumor tissues of GF mice administered AOM-3DSS was examined by RNA-seq analysis. Under GF conditions, the overall gene background was lower than under SPF conditions, making it easier to identify Dectin-1 regulatory genes. As a result, Clec7a - / - In mouse colon polyps, genes associated with T cells (Cd3e, Cd28, Cd69, Ccr6, Ccr9, etc.), Th1 cells (Cd4, Tbx21, Ifng, Il12a, Cxcr3, etc.), B cells (Cd19, Ig-associated, Cxcr5, etc.), and DCs (Itgax, H2-Ab1, Ccr7, etc.) were significantly increased, while Th17-related genes (Il17a, Il23a, etc.) were decreased (Figures 3a, 10a, 10b). Clec7a - / - In mouse colon polyps, the expression of tumor regulatory cytokines such as IL-22 (Il22) and IL-18 (Il18) remained unchanged, but the expression of IL-22-binding proteins (Il22bp or Il22ra2), which are IL-22 antagonist receptors, was significantly increased (Figures 3a, 10a). Furthermore, KEGG gene enrichment analysis revealed that immune-related genes such as those involved in antibody production, lymphocyte activation, NK cell-mediated cytotoxicity, and antigen presentation were increased in Clec7a - / -It was found that the levels were elevated in mouse tumor tissue compared to WT mice (Figure 2b). On the other hand, Clec7a - / - In the mouse colon, pathways such as Wnt signaling, IL-17 signaling, Ras signaling, and arachidonic acid metabolism were suppressed (Figure 3b). Clec7a - / - In mouse polyps, the expression of MDSC-related genes (reference 25) is suppressed, and Clec7a - / - This was consistent with impaired MDSC invasion in mouse colorectal tumors (Figure 2b). Also, Clec7a - / - In mouse polyps, the expression of Csf2, Il6, Il1b, and Tnf, which promote MDSC differentiation, was significantly impaired (References 23, 24) (Figures 3a, 10a, 10b). Interestingly, Clec7a - / - In mouse polyps, we also found that the expression of genes encoding prostaglandin E2 (PGE2) synthases, such as Pla2g2a (phospholipase A2), Ptgs1 (Cox1), and Ptgs2 (Cox2), was reduced (Figure 3d, 10a). In colorectal polyps, the expression of the dectin-1 gene Clec7a was elevated compared to non-polyp tissue (Figure 3e), which was thought to reflect the expansion and invasion of MDSCs, which are the major dectin-1 expressing cells in colorectal tumors (Figure 2b).

[0078] T cells and antibody-producing B cells are both involved in antitumor immunity (References 19-21), but Rag2 - / - When AOM-3DSS was administered to mice, mice lacking lymphocytes developed even fewer polyps compared to wild-type mice (Figures 10c, 10d), indicating that these lymphocytes are not involved in the suppression of colorectal cancer in mice.

[0079] 4. Dectin-1 deficiency suppresses the development of colorectal tumors by promoting the production of IL-22 binding protein. GF Clec7a - / - In polyps from AOM-3DSS mice, IL-22bp expression was significantly elevated (Figures 3a, 10a). Min / + Clec7a - / -In the intestines of -DSS mice, the expression of IL-22bp mRNA was enhanced not only in polyps but also in non-polyp tissues (Figure 4a, 11a). Although IL-18 has been reported as a direct suppressor of IL-22bp, GF Clec7a - / - and Apc Min / + Clec7a - / - In both mice, the expression of Il18 mRNA was normal (Figure 4a, 11a) (Reference 26).

[0080] Apc Min / + Clec7a - / - IL-22bp - / - mice were generated. As a result, the lifespan extension compared to Apc Min / + Clec7a - / - mice was completely canceled by IL-22bp deficiency, and even half IL-22bp deficiency was found to shorten the lifespan of Clec7a Min / + mice (Figure 4b). In Apc - / - Clec7a Min / + Clec7a - / - the number of polyps decreased compared to Apc Min / + mice (Figure 8c, 8d), but more polyps were observed in younger Apc Min / + Clec7a - / - Il-22bp - / - mice (Figure 4c). From these results, it was found that upregulation of IL-22bp is important for suppressing intestinal tumorigenesis in Clec7a - / - mice.

