Composition for treating colorectal cancer

Abstract

The present disclosure pertains to: a composition for preventing or treating colorectal cancer, which contains an IgA antibody or an antigen-binding fragment thereof; and a method for preventing or treating colorectal cancer using a composition containing an IgA antibody or an antigen-binding fragment thereof.

Description

Compositions for treating colorectal cancer

[0001] The present disclosure relates to a composition for preventing or treating colorectal cancer, comprising an IgA antibody or an antigen-binding fragment thereof, and a method for preventing or treating colorectal cancer using a composition comprising an IgA antibody or an antigen-binding fragment thereof.

[0002] Colorectal cancer (CRC) is the third most common cancer and ranks as one of the leading causes of death among cancer patients worldwide (Non-Patent Document 1). Both genetic and environmental risk factors contribute to the development of colorectal cancer. Genetic predisposition, inflammatory bowel disease (IBD), and lifestyle factors such as alcohol consumption and meat consumption are all thought to contribute to the development of CRC (Non-Patent Documents 2, 3). In particular, many studies have revealed that patients suffering from IBD are at a higher risk of developing CRC (Non-Patent Documents 4, 5).

[0003] The progression from inflammation to tumorigenesis is controlled by immune cells such as CD4+ T cells, IgA-producing plasma cells, and macrophages (ILCs) (Non-Patent Documents 4, 6, and 7). Tumorigenesis is also influenced by the composition of the gut microbiota (Non-Patent Document 8). Reactive oxygen species (ROS), lipid mediators generated by cyclooxygenase activity such as prostaglandin E2 (PGE2), and several proinflammatory cytokines are also involved in the development of CRC (Non-Patent Documents 6 and 9). These molecules promote epithelial cell transformation and tumor progression through the activation of genetic and signaling pathways such as nuclear factor-κB (NF-κB), pSTAT3 (phospho-signal transducer and activator of transcription 3), MARK-p38, and Wnt10-13 (Non-Patent Documents 10-13).

[0004] IgA is the most abundant antibody (Ab) produced in the human body (Non-Patent Documents 14-15). Monomeric IgA binds to J chains to form polymeric IgA, which is secreted into the intestinal lumen by poly-Ig receptors (Non-Patent Document 16). IgA maintains the separation between luminal antigens and underlying epithelial cells and forms the first line of defense for eliminating intestinal pathogens and toxins (Non-Patent Documents 17-18). Two types of IgA coexist in the intestine (Non-Patent Document 19). The first type is polyreactive and responds to commensals with low affinity in the absence of T cell help. The other type has high affinity and is specifically produced in germinal centers in response to pathogens (Non-Patent Document 20). Fab fragments of high-affinity IgA can bind to pathogens and endotoxins (Non-Patent Documents 21, 22). On the other hand, reduced or absent affinity IgA can cause gastrointestinal symptoms (Non-Patent Document 23). In humans, there are two subclasses of IgA, IgA1 and IgA2, which are encoded by two distinct α1 and α2 genes on chromosome 14 (Non-Patent Documents 24, 25). The main differences between IgA1 and IgA2 are the presence or absence of a 13-amino acid hinge region, which is present only in IgA1, and the number of glycosylation sites (Non-Patent Document 26). In serum, IgA1 predominates over IgA2 at a ratio of 9:1, whereas in mucosal tissues, IgA1 and IgA2 are more evenly distributed (Non-Patent Document 27).

[0005] The Mzb1 gene encodes a B cell-specific, endoplasmic reticulum (ER)-localized protein (MZB1) that is selectively expressed in marginal zone (MZ) B cells and B1 cells (NPL 28). MZB1 expression in B cells increases to higher levels during differentiation into Ab-secreting cells (ASCs) (NPL 29). Previous studies have identified MZB1 as a BiP and GRP94 co-chaperone that enables proper IgM synthesis and secretion (NPLs 30, 31). We recently discovered that MZB1 promotes efficient secretion of J-chain-containing dimeric IgA and plays an important role in suppressing intestinal inflammation (NPL 15).

[0006] Weitzz,4.et aln The al!anccet 394,1467-14480(2011) 0.444. The 3.3.&19tzkowitzz,3.8. Gastroenterolog 162,7115-730.1713(2022) .5515, 4.1.2.12.2.2.2.2.2.2.2.2.2.2.2.. Gut 48,566-535(2001)(aasryy, A,ZingerrrrA.&Ben-eriahhon Nat Immunol 17,230-240 (2016) 2016, 100,000 I 161,592-607(2021).301 nnan,C.｡.&Garrett4Wyｳ. Annu Rev Microbiol 70,395-4111 (2016) 3.&a.m., 8. Mediators Inflammm 2017,512260448(2017).77, 3.b.,b.b.,b.l.f.n.b.,..&b. Cell Mol Immunol 6,327-334 (2009) 2009, 300,000 69,1269-12482(2020). et al.Mol Cancer 21,144(20022) 0.0001,2001,2001,2001,100,000,000. 3ci 21(20020). Nat Rev Immunol 12,8811-8322(2012) 8.8) E.et al.Proc Natl Acad Sci Uｳ｡ 116,1344800-13489(2019) 1161100 annnJ.Y,,ojasssoooo Gut Microbes 5,6422-6662(2014). 9.&3lack,5. Mucosal ｩmmunol 13,120-21(2020) 300. ｡dv Exp Med Biol 1254,105-116(2000)ｳet aloｮat Microbioll 1,161103(2016).3.4.4.4.4.4.4.3.A. Immunity 49, 211-224 (2018). Mantis, N. J. , Rol, N. &Corthesy, B. Mucosal Immunol 4, 603-611 (2011). Brandtzaeg, P. Front Immunol 4, 222 (2013). Okay, S. et al. Gut Microbes 8, 486-492 (2017). Yel, L. J Clin Immunol 30, 10-16 (2010). Pakkanen, S. H. et al. Clin Vaccine Immunol 17, 393-401 (2010). van Egmond, M. et al. Trends Immunol 22, 205-211 (2001). Steffen, U. et al. Nat Commun 11, 120 (2020). Andreani, V. et al. Proc Natl Acad Sci USA 115, E9630-e9639 (2018). Shimizu, Y. , Meunier, L. &Hendershot, L. M. Proc Natl Acad Sci USA 106, 17013-17018 (2009). Rosenbaum, M. et al. Genes Dev 28, 1165-1178 (2014). Flach, H. et al. Immunity 33, 723-735 (2010). ,

[0007] CRC is ranked as one of the leading causes of death among cancer patients worldwide, and it is desirable to provide new prophylactic or therapeutic agents and methods for preventing or treating CRC.

[0008] The present inventors have intensively studied the mechanisms of CRC onset and progression, and using mouse models, further investigated the role of MZB1 in CRC onset and progression, revealing the important function of MZB1 in alleviating intestinal inflammation and CRC by promoting intestinal IgA secretion. Furthermore, they demonstrated that MZB1 deficiency accelerates the development and progression of AOM / DSS-induced colorectal cancer by exacerbating intestinal inflammation through reduced intestinal IgA production. They also found that exacerbated CRC can be alleviated by supplementing IgA into the intestinal lumen. Through this series of original studies, the present inventors discovered that colorectal cancer can be prevented or treated by administering a composition containing an IgA antibody as an active ingredient to a subject, thereby completing the present invention.

[0009] In one aspect, the present disclosure provides a composition for preventing or treating colorectal cancer, comprising an IgA antibody or an antigen-binding fragment thereof.

[0010] In one aspect, the present disclosure provides a method for preventing or treating colorectal cancer in a subject in need of such treatment, comprising administering to the subject a composition containing an IgA antibody or antigen-binding fragment thereof.

[0011] In one aspect, the present disclosure provides the following: [Item 1-1] A composition for preventing or treating colorectal cancer, comprising an IgA antibody or an antigen-binding fragment thereof. [Item 1-2] A composition comprising the IgA antibody or antigen-binding fragment thereof according to [Item 1-1], wherein the IgA antibody is an IgA antibody present in the intestinal lumen. [Item 1-3] A composition comprising the IgA antibody or antigen-binding fragment thereof according to [Item 1-1], wherein the IgA antibody or antigen-binding fragment thereof binds to and / or inhibits the growth of Fusobacterium and / or Clostridium difficile bacteria. [Item 1-4] The IgA antibody is: a) an antibody comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of the light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 8; b) an antibody comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 17, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of the light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 18; c) an antibody comprising a light chain variable region comprising: an antibody comprising: a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 27, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 28; d) an antibody comprising: a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 81, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 82;e) an antibody comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 93, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 94; f) an antibody comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 37, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 38; or g) an antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 37, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 38, A composition comprising the IgA antibody or antigen-binding fragment thereof according to [Item 1-1], which has at least one amino acid mutation in at least one region selected from heavy chain CDR1 to 3, light chain CDR1 to 3, and light chain FR1, and which binds to the amino acid sequence RQEEHIELIAS in Escherichia coli SHMT protein and the amino acid sequence VLDMMMKLEKPE in C. difficile iPGM protein. [Item 1-5] A composition comprising the IgA antibody or antigen-binding fragment thereof according to [Item 1-1], wherein the IgA antibody comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. [Item 1-6] The IgA antibody comprises a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13.and a light chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 16. [Item 1-7] A composition comprising the IgA antibody or an antigen-binding fragment thereof according to [Item 1-1], wherein the IgA antibody is an antibody comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26. [Item 1-8] A composition comprising the IgA antibody or antigen-binding fragment thereof according to [Item 1-1], wherein the IgA antibody is an antibody comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 83, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 84, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 85, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 86, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 87, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 88. [Item 1-9] A composition comprising the IgA antibody or antigen-binding fragment thereof according to [Item 1-1], wherein the IgA antibody is an antibody comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 95, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 96, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 97, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 98, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 99, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 100. [Item 1-10] The IgA antibody is an antibody comprising:A composition comprising the IgA antibody or antigen-binding fragment thereof according to [Item 1-1], wherein the IgA antibody comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36. [Item 1-11] The IgA antibody binds to Clostridium difficile bacteria, and has at least one amino acid mutation in at least one region selected from heavy chain CDR1-3, light chain CDR1-3, and light chain FR1 relative to a reference antibody comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 38, and has the amino acid sequence RQEEHIELIAS in the Escherichia coli SHMT protein and C. An antibody or antigen-binding fragment thereof that binds to the amino acid sequence VLDMMMKLEKPE in the iPGM protein of C. difficile, comprising: X １ heavy chain CDR1 comprising the amino acid sequence of YYIH, RIDPENX ２ X ３ a heavy chain CDR2 comprising the amino acid sequence of TTYAPKFQ; a heavy chain CDR3 comprising the amino acid sequence of YCARSTVL; and a heavy chain variable region comprising: RX ４ a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SQSIVHTNG; a light chain CDR2 comprising the amino acid sequence of KLLIYKV; and a light chain CDR3 comprising the amino acid sequence of GVYYCFQGS; and a light chain FR1 comprising the amino acid sequence of TPLSLPVSLGDQA or SPASX. ５ SVSLGDRX ６ X １ , X ２ , X ３ are each independently a neutral polar amino acid or an acidic polar amino acid, and X ４ is a nonpolar amino acid or a neutral polar amino acid, and X ５ , X６and each independently represent a non-polar amino acid. [Item 1-12] The IgA antibody comprises: (1) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO: 53; (2) a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO: 53, (3) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO: 53; (4) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36;(5) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO: 55; or (6) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO: 55. A composition comprising the IgA antibody or antigen-binding fragment thereof according to [Item 1-1], comprising a light chain variable region comprising: a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:36; and a light chain FR1 comprising the amino acid sequence of SEQ ID NO:53. [Item 1-13] The composition according to [Item 1-1], for administration to the gastrointestinal tract. [Item 1-14] The composition according to [Item 1-1], provided in the form of a pharmaceutical composition, food composition, or feed composition. [Item 2-1] A method for preventing or treating colorectal cancer in a subject in need of such treatment, comprising administering to the subject a composition containing an IgA antibody or antigen-binding fragment thereof. [Item 2-2] A method comprising the IgA antibody or antigen-binding fragment thereof according to [Item 2-1], wherein the IgA antibody is present in the intestinal lumen. [Item 2-3] The method according to [Item 2-1], wherein the IgA antibody or antigen-binding fragment thereof binds to and / or inhibits the growth of Fusobacterium and / or Clostridium difficile bacteria. [Item 2-4] The IgA antibody comprises: a)an antibody comprising: a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7; and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8; b) an antibody comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17; and an antibody comprising a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18; c) a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 27; an antibody comprising a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:28; d) an antibody comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:81, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:82; e) an antibody comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:93, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:94; f) an antibody comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:94; A heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37; andor g) an antibody comprising a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3, the light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 38; or g) an antibody which has at least one amino acid mutation in at least one region selected from heavy chain CDR1 to 3, light chain CDR1 to 3, and light chain FR1 relative to a reference antibody comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 38, and which binds to the amino acid sequence RQEEHIELIAS in Escherichia coli SHMT protein and the amino acid sequence VLDMMMKLEKPE in C. difficile iPGM protein. [Item 2-5] The method of item [2-1], wherein the IgA antibody comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. [Item 2-6] The method of item [2-1], wherein the IgA antibody comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16. [Item 2-7] The IgA antibody comprises a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23; and a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24.The method according to item [2-1], wherein the IgA antibody comprises a light chain variable region comprising a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26. [Item 2-8] The method according to item [2-1], wherein the IgA antibody comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 83, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 84, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 85, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 86, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 87, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 88. [Item 2-9] The method according to Item [2-1], wherein the IgA antibody comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 95, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 96, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 97, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 98, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 99, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 100. [Item 2-10] The method according to Item [2-1], wherein the IgA antibody is an antibody comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36. [Item 2-11] The IgA antibody binds to Clostridium difficile bacteria, and exhibits a high affinity for a reference antibody comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 38.An antibody or antigen-binding fragment thereof, which has at least one amino acid mutation in at least one region selected from heavy chain CDR1 to 3, light chain CDR1 to 3, and light chain FR1, and which binds to the amino acid sequence RQEEHIELIAS in Escherichia coli SHMT protein and the amino acid sequence VLDMMMKLEKPE in C. difficile iPGM protein, comprising: X １ heavy chain CDR1 comprising the amino acid sequence of YYIH, RIDPENX ２ X ３ a heavy chain CDR2 comprising the amino acid sequence of TTYAPKFQ; a heavy chain CDR3 comprising the amino acid sequence of YCARSTVL; and a heavy chain variable region comprising: RX ４ a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SQSIVHTNG; a light chain CDR2 comprising the amino acid sequence of KLLIYKV; and a light chain CDR3 comprising the amino acid sequence of GVYYCFQGS; and a light chain FR1 comprising the amino acid sequence of TPLSLPVSLGDQA or SPASX. ５ SVSLGDRX ６ X １ , X ２ , X ３ are each independently a neutral polar amino acid or an acidic polar amino acid, and X ４ is a nonpolar amino acid or a neutral polar amino acid, and X ５ , X ６and each independently represent a nonpolar amino acid. [Item 2-12] The IgA antibody comprises: (1) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO: 53; (2) a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO: 53, (3) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO: 53; (4) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36;(5) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO: 55; or (6) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO: 55. The method according to Item [2-1], wherein the composition comprises a light chain variable region comprising a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO: 53. [Item 2-13] The method according to Item [2-1], wherein the composition is administered to the gastrointestinal tract. [Item 2-14] The method according to Item [2-1], wherein the composition is provided in the form of a pharmaceutical composition, a food composition, or a feed composition.

[0012] The present disclosure has the effect of providing a new prophylactic or therapeutic agent and method for preventing or treating CRC.