[0081] To identify IL-22bp-expressing cells in the intestine, EpCAM + epithelial cells and CD45 + leukocytes were separated from intestinal polyps, and it was confirmed by qPCR that Il-22bp mRNA was expressed only in the CD45 + population (Figure 4d). By flow cytometry, the IL-22bp protein was found to be CD11b - CD103 + and CD11b + CD103 intCD11c was expressed in both DCs, but - CD11b + It was not expressed in MDSCs (Figure 4e). Il-22bp was expressed in both colorectal polyp and non-polyp tissues, and Thy1.2 + T cells, CD19 + B cells, EpCAM + Not epithelial cells, but CD11b + and CD11c + It is expressed only in myeloid cells (Figure 4f), and its expression was mainly detected in DCs, but CD11c - CD11b was not detected in MDSC (Figure 4g). + and CD11c + Since treating cells with dectin-1 ligand did not affect Il-22bp expression (Figure 4h), it is thought that dectin-1 is not directly involved in the regulation of Il-22bp expression.

[0082] Retinoic acid (RA) has been reported to regulate IL-22bp production (References 27, 28), and Clec7a - / - In mouse polyps, the expression of enzymes that catalyze RA synthesis, such as Adh1 and Aldh1a1, was upregulated (Figures 11b, 11c). However, β-glucan did not affect the expression of these enzymes (Figure 11d), suggesting that RA synthesis is not directly regulated by the Dectin-1 signaling pathway.

[0083] 5. Dectin-1 signaling directly promotes PGE2 production. RNA-seq data obtained from GF mice administered AOM-3DSS showed that the expression of genes involved in PGE2 synthesis, such as Pla2g2a / 2e (Phospholipase A2 group IIA / IIE), Ptgs1 / 2 (Cox1 / Cox2), and Ptges2 / 3 (PGE2synthase 2 / 3), was significantly elevated in polyps from WT mice, but Clec7a - / - The expression level was not elevated in mouse polyps (Figure 3d, 10a). The decrease in Ptgs2 expression in colon polyps and non-polyp tissue was observed under SPF conditions in Clec7a- / - This was also observed in mice (Figure 5a), and the PGE2 concentration in non-polyp tissue homogenate was Clec7a - / - It was significantly reduced in mice (Figure 5b). The enzyme involved in PGE2 synthesis is CD11c - CD11b + CD11c was preferentially expressed in MDSCs. + DCs also expressed these genes at low levels (Figure 5c).

[0084] Decreased expression of Ptgs2 in intestinal polyps is associated with Apc Min / + Clec7a - / - This was also observed in mice (Figure 5d), and this decrease is related to CD45 + This was more evident in intestinal leukocytes (Figure 5e). Apc Min / + When mouse polyp-infiltrating leukocytes were treated with curdlan (i.e., β-1,3-glucan, derived from the soil bacterium Alcaligenes faecalis var. myxogenes, weight-average molecular weight over 100K (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan)), an insoluble β-glucan and dectin-1 agonist, the expression of the gene encoding PGE synthase 3 (Ptges3), the gene encoding Cox1 (Ptgs1), and the gene encoding Cox2 (Ptgs2) was significantly induced, and this induction was suppressed by the dectin-1 antagonist (inhibitor) laminarin (Figure 5f). Purified polyp-infiltrating CD11b + Stimulation and blockade of the Dectin-1 signaling pathway in cells similarly altered PGE2 synthase expression (Figure 5g), indicating that the Dectin-1 signaling pathway directly induces PGE2 synthesis in intestinal tumor-infiltrating MDSCs.