[0013] Figure 1 shows the susceptibility of MZB1-deficient mice to AOM / DSS-induced colorectal cancer. MZB1-deficient mice have increased susceptibility to AOM / DSS-induced colorectal cancer. CRC was induced in Mzb1+ / + and Mzb1- / - mice (6 male mice per group) using an injection of AOM (10 mg / kg) followed by three rounds of 2.5% DSS treatment. a) Body weights of Mzb1+ / + and Mzb1- / - mice were monitored throughout the course of AOM / DSS-induced colorectal cancer. b) Disease Activity Index scores are shown. c) Colon and rectal tumor nodules observed 2 weeks after the completion of AOM / DSS treatment (day 82) are shown. d) Mzb1- / - mice exhibited a greater number and larger tumor nodules (diameter >3 mm). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Representative results from three independent experiments are shown. Figure 2 demonstrates more severe intestinal inflammation in MZB1-deficient mice. a) Colon length measured at the end of the experiment (day 82) is shown. b) Representative hematoxylin and eosin staining of colon sections from four pairs of Mzb1+ / + and Mzb1- / - mice is shown. c) Percentages of macrophages (CD45+F4 / 80+CD11b+7AAD-), neutrophils (CD45+CD11b+Ly6G+7AAD-), and B cells (CD45+B220+7AAD-) in the lamina propria and Peyer's patches, and GC B cells (B220+FAS+GL7+7AAD-) in Peyer's patches are shown. d) Expression of inflammatory cytokines in colon tissue. *p<0.05; **p<0.01; ***p<0.001; ***p<0.0001. These experiments used six pairs of male Mzb1+ / + and Mzb1- / - mice, of which six pairs were analyzed in a) and d) and four pairs were analyzed in b) and c). Representative results from three independent experiments are shown. Figure 3 shows that MZB1 deficiency reduces the percentage of IgA+ plasma cells and IgA secretion after CRC induction. Colonic lamina propria lymphocytes were isolated and flow cytometry analysis was performed (day 82). A) The percentage of IgA+ (CD45+IgA+B220-7AAD-) and IgM+ (CD45+IgM+B220-7AAD-) plasma cells in the colon after CRC induction is shown.b) Serum and fecal IgA levels at T1, T2, and T3 (measured 5 days after the first, second, and third cessation of DSS treatment, respectively) are shown. c) Swiss rolls of colonic tissue from Mzb1+ / + and Mzb1- / - mice stained for IgA are shown. Representative images from six pairs of Mzb1+ / + and Mzb1- / - mice are shown. Magnification: ×50. Scale bar: 200 μm. d) Fecal samples collected before and after CRC induction were processed to enrich for the microbiota and then stained for IgA. The microbiota were divided into high-IgA and low-IgA populations based on staining of control fecal samples from IgA- / - mice. *p<0.05; **p<0.01; ***p<0.001. These experiments used six pairs of male Mzb1+ / + and Mzb1- / - mice, of which four pairs were analyzed in a) and d), three pairs in b), and six pairs in c). Representative results from three independent experiments are shown. Figure 4 shows that MZB1 deficiency causes impaired gut microbiota after AOM / DSS-induced colorectal cancer. CRC induction was completed on day 68, and 10 days later, fecal samples were collected from Mzb1+ / + and Mzb1- / - mice. Three male mice per group were subjected to 16S V4 rRNA gene sequencing. a) Alpha diversity, showing the species richness of the microbiota in each group. b) Principal coordinate analysis (PCA), showing the level of variance between the indicated mice within each group. c) Relative abundance of bacterial phyla present in feces is shown. d) Differential abundance testing identifies distinct bacterial genera between the indicated groups. Independent experiments using five Mzb1+ / + mice and six Mzb1- / - mice were performed, and the results are shown in Figure 14. Figure 5 shows that IgA deficiency exacerbates the development of AOM / DSS-induced colorectal cancer. a) Body weight changes in Iga+ / + and Iga- / - mice throughout the course of AOM / DSS-induced colorectal cancer. b) Comparison of disease activity index between Iga+ / + and Iga- / - mice. c) Colon tumor nodules observed in Iga+ / + and Iga- / - mice 2 weeks after the completion of CRC induction (day 82) are shown. d-f) Ten days after the completion of AOM / DSS induction (day 78), feces were collected from each mouse and subjected to 16S rRNA sequencing.Figure 6 shows that W27 administration attenuates AOM / DSS-induced CRC in Mzb1- / - mice. Figure 7 shows that W27 administration reduces AOM / DSS-induced CRC in Mzb1- / - mice. Figure 8 shows that W27 administration reduces AOM / DSS-induced CRC in Mzb1- / - mice. Figure 9 shows that W27 administration reduces AOM / DSS-induced CRC in Mzb1- / - mice. Figure 10 shows that W27 administration reduces AOM / DSS-induced CRC in Mzb1- / - mice. Figure 11 shows that W27 administration reduces AOM / DSS-induced CRC in Mzb1- / - mice. Figure 12 shows that W27 administration reduces AOM / DSS-induced CRC in Mzb1- / - mice. Figure 13 shows that W27 administration reduces AOM / DSS-induced CRC in Mzb1- / - mice. Figure 14 shows that W27 administration reduces AOM / DSS-induced CRC in Mzb1- / - mice. Figure 15 shows that W27 administration reduces AOM / DSS-induced CRC in Mzb1- / - mice. Figure 16 shows that W27 administration reduces AOM / DSS-induced CRC in Mzb1- / - mice. Figure 17 shows that W27 administration reduces AOM / DSS-induced CRC in Mzb1- / - mice. Figure 18 shows that W27 administration reduces AOM / DSS-induced CRC in Mzb1- / - mice. Figure 19 ... c) Colon and rectal tumor nodules 2 weeks after completion of CRC induction (day 82). d) Tumor nodules in Mzb1- / - mice with and without W27 supplementation. Each group contained 6 male mice. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Representative results from two independent experiments are shown. Figure 7 shows that W27 administration reduces intestinal inflammation in Mzb1- / - CRC mice. a) Colon length at the end of the experiment (day 82). b) Percentages of macrophages (CD45+F4 / 80+CD11b+7AAD-), neutrophils (CD45+CD11b+Ly6G+7AAD-), and B cells (CD45+B220+7AAD-) in the colonic lamina propria of Mzb1- / - mice with and without W27 supplementation. c) Expression of inflammatory cytokines in colonic tissue. *p<0.05; **p<0.01; ***p<0.001; ***p<0.0001. Six pairs of male Mzb1+ / + and Mzb1- / - mice were used in the experiment. All six pairs were analyzed in a) and c), and four pairs were analyzed in b). Figure 8 shows that cohoused Mzb1+ / + and Mzb1- / - mice exhibited similar susceptibility to AOM / DSS-induced CRC development. Mzb1+ / + and Mzb1- / - mice were co-housed and shared nursing mothers. Mice underwent CRC induction from 8 weeks of age. a) Weight changes between the two groups are shown. b) Disease activity index is shown. c) Tumor number and size (day 82) are shown.d) Principal component analysis results analyzed on day 78 of CRC induction are shown. e) Relative abundance at the phylum level, and f) relative abundance at the genus level are shown. Eight pairs of male Mzb1+ / + and Mzb1- / - mice were analyzed. Figure 9 shows the method for generating Iga- / - mice. a) Targeting strategy. A gRNA targeting the mouse Igha gene (5'-TCACATTCATCGTGCCGGAAGGG-3'), a 28-bp insertion between c.107 and c.108 in the first exon of the Cα gene (5'-TGATAGACGTAAACGGCCACAAGTTCGA-3'), and Cas9 were co-injected into fertilized mouse eggs to generate targeted knock-in offspring. b) F0 founder animals were identified by PCR and subsequently sequenced. These founder animals were bred with wild-type mice to test for germline transmission and the generation of F1 animals. c) Serum IgA levels in Iga+ / + and Iga- / - mice are shown. Figure 10 shows the number of tumor nodules in Mzb1+ / + and Mzb1- / - mice. a) Examples of counted tumors are shown. They were counted as large (light gray arrows) and small (dark gray arrows). b) Absolute numbers of macrophages (CD45+F4 / 80+CD11b+7AAD-), neutrophils (CD45+CD11b+Ly6G+7AAD-), B cells (CD45+B220+7AAD-), and GCB cells (B220+Fas+GL7+7AAD-) in the intestinal lamina propria and Peyer's patches are shown. Figure 11 shows the gating strategy for immune cells in colonic LPL (day 82). (a) Gating strategy for macrophages (CD45+F4 / 80+CD11b+7AAD-). (b) Gating strategy for neutrophils (CD45+CD11b+Ly6G+7AAD-). (c) B cells (CD45+B220+7AAD-) Figure 12 shows increased phosphorylation of the MAPK signaling molecule p38 in the colon.a) Immunoblot analysis showing the phosphorylation levels of signaling molecules such as p38, ERK, and STAT3 in mouse colon tissues (day 82). B) ImageJ analysis of mean gray values. **p<0.01 (unpaired t-test). Figure 13 shows exacerbated systemic inflammation in MZB1-deficient mice analyzed on day 82 after CAC induction. a) Mzb1- / - mice showed enlarged spleens accompanied by increased body weight and splenocyte numbers. b) Percentages of macrophages (CD45+F4 / 80+CD11b+7AAD-), neutrophils (CD45+CD11b+Ly6G+7AAD-), T cells (CD45+CD3+7AAD-), B cells (CD45+B220+7AAD-), and germinal center B cells (CD45+B220+GL7+Fas+7AAD-) in Mzb1+ and Mzb1- / - mice. c) Serum levels of IgA, IgM, IgG1, IgG2b, IgG2c, and IgG3 in Mzb1+ / + and Mzb1- / - mice. *p<0.05 (unpaired t-test). Figure 14 shows the disrupted microbiota in Mzb1- / - CRC mice (day 78). a) Principal coordinate analysis (PCA) showing the level of variance between the indicated mice in each group. b) Relative abundance of bacterial bacterial infections is shown. c) Abundance difference testing identifies distinct bacterial genera between the Mzb1+ / + and Mzb1- / - groups. Figure 15 shows that W27 administration reduced systemic inflammation in Mzb1- / - CRC mice (analyzed 82 days after CAC induction). a) Spleen size, weight, and splenocyte count are shown. b) Percentages of macrophages, neutrophils, B cells, and T cells within the spleen are shown. c) Serum levels of IgA, IgM, IgG1, IgG2b, IgG2c, and IgG3 are shown in the Mzb1- / -Ctrl and Mzb1- / - W27 groups. *p<0.05; **p<0.01 (unpaired t-test). Figure 16 shows that W27 administration restores the disrupted microbiota in Mzb1- / - CRC mice (day 78). a) Principal coordinate analysis (PCA) showing the level of variance between designated mice within each group. b) Relative abundance of bacterial phyla present in feces. c) Differential abundance tests show that distinct bacterial genera were identified between the Mzb1- / - control:Ctrl group and the Mzb1- / - W27 group.FIG. 17 shows the lack of IgA in the serum of Iga- / - mice.

[0014] CRC is one of the leading causes of death worldwide. Because the incidence of CRC is increased in patients suffering from inflammatory bowel disease, intestinal inflammation is one of the important risk factors promoting the development of CRC. The role of IgA in maintaining intestinal homeostasis and preventing inflammation is well established. Our previous studies demonstrated that marginal zone and B1 cell-specific protein (MZB1) promotes intestinal IgA secretion, and its absence causes significant dextran sulfate sodium salt (DSS)-induced colitis. In this disclosure, we investigated the role of MZB1 in CRC development and observed an increase in both the number and size of tumor nodules in Mzb1- / - mice compared with Mzb1+ / + mice. The increased development and progression of CRC in Mzb1- / - mice was associated with reduced intestinal IgA levels, altered intestinal microbiota, and more severe intestinal inflammation and systemic inflammation. Oral administration of monoclonal IgA, W27, attenuated both intestinal inflammation and AOM / DSS-induced CRC. Remarkably, cohabitation of Mzb1+ / + and Mzb1- / - mice from postnatal day 10 resulted in similar gut microbiota composition and CRC development. Our findings highlight the crucial role of MZB1-mediated IgA secretion in suppressing the development and progression of colorectal cancer caused by intestinal inflammation. Furthermore, our study revealed that the microbiota composition, regulated by intestinal IgA levels, has a significant impact on the severity of intestinal inflammation and the resulting emergence of CRC.

[0015] (Definitions) In this specification, when multiple ranges of numerical values ​​are given, the same applies to ranges formed by any combination of the lower and upper limits of those multiple ranges.

[0016] As used herein, the term "antibody" is used in the broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and antibody fragments that exhibit the intended antigen-binding activity. A full-length antibody comprises a heavy chain and a light chain that are primarily composed of polypeptides. The heavy chain and light chain each contain a region called a variable region that recognizes an antigen, and these regions are generally referred to as the heavy chain variable region and the light chain variable region, respectively. The variable regions each contain CDRs 1 to 3, which are further specified as antigen-recognition sites, in order from the amino terminus. These CDRs 1 to 3 are also more specifically referred to as heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, light chain CDR3, etc. Furthermore, the regions other than CDRs 1 to 3 of the heavy chain and light chain are referred to as heavy chain FRs 1 to 4 and light chain FRs 1 to 4, respectively, in order from the amino terminus. The antibody may be in a form consisting of two heavy chains and two light chains, or in a form consisting of one heavy chain and one light chain (also called a single-chain antibody).

[0017] The antibody may be in the form of an antibody consisting of two heavy chains and two light chains, or in the form of an antibody consisting of one heavy chain and one light chain (also called a single-chain antibody).Furthermore, the antibody may be in the form of a dimer or multimer having, in addition to two heavy chains and two light chains, a J (joining) chain and, in some cases, an SC (secretary component).

[0018] There are various classes of antibodies, such as IgG, IgE, IgM, IgD, IgA, and IgY, and further subclasses thereof, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0019] The antibodies of the present disclosure may have amino acid sequences derived from the same species or from different species, such as human, mouse, rat, hamster, rabbit, goat, donkey, pig, cow, horse, chicken, monkey, chimpanzee, camel, and llama.

[0020] In the case of antibodies derived from different species, there is no particular limitation, and examples include antibodies derived from two or more of human, mouse, rat, hamster, rabbit, goat, sheep, donkey, pig, cow, horse, chicken, monkey, chimpanzee, camel, llama, etc.

[0021] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been found that the ability of an antibody to specifically bind to an antigen can also be maintained by fragments consisting of a portion thereof. In one embodiment, the "antigen-binding fragment" of an antibody may be, but is not limited to, a Fab fragment consisting of a light chain variable region (VL), a heavy chain variable region (VH), a light chain constant region (CL), and a CH1 domain that is part of the heavy chain constant region; an F(ab')2 fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single antibody arm; a dAb fragment containing a single variable domain; and an isolated complementarity-determining region (CDR).

[0022] The antibody of the present disclosure may be a CDR-grafted antibody. In one example, a CDR-grafted antibody has part or all of the CDR region sequence of an antibody derived from one animal species replaced with the CDR sequence of another animal species. For example, one or more CDRs of a mouse antibody are replaced with the CDR sequence of a human antibody.

[0023] Methods well known to those skilled in the art can be used to identify heavy chain CDR1 to 3 in the heavy chain variable region and light chain CDR1 to 3 in the light chain variable region of an antibody. For example, the "Kabat definition" (Kabat et al., Ann. NY Acad. Sci. 1971, Vol. 190, pp. 382-391 and Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, U.S. Department of Health and Human Services, NIH Publication, pp. 91-3242), the "Chothia definition" (Chothia et al., Nature, 1989, Vol. 342, pp. 877-883), and the "contact definition" (MacCallum et al., Nature, 1989, Vol. 342, pp. 877-883), all of which are well known to those skilled in the art, may be used. al., J. Mol. Biol., 1996, Vol. 262, pp. 732-745) can be used. To identify the CDR sequences in an antibody, the CDRs may be identified based on information in public databases (e.g., https: / / www.ncbi.nlm.nih.gov / igblast).

[0024] As used herein, "identity" refers to the degree to which two or more comparable amino acid sequences or nucleotide sequences are identical to each other. Thus, the higher the identity between two amino acid sequences or nucleotide sequences, the higher the identity or similarity between those sequences. The level of identity between amino acid sequences or nucleotide sequences is usually determined using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (e.g., Karlin S, Altschul S F. Proc. Natl Acad Sci USA. 87:2264-2268 (1990), Karlin S, Altschul S F. Natl Acad Sci USA. 90:5873-7 (1993), etc.). Programs called BLASTN and BLASTX based on such BLAST algorithms have been developed (e.g., Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. J Mol Biol. 215:403-10 (1990)). Specific techniques for these analysis methods are known, and can be found on the NCBI website. For example, when a certain amino acid sequence A is identical to another amino acid sequence B by a certain percentage, this means that amino acid sequence A and amino acid sequence B have the specified percentage of identity.

[0025] As used herein, "monoclonal" is a modifier indicating the character of an antibody or the like obtained from a population of substantially homogeneous antibodies, the individual antibodies within which are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0026] As used herein, the term "conservative substitution techniques" refers to techniques in which an amino acid residue is replaced with an amino acid residue having a similar side chain.

[0027] For example, conservative substitution techniques include substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine. Other conservative substitution techniques include substitutions between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0028] As used herein, "prevention" of a disease has the usual meaning in the art, such as preventing the onset of a disease or reducing the risk of developing a disease.

[0029] As used herein, "treatment" of a disease has its usual meaning in the art, such as preventing the onset of a disease, reducing the risk of developing a disease, inhibiting the progression of a disease that has already occurred, slowing the progression of a disease that has already occurred, eliminating a disease that has already occurred, ameliorating the symptoms of a disease that has already occurred, etc. When the disease is colorectal cancer, "treatment" includes improving the subject's survival, causing tumor regression, stopping or slowing tumor growth, eliminating a tumor, and inhibiting or preventing metastasis.

[0030] (Composition for preventing or treating colorectal cancer, comprising an IgA antibody or an antigen-binding fragment thereof) In one aspect, the present disclosure provides a composition for preventing or treating colorectal cancer, comprising an IgA antibody or an antigen-binding fragment thereof. The form of the composition of the present disclosure is not particularly limited, but it can be provided preferably as a pharmaceutical composition, an oral composition, or an enteral composition.

[0031] The colorectal cancer targeted for prevention or treatment by the composition of the present disclosure is also called colon cancer, and is a cancer occurring in the colorectum that is well known to those skilled in the art. The type of colorectal cancer targeted for prevention or treatment by the composition of the present disclosure is not particularly limited, and includes cancer that develops when a benign polyp becomes cancerous and cancer that develops directly from normal mucosa.

[0032] The colorectal cancer targeted for prevention or treatment by the composition of the present disclosure is also called colon cancer, and is a cancer occurring in the colorectum that is well known to those skilled in the art. The type of colorectal cancer targeted for prevention or treatment by the composition of the present disclosure is not particularly limited, and includes cancer that develops when a benign polyp becomes cancerous and cancer that develops directly from normal mucosa.

[0033] (IgA antibody or antigen-binding fragment thereof) The IgA antibody used in the composition for preventing or treating colorectal cancer of the present disclosure is not particularly limited as long as it has the effect of preventing or treating colorectal cancer. Whether an IgA antibody has the effect of preventing or treating colorectal cancer can be confirmed, for example, by tests using a model system shown in the examples of the present disclosure, or by tests in which the IgA antibody is administered to subjects suffering from or at risk of colorectal cancer.

[0034] In one aspect, the IgA antibody used in the composition for preventing or treating colorectal cancer of the present disclosure is an IgA antibody present in the intestinal lumen. The IgA antibody is not particularly limited, but an IgA antibody present in the intestinal lumen of a subject organism for preventing or treating colorectal cancer can be preferably used.

[0035] In one embodiment, the IgA antibody used in the composition for preventing or treating colorectal cancer of the present disclosure may be an IgA antibody that binds to and / or inhibits the growth of Fusobacterium and / or Clostridium difficile bacteria. Examples of such antibodies include the IgA antibodies and their variants described in WO 2023 / 277142 and Japanese Patent Application No. 2023-131537.

[0036] The IgA antibodies of the present disclosure may be produced from antibody-producing cells derived from B cells, such as hybridomas, or may be produced by using genetic recombination technology to introduce nucleic acids encoding the antibodies into cells outside the immune system, and used as recombinant antibodies.

[0037] In one embodiment, the IgA antibody or antigen-binding fragment thereof comprised in the composition of the present disclosure is the following antibody disclosed in WO 2023 / 277142: an IgA antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3, the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3, the light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 8.

[0038] In one aspect, the IgA antibody or antigen-binding fragment thereof comprised in the composition of the present disclosure comprises the following antibody: a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6.

[0039] In one embodiment, the composition of the present disclosure comprises an IgA antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0040] In one embodiment, the IgA antibody or antigen-binding fragment thereof contained in the composition of the present disclosure is a purified recombinant antibody SNK0001AR obtained by determining the nucleic acid sequence encoding the IgM antibody SNK0001M produced by a hybridoma obtained from spleen-derived B cells and then applying recombinant technology to convert the IgA antibody into SNK0001AR. The antibody SNK0001AR has the following amino acid sequence:

[0041] In one embodiment, the IgA antibody or antigen-binding fragment thereof comprised in the composition of the present disclosure comprises a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 17, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of the light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 18.

[0042] In one embodiment, the IgA antibody or antigen-binding fragment thereof comprised in the composition of the present disclosure comprises a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 11; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 12; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13; and a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 14; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 15; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 16.

[0043] In one embodiment, the IgA antibody or antigen-binding fragment thereof comprised in the composition of the present disclosure comprises: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0044] In one embodiment, the IgA antibody or antigen-binding fragment thereof contained in the composition of the present disclosure is a purified recombinant antibody SNK0002AR, which is an IgA antibody obtained by determining the nucleic acid sequence encoding the IgM antibody SNK0002M produced by a hybridoma obtained from spleen-derived B cells and applying recombinant technology. The antibody SNK0002AR has the following amino acid sequence:

[0045] In one embodiment, the IgA antibody or antigen-binding fragment thereof comprised in the composition of the present disclosure comprises a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:27, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of the light chain variable region comprising the amino acid sequence represented by SEQ ID NO:28.

[0046] In one embodiment, the IgA antibody or antigen-binding fragment thereof comprised in the composition of the present disclosure comprises a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23; and a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 24; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 25; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26.

[0047] In one embodiment, the composition of the present disclosure comprises an IgA antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:27, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:28, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0048] In one embodiment, the IgA antibody or antigen-binding fragment thereof contained in the composition of the present disclosure is a purified recombinant antibody, SNK0003A, obtained by applying recombinant technology to the IgA antibody SNK0003M produced by a hybridoma obtained from B cells derived from the intestinal lamina propria mucosa. The antibody SNK0003A has the following amino acid sequence:

[0049] In one embodiment, the IgA antibody or antigen-binding fragment thereof comprised in the composition of the present disclosure is the following IgA antibody described in Japanese Patent Application No. 2023-131537: an IgA antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3, the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 81, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3, the light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 82.

[0050] In one aspect, the IgA antibody or antigen-binding fragment thereof comprised in the composition of the present disclosure comprises a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 83; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 84; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 85; and a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 86; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 87; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 88.

[0051] In one embodiment, the composition of the present disclosure comprises an IgA antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:81, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:82, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0052] In one aspect, the IgA antibody or antigen-binding fragment thereof comprised in the composition of the present disclosure comprises: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 81, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 82, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and the IgA antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 83; a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 84; and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 85; and a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 86; a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 87; and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 88.

[0053] In one embodiment, the IgA antibody of the present disclosure is SNK0004, which was produced by determining the nucleic acid sequence encoding an IgA antibody produced by a hybridoma obtained from mouse-derived B cells and then applying recombinant technology to produce the IgA antibody. Antibody SNK0004 has the following amino acid sequence:

[0054] In one embodiment, the antibody SNK0004 is encoded by the following nucleic acid sequence:

[0055] In one embodiment, the IgA antibody or antigen-binding fragment thereof contained in the composition of the present disclosure is the following IgA antibody described in Japanese Patent Application No. 2023-131537: an IgA antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3, the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 93, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3, the light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 94.