[0085] 6. Administration of dectin-1 antagonists suppresses the development of intestinal polyps by reducing PGE2 in the intestines. To investigate the therapeutic effect of dectin-1 blockade on intestinal tumor formation, wild-type mice were administered AOM-3DSS, and during three cycles of DSS administration, the mice were fed a diet containing 5% by mass of laminarin (laminarin derived from arame seaweed (Tokyo Chemical Industries, Ltd., Japan, Tokyo), weight-average molecular weight less than 50K) which is a soluble β-glucan and a dectin-1 antagonist. Clec7a - / - Similar to observations in mice, mice treated with laminarin showed significantly fewer colon polyps compared to control mice (Figures 6a, 6b). Blocking the Dectin-1 signaling pathway suppressed PGE2 production in the mouse intestinal tract (Figure 6c), and consequently, CD11b (Itgam) expression also decreased (Figure 6d). On the other hand, DSS-treated Apc Min / + Clec7a - / - When mice were intermittently administered PGE2 for 4 weeks, the number of polyps decreased. Min / + The increase was comparable to that in mice (Figures 6e, 6f). In polyps administered with PGE2, CD11c + DC and T cell infiltration was suppressed, but CD11b +MDSC invasion was promoted (Figure 6g, 12a). Furthermore, PGE2 administration increased the expression of MDSC markers CD11b (Itgam) and arginase-1 (Arg1), and oncogenes Cyclin D1 (Ccnd1) and c-Myc (Myc), while suppressing Il-22bp expression (Figure 6h). This suggests that MDSC invasion, tumor growth, and IL-22bp production in colorectal cancer are regulated by PGE2. To confirm the regulatory function of PGE2, intestinal polyp-infiltrating cells were stimulated with PGE2, and it was found that the expression of MDSC markers such as Arg1, CD11b, and Nos2 was directly induced, and IL-22bp was directly suppressed by PGE2 administration (Figure 6i). Interestingly, in colorectal polyps, Cox2 mRNA levels were found to be negatively correlated with Il-22bp expression under both GF and SPF conditions (Figure 6j). These results suggest that inhibiting the Dectin-1 signaling pathway suppresses PGE2 synthesis from MDSCs, further inhibiting MDSC proliferation, promoting IL-22bp, and ultimately suppressing intestinal tumor formation.

[0086] 7. Dectin-1 signaling promotes PGE2 synthesis in colorectal cancer patients. To evaluate the role of Dectin-1 signaling in the development of CRC in humans, we first collected CRC specimens and examined CLEC7A expression. The results showed that patients with high CLEC7A expression levels (relative expression level ≥2, normalized by GAPDH) had significantly lower postoperative survival rates than patients with low CLEC7A expression levels (<2) (Figure 7a). Next, we noticed a positive correlation between CLEC7A expression levels and the pathological malignancy of the tumors (Figures 7b, 7c). Furthermore, while disease rates and progression rates were relatively lower in CRC patients with CLEC7A mutations, this trend was not statistically significant due to the small number of CRC patients with CLEC7A mutations in the TCGA (Cancer Genome Atlas) database (Figure 13a). These observations suggest that Dectin-1 signaling promotes the development of CRC in humans.

[0087] To determine whether the "dectin-1-PGE2-IL-22bp axis," discovered in mice, may also exist in humans, we first examined the mRNA expression of IL-22BP and COX2 in tumors of CRC patients. In tumor tissue, IL-22BP was relatively decreased and PTGS2 was significantly increased compared to non-tumor tissue (Figure 7d), and this trend was even more pronounced when comparing samples from the same individual (23 pairs of tumor and non-tumor tissue) (Figure 7e). Furthermore, we found a strong positive correlation between the expression levels of CLEC7A in the intestines of CRC patients and the expression levels of PLA2G2A, PTGS2, and PTGES (Figures 7f, 13b), and confirmed that administering curdlan to CRC tissue and cancer-infiltrating leukocytes induced the expression of the PGE2 synthase gene (Figures 7g, 13c). Furthermore, Curdran increased the expression of CD33, a common marker for human MDSCs (Reference 29), in both tumor and normal tissue (Figure 7h), suggesting that Dectin-1 signaling also promotes MDSCs in CRCs. The expression of CD33 and CD11B (ITGAM) in CRCs was directly induced in vitro by PGE2, and human IL-22BP was also dose-dependently suppressed by PGE2 (Figure 7i). These results suggest the existence of a "Dectin-1-PGE2-IL-22BP axis" that may be involved in the regulation of human colorectal carcinogenesis.