[0056] In one aspect, the composition of the present disclosure comprises an IgA antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 95; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 96; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 97; and a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 98; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 99; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 100;

[0057] In one embodiment, the composition of the present disclosure comprises an IgA antibody or antigen-binding fragment thereof. The IgA antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 93, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 94, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0058] In one aspect, the IgA antibody or antigen-binding fragment thereof comprised in the composition of the present disclosure is an IgA antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 93 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 94 or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 95; a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 96; and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 97; and a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 98; a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 99; and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 100.

[0059] In one aspect, the IgA antibody of the present disclosure is SNK0005, which was produced by determining the nucleic acid sequence encoding an IgA antibody produced by a hybridoma obtained from mouse small intestine-derived B cells and then applying recombinant technology to produce the IgA antibody. Antibody SNK0005 has the following amino acid sequence:

[0060] In one embodiment, the antibody SNK0005 is encoded by the following nucleic acid sequence:

[0061] In one aspect, the IgA antibody or antigen-binding fragment thereof comprised in the composition of the present disclosure is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; and a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36.

[0062] In one embodiment, the composition of the present disclosure comprises an IgA antibody or antigen-binding fragment thereof, which is an antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 37, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 38, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0063] In one embodiment, the IgA antibody or antigen-binding fragment thereof comprised in the composition of the present disclosure has at least one amino acid mutation in at least one region selected from heavy chain CDR1 to 3, light chain CDR1 to 3, and light chain FR1, relative to a reference antibody comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 38, and is an antibody or antigen-binding fragment thereof that binds to the amino acid sequence RQEEHIELIAS (SEQ ID NO: 72) in the Escherichia coli SHMT protein and the amino acid sequence VLDMMMKLEKPE (SEQ ID NO: 73) in the iPGM protein of C. difficile.

[0064] The at least one amino acid mutation in the antibody can be identified by applying techniques available to those skilled in the art, utilizing the results of structural analysis of the complexes of an antibody having the amino acid sequence of the reference antibody with the E. coli SHMT protein, and an antibody having the amino acid sequence of the reference antibody with the C. difficile iPGM protein. The number of amino acid mutations relative to the reference antibody may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more.

[0065] Such a reference antibody is not particularly limited as long as it has a heavy chain variable region of W27G2_HV (SEQ ID NO: 37) and a light chain variable region of W27G2_LV (SEQ ID NO: 38). For example, an IgG antibody (W27RS_H000_L000GR) can be used. The antibody W27RS_H000_L000GR has the same heavy chain variable region and light chain variable region as the antibody W27G2 produced by a hybridoma obtained from B cells derived from the intestinal lamina propria mucosa.

[0066] Whether an antibody having the specified mutation actually binds to the amino acid sequence RQEEHIELIAS in the E. coli SHMT protein and the amino acid sequence VLDMMMKLEKPE in the iPGM protein of C. difficile can be confirmed by methods well known to those skilled in the art, such as ELISA or Western blot. For example, the test method disclosed in WO2023 / 277142 can be used.

[0067] In one embodiment, the composition of the present disclosure comprises an IgA antibody or antigen-binding fragment thereof comprising: X １ heavy chain CDR1 comprising the amino acid sequence of YYIH, RIDPENX ２ X ３ a heavy chain CDR2 comprising the amino acid sequence of TTYAPKFQ; a heavy chain CDR3 comprising the amino acid sequence of YCARSTVL; and a heavy chain variable region comprising: RX ４ a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SQSIVHTNG; a light chain CDR2 comprising the amino acid sequence of KLLIYKV; and a light chain CDR3 comprising the amino acid sequence of GVYYCFQGS; and a light chain FR1 comprising the amino acid sequence of TPLSLPVSLGDQA or SPASX. ５ SVSLGDRX ６ X １ , X ２ , X ３ are each independently a neutral polar amino acid or an acidic polar amino acid, and X ４ is a nonpolar amino acid or a neutral polar amino acid, and X ５ , X ６is an antibody or antigen-binding fragment thereof, each independently a nonpolar amino acid.

[0068] The above antibody was designed, based on three-dimensional structural analysis, to have a binding mode similar to that of the W27G2 antibody with respect to the amino acid sequence RQEEHIELIAS in the E. coli SHMT protein and the amino acid sequence VLDMMMKLEKPE in the iPGM protein of C. difficile, and actually exhibits a binding mode and bacterial growth inhibitory effect similar to that of the W27G2 antibody with respect to these bacteria.

[0069] In one embodiment, the composition of the present disclosure comprises an IgA antibody or antigen-binding fragment thereof comprising: X １ heavy chain CDR1 comprising the amino acid sequence of YYIH, RIDPENX ２ X ３ a heavy chain CDR2 comprising the amino acid sequence of TTYAPKFQ; a heavy chain CDR3 comprising the amino acid sequence of YCARSTVL; and a heavy chain variable region comprising: RX ４ a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SQSIVHTNG; a light chain CDR2 comprising the amino acid sequence of KLLIYKV; and a light chain CDR3 comprising the amino acid sequence of GVYYCFQGS; and a light chain FR1 comprising the amino acid sequence of TPLSLPVSLGDQA or SPASX. ５ SVSLGDRX ６ X １ , X ２ are each independently asparagine or aspartic acid, and X ３ is glutamine or glutamic acid, and X ４ is alanine or serine, and X ５ is leucine or methionine, and X ６ is an alanine or valine.

[0070] In one embodiment, the heavy chain of the IgA antibody comprised in the composition of the present disclosure comprises: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31 or 41; a heavy chain CDR2 comprising the amino acid sequence represented by any one of SEQ ID NOs: 32, 42 to 44; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33.

[0071] In one aspect, the heavy chain of the IgA antibody comprised in the composition of the present disclosure comprises: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33.

[0072] In one aspect, the heavy chain of the IgA antibody comprised in the composition of the present disclosure comprises: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33.

[0073] In one aspect, the heavy chain of the IgA antibody comprised in the composition of the present disclosure comprises: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 43; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33.

[0074] In one aspect, the heavy chain of the IgA antibody comprised in the composition of the present disclosure comprises: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33.

[0075] In one aspect, the heavy chain of the IgA antibody comprised in the composition of the present disclosure comprises: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33.

[0076] In one aspect, the heavy chain of the IgA antibody comprised in the composition of the present disclosure comprises: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 42; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33.

[0077] In one aspect, the heavy chain of the IgA antibody comprised in the composition of the present disclosure comprises: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 43; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33.

[0078] In one aspect, the heavy chain of the IgA antibody comprised in the composition of the present disclosure comprises: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44; and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33.

[0079] In one embodiment, the heavy chain of the IgA antibody comprised in the composition of the present disclosure comprises: a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34 or 52; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36.

[0080] In one aspect, the light chain of the IgA antibody comprised in the composition of the present disclosure comprises: a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36.

[0081] In one aspect, the light chain of the IgA antibody comprised in the composition of the present disclosure comprises: a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35; and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36.

[0082] In one embodiment, the antibody or antigen-binding fragment thereof comprised in the composition of the present disclosure comprises in its light chain variable region a sequence that binds to Protein L. The antibody molecule or antigen-binding fragment thereof comprising the sequence that binds to Protein L can be purified using a Protein L column.

[0083] In one aspect, the antibody or antigen-binding fragment thereof comprising a sequence that binds to Protein L of the present disclosure has the amino acid sequence SPASX ５ SVSLGDRX６ a light chain variable region comprising X ５ , X ６ are each independently a nonpolar amino acid.

[0084] In one aspect, the antibody or antigen-binding fragment thereof comprising a sequence that binds to Protein L of the present disclosure has the amino acid sequence SPASX ５ SVSLGDRX ６ a light chain variable region comprising X ５ is leucine or methionine, and X ６ is alanine or valine.

[0085] In one embodiment, the antibody or antigen-binding fragment thereof comprising a sequence that binds to Protein L of the present disclosure comprises a light chain variable region comprising an amino acid sequence represented by one selected from the group consisting of SEQ ID NOs: 53-55.

[0086] In one aspect, the antibody or antigen-binding fragment thereof comprising a sequence that binds to Protein L of the present disclosure comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 54. This sequence is obtained by introducing a mutation into the sequence TPLSLPVSLGDQA (SEQ ID NO: 56) of the light chain FR1 region of the W27G2 antibody so that the antibody molecule binds to Protein L.

[0087] In one aspect, the antibody or antigen-binding fragment thereof comprising a sequence that binds to Protein L of the present disclosure comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 55. This sequence is obtained by further introducing a mutation into the light chain FR1 region of the light chain variable region comprising the amino acid sequence of SEQ ID NO: 54, so that the antibody molecule binds to Protein L more tightly.

[0088] In one aspect, the antibody or antigen-binding fragment thereof comprising a sequence that binds to Protein L of the present disclosure comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 58. This sequence is obtained by further introducing a mutation into the light chain FR1 region of the light chain variable region comprising the amino acid sequence of SEQ ID NO: 54, so that the antibody molecule binds to Protein L more tightly.

[0089] The antibody or antigen-binding fragment thereof contained in the composition of the present disclosure may have any combination of the heavy and light chains described above, and may or may not contain a sequence that binds to Protein L.

[0090] In one aspect, the antibody or antigen-binding fragment thereof comprised in the composition of the present disclosure is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31 or 41, a heavy chain CDR2 comprising the amino acid sequence represented by any one of SEQ ID NOs: 32, 42 to 44, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34 or 52, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and a light chain FR1 comprising the amino acid sequence represented by any one of SEQ ID NOs: 53 to 56, or an antibody or antigen-binding fragment thereof comprising: a light chain variable region comprising: a light chain FR1 comprising the amino acid sequence represented by any one of SEQ ID NOs: 53 to 56.

[0091] For example, the antibody or antigen-binding fragment thereof contained in the composition of the present disclosure is an antibody or antigen-binding fragment thereof that includes a combination of any of the heavy chain variable regions and any of the light chain variable regions described below.

[0092] In one embodiment, the IgA antibody or antigen-binding fragment thereof preferably comprised in the composition of the present disclosure comprises the following combination of heavy chain variable regions and light chain variable regions:

[0093] In one aspect, the IgA antibody or antigen-binding fragment thereof preferably comprised in the composition of the present disclosure includes the antibodies described below or modified versions thereof to IgA.