[0088] ≪Consideration≫ Currently, β-glucans are known to have no chemical cytotoxic effects. Most studies suggesting β-glucan cytotoxicity have used crude extracts of β-glucan-containing herbs such as Ganoderma lucidum, but among these, other active ingredients such as ganoderic acid from its mycelium (Reference 30) and triterpenes from its spores (Reference 31) are known to actually have direct anticancer effects. Previous studies have shown that oral administration of β-glucans enhances the antitumor effect of antitumor agents (Reference 32), and that oral administration of yeast-derived β-glucan particles suppresses the growth of Lewis lung cancer subcutaneously inoculated (Reference 33). However, Cheung et al. tracked the in vivo processing of glucans after oral ingestion by labeling them with fluorescein and revealed that large β-glucan molecules are taken up by macrophages via dectin-1 and then broken down into smaller, soluble β-1,3-glucan fragments (Reference 34). Since laminarin, a representative soluble β-glucan, is an antagonistic ligand for dectin-1, it is thought that after oral ingestion, some of the large molecular weight insoluble β-glucans are broken down into laminarin, inhibiting the dectin-1 signaling pathway in the intestinal tract, and consequently exhibiting anti-intestinal tumor formation.

[0089] In this study, inhibiting Dectin-1 was used to study AOM-DSS-induced CRC models and Apc Min We demonstrated that Clec7a effectively suppresses the development of colorectal tumors in a mouse model of familial adenomatous polyposis colorectal tumors. - / - In mice, the expression of PGE2 synthases such as Cox2, Cox1, and Ptges3 was reduced, resulting in decreased levels of PGE2, which promotes the proliferation of MDSCs and tumor development. Furthermore, the expression of IL-22bp, which can suppress the development of colorectal cancer by inhibiting IL-22 activity, was reduced in Clec7a. - / -Dectin-1 expression levels were significantly increased in mice. Furthermore, CRC patients with high Dectin-1 expression levels had shorter survival times compared to CRC patients with low expression levels. A positive correlation was observed between Dectin-1 expression levels and the stage of colorectal cancer. In CRC patients, IL-22BP expression was decreased and PTGS2 expression was increased in tumors compared to normal tissue. These results suggest that Dectin-1 plays an important role in the development of colorectal cancer in mice and humans by regulating PGE2 concentration and IL-22bp expression.

[0090] PGE2, a well-known major mediator of inflammatory diseases, binds to the receptor EP2 and activates the β-catenin axis, thereby promoting the proliferation of colorectal cancer cells and the expression of inflammation and growth-related genes such as Tnf, Il6, Cxcl1, and Cox2, and playing a crucial role in the development of AOM-DSS-induced colorectal tumors (References 36, 11, 37). In fact, intestinal polyposis in Apc-deficient mice is suppressed in Ptgs2-deficient mutant mice, and COX-2 inhibitors, such as NSAIDs like aspirin, can suppress the development and progression of colorectal cancer and polyps (References 38, 39). In this study, we found that dectin-1 deficiency affects the expression of a series of enzymes involved in PGE2 synthesis, particularly phospholipase A2 and Cox2, in relation to AOM and Apc Min We revealed that this is impaired in both induced intestinal tumors. We also showed that Dectin-1 signaling directly induces the expression of these PGE2 synthases in MDSCs that have infiltrated the tumor.

[0091] In fact, Clec7a - / - In mice, intestinal levels of PGE2 were reduced, and colonic polyposis were reversed by administration of exogenous PGE2. These results indicate that Dectin-1 signaling promotes PGE2 synthesis, thereby promoting intestinal tumorigenesis. Previous reports have suggested the involvement of Dectin-1 in PGE2 production by human neutrophils in allergic airway inflammation caused by house dust (Reference 40) and the fungus Paracoccidioides brasiliensis (Reference 41).

[0092] IL-22BP is a soluble inhibitory receptor for IL-22 (References 42, 43). IL-22BP plays a crucial role in regulating tumorigenesis, and deficiency of this cytokine promotes the development of colorectal tumors (Reference 26). This study demonstrates that deficiency of IL-22bp completely eliminates polyp formation impairment caused by dectin-1 deficiency, and Clec7a - / - This suggests that IL-22bp plays a significant role in suppressing tumor formation in mice. It was also found that IL-22bp is produced only in intestinal dendritic cells (DCs) and not in mediolytic cell clusters (MDSCs), and that IL-22bp expression in tumor-infiltrating cells is directly suppressed by PGE2, an enzyme mainly secreted by MDSCs.