[0094] Antibodies produced in CHO cells by introducing mutations into the heavy or light chain of the W27G2 antibody so as not to substantially alter its antigen specificity are referred to as RS mutant recombinant purified antibodies. In one embodiment, these RS mutant recombinant purified antibodies further comprise a mutation for binding to Protein L.

[0095] The antibodies of the present disclosure can have mutations in their constituent amino acid sequences, such as their constant region, heavy chain variable region, light chain variable region, heavy chain CDR1-3, or light chain CDR1-3, to the extent that their antigen-binding properties and physiological activity are not lost. Such mutations may be, but are not limited to, substitutions, deletions, insertions, and the like. For example, conservative substitution techniques can be employed for substitutions. Furthermore, by analyzing the binding mode between the antibody and the antigen in detail using three-dimensional structural analysis or the like, various mutations can be introduced into the antibodies of the present disclosure to the extent that their antigen-binding properties are not lost.

[0096] In one embodiment, the IgA antibody or antigen-binding fragment thereof contained in the composition of the present disclosure may be provided in the form of an antibody consisting of one heavy chain and one light chain (also called a single-chain antibody), in addition to a form consisting of two heavy chains and two light chains. Furthermore, the antibody according to the present disclosure may be provided in the form of a dimer or multimer having, in addition to two heavy chains and two light chains, a J (joining) chain and, optionally, an SC (secretary component).

[0097] In one embodiment, the IgA antibody or antigen-binding fragment thereof contained in the composition of the present disclosure includes an antibody whose constant or variable region has been modified from its native counterpart, provided that its antigen-binding properties and biological activity are not lost. In one embodiment, such modifications include amino acid substitution, deletion, insertion, etc. (terminal addition). In one embodiment, such modifications include fusion or conjugation with other peptide or non-peptide structures. In one embodiment, the modified constant or variable region has at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity in its amino acid portion with the native amino acid sequence.

[0098] (Nucleic Acid and Nucleic Acid Set) In one aspect, the present disclosure provides a nucleic acid encoding an IgA antibody or antigen-binding fragment thereof contained in the composition of the present disclosure. The nucleic acid encoding the IgA antibody or antigen-binding fragment thereof may be ribonucleotides or deoxynucleotides. Furthermore, the form of the nucleic acid is not particularly limited, and may be single-stranded or double-stranded. The codons used in the nucleic acid sequence are not particularly limited, and various codons can be appropriately selected and used depending on the purpose. For example, appropriate codons can be selected taking into account codon frequency and the type of host cell used during production. In one aspect, a nucleic acid encoding an antibody or antigen-binding fragment thereof of the present disclosure is used to express and produce an antibody or antigen-binding fragment thereof according to the present disclosure. In one aspect, a nucleic acid encoding an antibody or antigen-binding fragment thereof of the present disclosure is used to express an antibody or antigen-binding fragment thereof according to the present disclosure in vivo and prevent or treat colorectal cancer.

[0099] When a nucleic acid encoding an antibody or antigen-binding fragment thereof of the present disclosure encodes an antigen-binding fragment, for example, the two domains of the Fv fragment, VL and VH, may be encoded by separate nucleic acid molecules, or, using recombinant techniques, a single protein chain in which the pair of VL and VH domains form a monovalent molecule (single-chain Fv (scFv)) may be encoded by a single nucleic acid.

[0100] In one embodiment, a nucleic acid encoding an antibody or antigen-binding fragment thereof of the present disclosure may be in the form of a vector. For example, the vector may be an expression vector or a vector for use in genetic recombination. The configuration of the vector is not particularly limited and may include various components used in known vectors.

[0101] In one embodiment, a nucleic acid encoding an antibody or antigen-binding fragment thereof of the present disclosure encodes a portion of the peptide fragment that constitutes the antibody or antigen-binding fragment, and the nucleic acid may be provided as a set together with nucleic acids encoding other peptide fragments that constitute the antibody or antigen-binding fragment. The set of nucleic acids may include all of the nucleic acids encoding the peptide fragments that constitute the antibody or antigen-binding fragment, or may include only a portion of the nucleic acids encoding the peptide fragments that constitute the antibody or antigen-binding fragment. A set of nucleic acids containing only a portion of the nucleic acids can be used in combination with another nucleic acid or set of nucleic acids that encodes another portion of the peptide fragment that constitutes the antibody or antigen-binding fragment.

[0102] (Form of composition) The form of the composition for preventing or treating colorectal cancer of the present disclosure is not particularly limited. In addition to being used as a medicine, the composition of the present disclosure can also be suitably used in the fields of food and feed, with the expectation of its preventive or therapeutic effect on colorectal cancer. Therefore, the composition of the present disclosure can be a pharmaceutical composition, a food composition, or a feed composition.

[0103] The composition of the present disclosure may contain an effective amount of an IgA antibody or an antigen-binding fragment thereof. For example, when used as a pharmaceutical composition, the antibody content can be appropriately determined so that it falls within the range of 0.001 to 99.99% by weight of 100% by weight of the composition, taking into consideration the type and condition of the target colorectal cancer, symptoms of the recipient, the degree of effect exerted by administration, dosage form, administration method, recipient, and gender of the recipient, etc.

[0104] As used herein, the term "effective amount" refers to an amount that can exert a preventive or therapeutic effect on colorectal cancer.

[0105] The composition of the present disclosure may contain a pharmaceutically acceptable carrier or additive together with the IgA antibody. For example, in the case of a pharmaceutical composition, the pharmaceutically acceptable carrier or additive means any carrier, diluent, excipient, suspending agent, lubricant, adjuvant, vehicle, delivery system, emulsifier, tablet disintegrant, absorbent, preservative, surfactant, colorant, flavoring, or sweetener, and any known pharmaceutically acceptable carrier or additive may be used.

[0106] The organisms to which the composition of the present disclosure is administered are not particularly limited, and examples include mammals such as humans, primates, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, and pigs, and birds such as chickens.

[0107] The dosage and administration method of the composition of the present disclosure can be appropriately determined taking into consideration the type and condition of the target colorectal cancer, the symptoms of the subject, the degree of effect exerted by administration, the dosage form, the administration method, the subject, the gender of the subject, etc. The dosage may generally be appropriately determined within the range of 0.001 to 100 mg / kg / day in terms of IgA.

[0108] The administration method is not particularly limited, but direct administration into the digestive tract is preferred, and examples of such administration methods include oral administration, nasal administration, transmucosal administration, and enteral administration.

[0109] Enteral administration is not limited to administration via the anus, but also includes administration via a tube or the like inserted into the digestive tract from outside the individual, such as a gastrostomy. The location into which the digestive tract is inserted is not limited to the intestine, but includes the esophagus, stomach, small intestine (including the duodenum, jejunum, ileum, etc.), large intestine (including the cecum, colon, rectum, etc.), etc.

[0110] The composition according to the present disclosure may be administered in the above amount once a day or in divided doses. Furthermore, the administration interval may be daily, every other day, weekly, biweekly, every 2-3 weeks, monthly, bimonthly, or every 2-3 months, as long as it has a therapeutic effect against the above-mentioned diseases.

[0111] The food composition described above is suitable for use exclusively in the food industry, and can be provided as a food composition that displays, as a function, an effect related to the prevention or treatment of colorectal cancer, for example.

[0112] The food compositions described above can be provided as general foods, as well as foods for specified health uses including conditional foods for specified health uses, nutritional supplements, functional foods, foods for the sick, and the like.

[0113] The specific form of the food composition is not particularly limited, and examples thereof include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, and dairy drinks; frozen desserts such as ice cream, ice sherbet, and shaved ice; sweets such as candy, candy, gum, chocolate, tablet candy, snacks, biscuits, jelly, jam, cream, and baked goods; noodles such as soba, udon, harusame, Chinese noodles, and instant noodles; processed seafood and livestock foods such as kamaboko, ham, and sausage; dairy products such as processed milk and fermented milk; oils and fats and processed oil foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressing; seasonings such as sauces and sauces; soups, stews, salads, side dishes, sprinkles, pickles, bread, and cereals. In the case of foods for specified health uses, nutritional supplements, and functional foods, examples of the food composition include powders, granules, capsules, lozenges, tablets, and syrups.

[0114] The feed composition described above is suitable for use exclusively in the field of feed, and can be provided as a feed composition that exhibits, for example, an effect related to the prevention or treatment of colorectal cancer as a function.

[0115] The specific form of the above-mentioned feed composition is not particularly limited, and for example, as long as the effects exerted by the feed composition according to the present disclosure are not impaired, the feed composition may be prepared by mixing it with ordinary feed or, if necessary, by mixing it with components that can be incorporated into ordinary feed, or the feed composition itself may be used as feed.

[0116] (Method of preventing or treating colorectal cancer) In one aspect, the present disclosure provides a method of preventing or treating colorectal cancer. Typically, the method is a method for preventing or treating colorectal cancer in a subject in need of treatment, comprising administering to the subject a composition containing an IgA antibody or antigen-binding fragment thereof.

[0117] The IgA antibody or antigen-binding fragment thereof used in the method for preventing or treating colorectal cancer of the present disclosure can be the IgA antibody or antigen-binding fragment thereof described above as being used in the composition for preventing or treating colorectal cancer, comprising an IgA antibody or antigen-binding fragment thereof.

[0118] The composition containing an IgA antibody or its antigen-binding fragment used in the method for preventing or treating colorectal cancer of the present disclosure can have the form (form of the composition) described above in (Composition for preventing or treating colorectal cancer containing an IgA antibody or its antigen-binding fragment). In the method of the present disclosure, the composition can be used in the form of a pharmaceutical composition, food composition, or feed composition.

[0119] The target organisms to which the method of the present disclosure can be applied are not particularly limited, but examples include mammals such as humans, primates, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cows, and pigs, and birds such as chickens.

[0120] The dosage and administration method of the composition in the method of the present disclosure can be appropriately determined taking into consideration the type and condition of the target colorectal cancer, the symptoms of the subject, the degree of effect exerted by administration, the dosage form, the administration method, the subject, the gender of the subject, etc. The dosage may generally be appropriately set within the range of 0.001 to 100 mg / kg / day in terms of IgA.

[0121] The administration method is not particularly limited, but direct administration into the digestive tract is preferred, and examples of such administration methods include oral administration, nasal administration, transmucosal administration, and enteral administration.

[0122] Enteral administration is not limited to administration via the anus, but also includes administration via a tube or the like inserted into the digestive tract from outside the individual, such as a gastrostomy. The location into which the digestive tract is inserted is not limited to the intestine, but includes the esophagus, stomach, small intestine (including the duodenum, jejunum, ileum, etc.), large intestine (including the cecum, colon, rectum, etc.), etc.

[0123] The composition according to the present disclosure may be administered in the above amount once a day or in divided doses. Furthermore, the administration interval may be daily, every other day, weekly, biweekly, every 2-3 weeks, monthly, bimonthly, or every 2-3 months, as long as it has a therapeutic effect against the above-mentioned diseases.

[0124] The food compositions described above can be provided as general foods, as well as foods for specified health uses including conditional foods for specified health uses, nutritional supplements, functional foods, foods for the sick, and the like.

[0125] When the composition according to the present disclosure is a food composition, its specific form is not particularly limited, and examples thereof include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, and milk drinks; frozen desserts such as ice cream, ice sherbet, and shaved ice; sweets such as candy, candy, gum, chocolate, tablet candy, snacks, biscuits, jelly, jam, cream, and baked goods; noodles such as soba, udon, harusame, Chinese noodles, and instant noodles; processed seafood and livestock foods such as kamaboko, ham, and sausage; dairy products such as processed milk and fermented milk; oils and fats and oil-and-fat processed foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressing; condiments such as sauces and dressings; soups, stews, salads, side dishes, furikake (seasonings), pickles, bread, and cereals. In the case of foods for specified health uses, nutritional supplements, functional foods, etc., the formulations include powder, granules, capsules, lozenges, tablets, syrup, etc.

[0126] When the composition according to the present disclosure is a feed composition, its specific form is not particularly limited, and as long as the effect of the composition is not impaired, the composition may be mixed with ordinary feed or, if necessary, mixed with components that can be incorporated into ordinary feed to form a feed composition, or the feed composition itself may be used as feed.

[0127]

[0128] Hereinafter, the present disclosure will be described in more detail with reference to examples, but these are merely illustrative and do not limit the present disclosure.

[0129] (1) Materials and Methods (1-1) Generation of Mzb1- / - and Iga- / - Mice MZB1-deficient mice were generated as previously described (Xiong, E. et al. Proc Natl Acad Sci USA 116, 13480-13489 (2019)). To generate Iga- / - mice, a gRNA targeting the mouse Igha gene (5'-TCACATTCATCGTCCGGGAAGGG-3'), a donor oligo containing a 28-bp insertion between c. 107 and c. 108 of the first exon of the Cα gene (5'-TGATAGACTAAACGGCCACAAGTTCGA-3'), and Cas9 were co-injected into mouse fertilized eggs to generate targeted knock-in offspring (Figure 9a). F0 founder animals were identified by PCR using F1 (5'-GGAGTCATTTGGCTAGATCCTTGT-3') and R1 (5'-ATAAGGTAGAACTTATCCCACCCC-3') primers, followed by sequence analysis (Fig. 9b). These animals were mated with wild-type mice to test for germline transmission and the generation of F1 animals. ELISA confirmed the absence of IgA in the serum of Iga- / - mice (Fig. 9c). Heterozygous mice were mated to generate + / + and - / - mice, which were housed in separate cages. Mice were maintained under specific pathogen-free conditions in the animal facility at Fudan University. All animal experiments and procedures were approved by the Fudan University Animal Care and Use Committee.

[0130] (1-2) AOM / DSS-Induced Colorectal Cancer. Colorectal cancer was induced by a single intraperitoneal injection of the mutagen AOM (10 mg / kg, Sigma-Aldrich) on day 1, followed by three cycles of drinking water containing 2.5% DSS (MP Biomedicals) for one week and regular drinking water for two weeks. Supplementation with W27 monoclonal IgA antibody was performed according to (Okai, S. et al. Nat Microbiol 1, 16103 (2016)). Briefly, throughout the CRC induction process, mice received drinking water containing 2.5 μg / ml of W27. The control group received regular drinking water without IgA antibody. Fecal samples were collected and sent to Sangon Biotech for sequencing of the V4 region of the 16S rRNA gene. In all AOM / DSS experiments, male mice aged 8-10 weeks were used.

[0131] (1-3) Disease Activity Index (DAI) Clinical symptoms of mice during AOM / DSS induction were monitored using the DAI. DAI was calculated as a total score according to the following indices: weight loss (0, <1%; 1, 1-5%; 2, 5-10%; 3, 10-15%; 4, >15%); diarrhea (0, normal stool; 2, loose stool; 4, watery diarrhea); and rectal bleeding (0, no bleeding; 2, slight bleeding; 4, severe bleeding).

[0132] (1-4) Isolation of Colonic Lamina Propria Cells. The colon was excised, opened longitudinally, and washed twice with PBS to remove feces and fat. The washed colon was then cut into 5 mm segments and stirred at 37°C for 30 minutes in D-Hank's Buffer (Sangon) containing 2% FBS, 1 mM DTT, 1 nM EDTA (Invitrogen), and 25 nM sodium bicarbonate (Meilunbio). The segments were filtered through a 70 μm cell filter and cut into fragments. These fragments were further digested at 37°C for 1 hour in RPMI-1640 medium containing 10% FBS, 1 mg / ml collagenase IV (Sigma), and 0.08 mg / ml DNase I (STEMCELL). The digestion process was stopped by adding PBS. Finally, the digested cells were filtered through a 70 μm cell filter and collected.

[0133] (1-5) Flow Cytometry Single-cell suspensions were first incubated with anti-CD16 / 32 (catalog number 2.4G2; BD Biosciences) to block FcγR and then stained with the following fluorochrome-conjugated antibodies: α-B220 (catalog number 552772; BD Biosciences), α-IgM (catalog number AF6-78; BD Biosciences), α-IgA (catalog number 12-4204-82; eBioscience), α-CD11b (catalog number 101207; Biolegend), α-F4 / 80 (catalog number 25-4801-82; eBioscience), and α-Ly6G (catalog number 560599; BD Biosciences). Biosciences), α-CD45 (catalog no. 11-0451-81; eBioscience), α-CD3 (catalog no. 152323; Biolegend), α-GL7 (catalog no. 144604; Biolegend), and α-FAS (catalog no. 13-5911-81; eBioscience). 7-AAD Viability Staining Solution (eBioscience) was used to distinguish between live and dead cells. Samples were analyzed on a FACSVerse flow cytometer (BD Biosciences) using FACSsuite software. Data analysis was performed using FlowJo software (Treestar).

[0134] Flow cytometry analysis of IgA+ fecal microbiota was performed as previously reported (Kimura, S. et al. J Exp Med 216, 831-846 (2019)). Briefly, mouse fecal samples were diluted with PBS, homogenized, and centrifuged (90 x g, 15 minutes, 4°C) to remove large particles. The supernatant was filtered and further centrifuged (8000 x g, 5 minutes, 4°C) to enrich the microbiota. The bacterial pellet was washed and incubated with anti-CD16 / 32 (catalog no. 2.4G2; BD Biosciences) and then stained with α-IgA (catalog no. 12-4204-82; eBioscience). Samples were then washed twice and filtered again before flow cytometry analysis.

[0135] (1-6) ELISA Analysis. To measure serum Ig, plates were coated with 5 μg / mL anti-Ig (H+L) Ab (SouthernBiotech) and blocked with 1% BSA in PBS for 1 hour. Diluted samples were then added and incubated at room temperature for 1 hour. After washing, HRP-conjugated goat anti-mouse IgM, IgG1, IgG2b, IgG2c, IgG3, or IgA (SouthernBiotech) antibodies were added and developed with 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid solution. For fecal antibody detection, fecal pellets were weighed and dissolved in PBS containing a protease inhibitor mixture (Sigma-Aldrich), and debris was removed by centrifugation. Fecal IgA was measured using a mouse IgA ELISA quantification set (Bether Laboratories).

[0136] (1-7) Immunoblotting. Colon tissues were frozen, pulverized, and then resuspended in radioimmunoprecipitation assay lysis buffer (Cwbiotech) containing a protease inhibitor mixture. Lysates were mixed with sample buffer, heated at 95°C for 10 minutes, and then separated by SDS-PAGE. Immunoblotting was performed overnight using specific antibodies, including α-phospho-p38 (Cat. No. 9211; CSR), α-p38 (Cat. No. 9212; CST), α-phospho-ERK (Cat. No. 9101; CST), α-phospho-p38 (Cat. No. 9101; CST), ERK (Cat. No. 4695; CST), α-phospho-STAT3 (Cat. No. 9145; CST), and α-STAT3 (Cat. No. 9139; CST), at a 1:2000 dilution. This was followed by incubation with a secondary α-mouse or rabbit antibody conjugated to HRP. Finally, the membrane was developed using chemiluminescence.

[0137] (1-8) Immunoblotting Quantitative Real-Time PCR RNA was isolated using RNAiso Plus (TaKaRa) according to the manufacturer's instructions and then converted to cDNA using Reverse Transcriptase (Yeasen). Gene expression was assessed using 2x SYBR Green Master Mix according to the manufacturer's instructions (TaKaRa). RT-qPCR data were analyzed using the 2-ΔΔCT method with GAPDH as the housekeeping gene. The following primers were used for RT-qPCR: GAPDH (Forward: 5'-CCCACTAACATCAAATGGGG-3', Reverse: 5'-CCTTCCACAATGCCAAAGTT-3'); TNF-α (Forward: 5'-CCCTCACACTCACAAAC CAC-3', Reverse: 5'-ACAAGGTACA ACCCATCGGC-3'); IL-6 (Forward: 5'-GGATACCACTCCCAACAGACC-3', Reverse: 5'-TTCTGCAAGTGCA TCATCGT-3'); IFN-γ (Forward: 5'-GGAGGAACTGGCAAAAGGATG-3', Reverse: 5'-GTTGCTGATGGCCTGA TTGT-3'); IL-1β (Forward: 5'-TGCCACCTTTTGACAGTGATG-3', Reverse: 5'-ATGTGCTGCTGCGAGATT TG-3'); IL-4 (Forward: 5'-CCAGGAGCCATATCCACGG-3', Reverse: 5'-ACTCTCTGTGGTGTTCTTCGTT-3 '); IL-17A (Forward: 5'-CCCTCAGACTACCTCAACCG-3', Reverse: 5'-CATGTGGTGGTCCAGCTTTC-3'); IL-10 (Forward: 5'-AGCTGAAGACCCTCAGGATGC-3', Reverse: 5'-TCATGGCCTTGTAGACACCTTGG-3').