[0093] Dectin-1 expression in colorectal tumors was also mainly detected in MDSCs, suggesting that impaired intestinal PGE2 production due to dectin-1 deficiency is related to Clec7a - / - It is thought that this leads to upregulation of Il-22bp expression in mouse DCs, and consequently suppresses tumor formation. Supporting this idea, Clec7a - / - When PGE2 was administered to mice, the expression of Il-22bp in the colon was suppressed, and the tumor development impairment caused by Dectin-1 deficiency was eliminated.

[0094] The accumulation of MDSCs in tumors is thought to promote cancer progression by facilitating invasion, angiogenesis, and metastasis, and by inhibiting anti-tumor immunity (References 44, 45). In this study, we discovered that cytokines such as GM-CSF, IL-6, IL-1, and TNF (References 23, 24), which are known to promote or enhance the differentiation and inhibitory activity of MDSCs in mouse intestinal tumors, are downstream of the Dectin-1 signaling pathway. - / -This study explains the decrease in MDSC population and MDSC-related gene expression in mouse colon polyps. Furthermore, it shows that MDSCs are major producers of PGE2 synthase, and that PGE2 promotes MDSC differentiation by inducing Itgam and Arg1 in mice, and ITGAM and CD33 in human intestinal tumor-infiltrating cells. These results suggest that Dectin-1 may promote MDSC expansion and differentiation, thereby accelerating intestinal tumorigenesis, through an auto-amplification loop of inflammatory cytokine and PGE2 production. Clec7a - / - In mice, MDSCs suppress the antitumor response, suggesting that a decrease in the number of MDSCs promotes lymphocyte-mediated antitumor immunity.

[0095] It has been suggested that the gut environment, including the gut microbiota and inflammatory environment, influences the development of colorectal tumors. In this study, it took 36 weeks for obvious colorectal tumors to be induced in GF mice administered AOM-DSS, compared to 16 weeks in SPF mice. Chronic intestinal inflammation, such as inflammatory bowel disease (IBD), is considered a risk factor for intestinal tumor formation (Reference 46), but several clinical studies have shown only a weak correlation between IBD and gastrointestinal malignancies (Reference 47). We previously showed that Dectin-1 signaling suppresses the growth of Treg-inducing bacteria such as Lactobacillus and Clostridium species by directly inducing antimicrobial calprotectin on neutrophils or by inducing antimicrobial peptides via IL-17F (References 11-13). Thus, blocking Dectin-1 signaling can increase colorectal Treg cells and suppress colitis in mice (References 11-13). However, in this study, Clec7a - / - When mice are housed together with WT mice, or under GF conditions, Clec7a - / - Even when mice are raised, the number of polyps is lower than in WT mice, therefore Clec7a - / - It was concluded that commensal bacteria are not involved in the reduction of polyp formation in mice. Clec7a - / - In GF mice, Tregs did not increase, and Rag2 lacked lymphocytes. - / -Since it also demonstrates sufficient ability to prevent the development of intestinal tumors in mice, Clec7a - / - In the mouse gut, it is thought that a decrease in PGE2 and an increase in IL-22BP, rather than Tregs, suppress polyp formation.

[0096] We demonstrated that oral administration of laminarin, an antagonistic ligand for dectin-1, suppresses colorectal polyposis induced by AOM-DSS in mice. Regarding the source of the dectin-1 ligand, the mouse diet contains more than 10% by mass of yeast extract, which is rich in glucans. Furthermore, our diets also contain many foods rich in glucans, such as mushrooms, yeast, and seaweed. Recent reports have confirmed the presence of galectin-9, an endogenous ligand, in pancreatic cancer tissue (Reference 17). In this study, we investigated the expression level of galectin-9 and found that Clec7a - / - In mice, expression was increased in polyps compared to other tissues, but not in wild-type mice (Figures 12b, 12c). In summary, this study revealed that Dectin-1 plays an important role in intestinal tumorigenesis by promoting the suppression of IL-22bp via MDSC-PGE2. This suggests that Dectin-1 is a promising target for the prevention and treatment of CRC.

[0097] Materials and Methods 1. Mouse Clec7a - / - Mice were used after backcrossing C57BL / 6J for nine generations. Wild-type (WT) C57BL / 6J mice purchased from Sankyo Lab Service were used as a control. Apc was provided by Professor Ryo Abe of Tokyo University of Science. Min / + Mouse Clec7a - / - By crossbreeding with mice, Apc Min / + Clec7a - / - A mouse was created. Apc Min / + Clec7a - / - Il-22bp - / - The mouse is Apc Min / + Clec7a - / -The mice were prepared using the CRISPR-Cpf1 method with conjugates. Age- and sex-matched wild-type mice were weaned at 4 weeks of age and then given Clec7a - / - Mice were either isolated or housed together. All mice were raised in controlled cleanrooms at the experimental animal facilities of the Institute of Biomedical Sciences, Tokyo University of Science and the Faculty of Medicine, Sun Yat-sen University, under conditions free from specific pathogens, using X-ray sterilized normal food, acidified tap water (0.002N HCl, pH 2.5), and autoclaved wooden chip beds. All animal experiments were conducted using protocols approved by the Animal Experiment Committee of Tokyo University of Science and the Committee for the Management and Use of Experimental Animals of Sun Yat-sen University, in accordance with the institutions' ethical codes and guidelines.

[0098] 2. Human CRC sample collection Prior to collecting human samples from CRC patients, written informed consent was obtained from each patient before colorectal tumor resection surgery. All experiments were conducted in accordance with protocols approved by the Clinical Trials Committee of the First Affiliated Hospital of Sun Yat-sen University of China, and in accordance with the institution's ethical codes and guidelines. For the purification of tissue-infiltrating cells or in vitro culture of tissues and cells, tumor specimens and / or surrounding normal tissue were excised after each surgery, transferred to ice-cold PBS, and kept on ice until use. For the analysis of mRNA levels and relative survival rates, 47 tumor specimens and 43 non-tumor specimens were collected from 70 CRC patients, including 23 sets of tumor and non-tumor specimens from the same individual. Detailed clinical information of the patients, including sex, age, and severity, is shown in Tables 1-3.

[0099] [Table 1]

[0100] [Table 2]

[0101] [Table 3]

[0102] 3. In vitro culture of cells and tissues Mouse / Human Colon Tumor / Non-tumor Tissue (2×10) 5 Cells infiltrating mouse / human colorectal tumor / non-tumor tissue pieces (3mm x 3mm cuts) were cultured for 20 hours at 37°C under 5% CO2 using dectin-1 ligands such as curdlan, OXCA, and laminarin, or with PGE2 stimulation, in 96-well flat-bottom plates (Falcon, Becton Dickinson). 0.2 ml of RPMI was then used, supplemented with 10% FBS and 1% streptomycin + penicillin. After culturing, cells or tissues were harvested, and mRNA expression of tumor-related / anti-tumor-related genes was measured by the qPCR method described in the "Quantitative Reverse Transcription PCR" section below.

[0103] 4. Chemical induction of colorectal tumors Six-to-seven-week-old mice were intraperitoneally administered 10 μg of AOM (Fujifilm Wako Pure Chemical Corporation, Osaka) per gram of body weight. One week after administration, these mice were given 1% by mass DSS (36-50 kDa; MP Biomedicals, Illkirch, France) in their drinking water for 7 days, followed by 14 days of normal water administration, which constituted one cycle. After three cycles of DSS / normal water treatment, these mice were kept for a further 2-6 weeks before being euthanized.

[0104] 5. Induction of spontaneously occurring small intestinal polyps and colorectal tumors Apc Min / + or Apc Min / + Clec7a - / - When mice were raised normally in an SPF (Specific Pathogen Free) state for 19-21 weeks after birth, polyps spontaneously developed in the small intestine. To induce colon tumors, the same mice, 6-7 weeks old, were given 1% by mass DSS orally for one week, then raised normally for another 4-5 weeks before being euthanized.

[0105] 6. Cell preparation Cells infiltrating tumor / non-tumor tissue of the colon were isolated using the following method: Tissue was cut to a thickness of 1 mm and shaken for 40 minutes at 37°C in HBSS (Hanks' Balanced Salt Solution) containing 3 mM EDTA. The intraepithelial lymphocytes and colonic epithelial cells suspended in the culture supernatant were discarded, and the intestinal sections were washed twice with HBSS. Then, they were shaken for 120 minutes at 37°C in RPMI containing 10% FBS and 1% streptomycin + penicillin, 200 U / ml collagenase (C2139; Sigma-Aldrich), and 5 U / ml DNase 1 (Sigma-Aldrich). After culturing, the samples were vortexed for 10 seconds, filtered through sterile gauze, and single-cell suspensions were collected. There were also experiments in which tissue-infiltrating cells were purified to lymphocytes using a 45% / 66.6% discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient for 20 minutes.

[0106] 7. Flow cytometry The mouse antibodies CD4 (GK1.5), CD8α (53-6.7), TCRγδ (GL3), CD45 (30-F11), CD19 (6D5), CD11b (M1 / 70), CD11c (N418), IA / IE (M5 / 114.15.2), CD103 (2E7), IgG1 (RMG1-1), IFN-γ (XMG1.2), and IL-17 (TC11-18H10.1) were purchased from Biolegend (San Diego, USA). The antibody against mouse dectin-1 (2A11) was purchased from Abcam (Cambridge, UK). The anti-mouse IL-22bp polyclonal antibody was purchased from R&D systems (Biotechne, Minnesota, USA). The 7-AAD (7-amino-actinomycin D) viability staining solution was purchased from eBioscience (San Diego, USA). 2.4G2 (anti-FCγRII / III-specific mAb) was obtained from the American Type Culture Collection (Manassas, VA). All antibodies were used at a 1:250 dilution. Cells isolated from mouse intestines were washed twice with FACS Hanks buffer (HBSS containing 2% FCS and 0.1% sodium azide) and then treated with 2.4G2 to block FcR binding. Cells were then surface-stained with the mAb for 25-30 minutes. For cytokine FACS, cells were first stimulated with 50 ng / ml PMA (Sigma Aldrich) + 500 ng / ml ionomycin (Sigma Aldrich), or unstimulated, with 1 mM monensin (Sigma Aldrich) for 4 hours. After stimulation, cells were washed twice with FACS Hanks buffer (HBSS containing 2% FCS and 0.1% sodium azide), and then treated with 2.4G2 to block FcR binding. Subsequently, cells were surface-stained with mAbs for 30 minutes, fixed in Cytofix / Cytoperm solution (BD Biosciences) for 20 minutes, and then permeabilized. After washing with 1× wash buffer, cells were incubated with anti-mouse IFN-γ mAb and IL-17 mAb for a further 30 minutes.For the analysis, we used a Canto II or FACSCalibur flow cytometer (BD Biosciences) and FlowJo 10.0 FACS software.

[0107] 8. Purification of epithelial cells, bone marrow-derived cells, and other immune cells from the colon. Whole single cells, isolated from the entire intestine or polyps of mice, were labeled with biotin-labeled anti-mouse CD326 (G8.8) (Biolegend), and then labeled with anti-biotin microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD326 + Epithelial cells were positively purified using autoMACS (Miltenyi Biotec). The negative fraction was stained with anti-mouse CD45 microbeads for subsequent leukocyte purification, anti-mouse CD11b and CD11c microbeads for myeloid-derived cells, Thy1.2 for T cells, or CD19 for B cell purification using autoMACS selection.

[0108] 9. Quantitative reverse transcription PCR Total RNA was extracted using the Mammalian Total RNA Miniprep kit (Sigma Aldrich). The RNA was denatured in the presence of oligo-dT primers and then reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, San Francisco, USA). qPCR was performed using the SYBR Green qPCR kit (Carlsbad, USA) and the C1000 Thermal Cycler system (Bio-Rad, Hercules, USA) with the primer sets listed in Table 4. The relative expression levels of each mRNA were calculated using the comparative cycle threshold (Ct value) method and normalized by the expression level of Gapdh mRNA.

[0109] [Table 4]

[0110] 10.PGE2 concentration measurement Polyp and non-polyp tissue were collected from the mouse colon and placed in 2 ml tubes containing 500 μl of protein lysis buffer (RIPA buffer: 50 mM Tris (pH 8.0) + 100 mM NaCl + 0.1% Triton X-100) and a protease inhibitor cocktail (Takara Bio, Shiga, Japan). The tissue was homogenized using beads and a Micro Smasher (MS-100R, TOMY). After centrifugation, the supernatant of the tissue homogenate was collected, and the PGE2 concentration was measured using the ELISA Development Kit for PGE2 (R&D systems, Minneapolis, USA). The final PGE2 concentration was normalized by the total protein concentration of each individual.

[0111] 11. RNA sequencing and analysis RNA was isolated from colorectal tumors or non-tumor tissues using Sigma Aldrich's total RNA-prep kit. RNA was prepared using the Contech SmartSeq2 library prep kit and sequenced using NovaSeq6000. Data were normalized, and differential gene expression was identified using usegalaxy.org. Pathway and process enrichment analysis using relative gene expression data was performed using metascape.org.

[0112] 12.Statistical analysis Differences in parametric data were assessed using unpaired, two-sided Student's t-tests. In experiments involving two or more relative groups, Dunnett's multiple comparison test or Tukey's multiple comparison test was performed following a one-way ANOVA. Statistical significance of survival rates between groups was assessed using the log-rank test. Statistics were calculated using PRISM8 software (GraphPad Software). In studies of CRC patients, categorical variables were compared using Fisher's test, and correlations were analyzed using Spearman's correlation test. Statistical analysis was performed using R software (version 4.0.3, R Foundation). A difference of p-value < 0.05 was considered statistically significant.

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[0114] The disclosure of Japanese Patent Application No. 2021-204572 is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually noted to be incorporated by reference.

Claims

1. An intestinal tumor suppressant containing β-glucan with dectin-1 inhibitory activity, which suppresses intestinal tumors through dectin-1 inhibition.

2. The intestinal tumor inhibitor according to claim 1, wherein the β-glucan is a β-glucan containing β-1,3 linkages or a β-glucan containing a β-1,6 linkage branched chain in a β-1,3 linkage main chain.

3. The intestinal tumor inhibitor according to claim 1 or claim 2, wherein the molecular weight of the β-glucan is 0.2K to 100K.

4. The intestinal tumor inhibitor according to any one of claims 1 to 3, wherein the β-glucan is laminarin.

5. PGE contains β-glucan which has dectin-1 inhibitory activity. ２ Production inhibitor.

6. The PGE according to claim 5, wherein the β-glucan is a β-glucan containing β-1,3 linkages or a β-glucan containing a β-1,6 linkage branched chain in a β-1,3 linkage main chain. ２ Production inhibitor.

7. The PGE according to claim 5 or claim 6, wherein the molecular weight of the β-glucan is 0.2K to 100K. ２ Production inhibitor.

8. The PGE according to any one of claims 5 to 7, wherein the β-glucan is laminarin. ２ Production inhibitor.

9. An IL-22BP production promoter containing β-glucan with dectin-1 inhibitory activity.

10. The IL-22BP production promoter according to claim 9, wherein the β-glucan is a β-glucan containing β-1,3 linkages or a β-glucan containing a β-1,6 linkage branched chain in a β-1,3 linkage main chain.

11. The IL-22BP production promoter according to claim 9 or claim 10, wherein the molecular weight of the β-glucan is 0.2 K to 100 K.

12. The IL-22BP production promoter according to any one of claims 9 to 11, wherein the β-glucan is laminarin.

13. An intestinal tumor suppressant containing a dectin-1 inhibitor, which suppresses intestinal tumors through dectin-1 inhibition.

14. The intestinal tumor suppressor according to claim 13, wherein the dectin-1 inhibitor is a dectin-1 antagonist.

15. The intestinal tumor suppressor according to claim 13, wherein the dectin-1 inhibitor is an anti-dectin-1 inhibitory antibody or a dectin-1 inhibitory small molecule compound.

16. The aforementioned intestinal tumor is PGE ２ An intestinal tumor inhibitor according to any one of claims 1 to 4, 13, and 14, wherein the intestinal tumor is accompanied by an increased expression level of or an intestinal tumor is accompanied by a decreased expression level of IL-22BP.

Images