[0138] (1-9) Immunofluorescence. Colon samples were sectioned from the ileocecal valve to the anus, washed with ice-cold PBS, fixed in 4% paraformaldehyde, and dehydrated. These 7-μm frozen colon samples were incubated with AF488 rat anti-mouse IgA (Cat. No. 1040-30; Southernbiotech) overnight at 4°C. Nuclei were then counterstained with 4'6-diamidino-2-phenylindole (Cat. No. C1005; Beyotime) for 5 minutes. Images were acquired using a fluorescence microscope.

[0139] (1-10) Expression and Purification of Monoclonal IgA, W27. The cDNAs for the W27 heavy chain (HC), W27 light chain (LC), and J chain were separately subcloned into the pcDNA3.1 vector. 293T cells were cultured without antibiotics and cotransfected with pcDNA3.1 vectors expressing the HC, LC, and J chain. Transfection was performed using Hieff Trans™ liposomal transfection reagent (Yeasen) according to the manufacturer's protocol. After 48 and 96 hours, the culture supernatants were collected and filtered. An equal volume of saturated ammonium sulfate (Sangon) solution was added to the filtrate, stirred overnight, and then centrifuged at 10,000 g for 30 minutes. The resulting precipitate was dialyzed, filtered, and subjected to ELISA to measure the W27 concentration.

[0140] (1-11) Statistical Analysis Statistical analysis was performed using GraphPad Prism version 8. The Mann-Whitney test was used to determine statistical significance. A sample size of at least four animals per group was required for a valid Mann-Whitney test. Due to variability within each experiment, 5 to 8 mice per group were used to ensure robust statistical results. In some experiments, only triplicate mice were analyzed; in these cases, a Student's t-test was performed to determine statistical significance. Data from all animals were included in the analysis; none were excluded. A p value of <0.05 was considered statistically significant. ns represents no significance; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Additional data from this study can be provided upon request.

[0141] (2) Test results

[0142] (2-1) MZB1 Deficiency Accelerates the Development and Progression of AOM / DSS-Induced Colorectal Cancer. To evaluate the role of MZB1 in CRC development, we utilized an established CRC model in which mice were injected with the DNA-damaging agent azoxymethane (AOM), followed by three cycles of 2.5% DSS treatment. As shown in Figure 1a, Mzb1- / - mice exhibited significantly greater weight loss during each round of DSS treatment compared with Mzb1+ / + mice. Consistently, the disease activity index (DAI), a composite assessment of weight loss rate, stool viscosity, and stool bleeding, was also higher in Mzb1- / - mice than in Mzb1+ / + mice, especially toward the end of the DSS period (Figure 1b). Upon sacrifice, significant differences in the number of tumor nodules were observed between Mzb1+ / + and Mzb1- / - mice (Figure 1c and Figure 10a). Mzb1- / - mice developed significantly more tumor nodules than control mice (Fig. 1d, left panel). Furthermore, tumor nodules in mutant mice were significantly larger than those in Mzb1+ / + mice (Fig. 1d, center panel), and tumor nodules larger than 3 mm in size were significantly increased (Fig. 1d, right panel). These results suggest that MZB1 deficiency accelerates both the onset and progression of CRC.

[0143] (2-2) MZB1 Reduces Intestinal Inflammation During Colitis-Associated Colorectal Cancer (CAC) Development. To investigate the mechanisms underlying the accelerated development of CAC in Mzb1- / - mice, we next analyzed intestinal inflammation 2 weeks after the completion of CAC induction. Mzb1- / - mice exhibited more severe colon shortening (Fig. 2a) and more pronounced crypt destruction and cytological atypia compared with Mzb1+ / + mice (Fig. 2b). Analysis of immune cells within the colonic lamina propria revealed significantly increased percentages and absolute cell numbers of both macrophages and neutrophils, and decreased percentages and numbers of B cells, in Mzb1- / - mice compared with Mzb1+ / + mice (Fig. 2c, Fig. 10b, and Fig. 11). However, no differences were observed in the percentages and cell numbers of macrophages, neutrophils, B cells, and germinal center (GC) B cells in Peyer's patches between Mzb1+ / + and Mzb1- / - mice. Increased infiltration of macrophages and neutrophils in the lamina propria may enhance the intestinal inflammatory response, thereby further accelerating colorectal tumorigenesis. Supporting this hypothesis, quantitative PCR revealed increased expression of pro-inflammatory cytokines, such as TNF-α and IL-6, and decreased expression of the anti-inflammatory cytokine IL-10, in colonic tissues from Mzb1- / - mice compared with Mzb1+ / + mice (Figure 2d).

[0144] (2-3) Increased phosphorylation of p38 in Mzb1- / - CRC mice. To determine whether the observed changes in macrophages, neutrophils, and inflammatory cytokines were associated with activation of downstream signaling pathways, we performed immunoblot analysis of signaling molecules previously shown to be activated during intestinal inflammation. Specifically, phosphorylation of p38, ERK, and STAT3 may be involved in epithelial cell transformation and tumor progression. Consistent with the increased proportion of lamina propria macrophages and increased cytokine production in the colon, we observed a significant increase in phosphorylation of the MAPK signaling molecule p38 in Mzb1- / - CRC mice (Figure 12). However, the phosphorylation levels of STAT3 and ERK were not significantly elevated in Mzb1- / - mice compared with Mzb1+ / + mice.

[0145] (2-4) Mzb1- / - Mice Show Exacerbated Systemic Inflammation After CRC Induction We also noted enlargement of the spleens in Mzb1- / - mice. This correlated with increased spleen weight and cell counts compared with Mzb1+ / + mice after CRC induction (day 82, Figure 13a). These results demonstrate systemic inflammation in Mzb1- / - mice. Consistent with the observations of the lamina propria, the spleens of Mzb1- / - mice showed increased proportions of macrophages and neutrophils, but not T cells, B cells, or germinal center B cells (Figure 13b). Serum Ig levels (day 82), including IgA, IgM, IgG3, IgG1, IgG2b, and IgG2c, were comparable between Mzb1+ / + and Mzb1- / - mice (Figure 13c). The results suggest that the absence of MZB1 primarily affects myeloid cells, with minimal effects on lymphoid cells and humoral immune responses.

[0146] (2-5) MZB1 deficiency leads to reduced intestinal IgA levels. Previously, MZB1 has been shown to promote intestinal IgA secretion, thereby attenuating DSS-induced colitis. We hypothesized that the worsening of CRC development in the absence of MZB1 may be due to reduced intestinal IgA production. Flow cytometry analysis revealed a moderate decrease in the percentage and absolute number of IgA-positive plasma cells in the colonic lamina propria of Mzb1- / - mice after CRC induction (Figures 3a and 10c). The proportion of IgM-positive cells was comparable between Mzb1+ / + and Mzb1- / - mice. Five days after each round of DSS (T1, T2, and T3), serum and fecal IgA levels were measured using ELISA. Serum IgA levels in Mzb1+ / + mice increased significantly at T3 (day 59) and were significantly higher than those in Mzb1- / - mice (Fig. 3b). In parallel, fecal IgA levels increased from T1 to T3 in Mzb1+ / + mice but remained low in Mzb1- / - mice (Fig. 3b). Immunofluorescence staining of colonic Swiss rolls revealed bright IgA staining in Mzb1+ / + mice but not in Mzb1- / - mice (Fig. 3c). These results consistently demonstrate that MZB1 deficiency causes reduced IgA levels in both the intestine and serum.

[0147] Next, we investigated the potential relationship between changes in IgA levels and the gut microbiota in Mzb1+ / + and Mzb1- / - mice during CAC. We assessed the ability of fecal IgA to coat the microbiota by quantifying IgA-bound (IgA+) bacteria in the feces of AOM / DSS-treated mice using flow cytometry. Using feces from IgA- / - mice as a staining control, we found that the frequency of fecal bacteria coated with high levels of IgA was much lower in Mzb1- / - mice (1.55 ± 0.71%) compared with Mzb1+ / + mice (8.99% ± 2.45%) after AOM / DSS treatment (Fig. 3d). These observations suggest a decrease in the average amount of bound IgA per bacterium in Mzb1- / - mice, which could be due to either a reduced amount and / or lower binding affinity of intestinal IgA in the mutant mice compared with Mzb1+ / + mice.

[0148] (2-6) Mzb1- / - Mice Show Disrupted Gut Microbiota After CRC Induction. Previous research (Xiong, E. et al. Proc Natl Acad Sci USA 116, 13480-13489 (2019)) demonstrated that the fecal microbiota composition of Mzb1+ / + and Mzb1- / - mice was similar at steady state before AOM / DSS administration. However, 10 days after the end of CRC induction, the microbiome of Mzb1- / - mice showed reduced alpha diversity compared to Mzb1+ / + mice (Figure 4a) and was clearly different in composition from Mzb1+ / + mice (Figure 4b). Compared with Mzb1+ / + mice, Mzb1- / - mice showed significant changes in the relative abundance of four different bacterial phyla, with increases in Bacteroidetes and Deferibacteres and decreases in Verrucosa and Actinomycetes (Fig. 4c, Fig. S14). At the genus level, significant differences were detected in the average proportions of 16 different bacterial genera, with a marked reduction in Akkermansiosis in Mzb1- / - mice (Fig. 4d).

[0149] (2-7) IgA deficiency exacerbates AOM / DSS-induced colon cancer. To further emphasize the important role of IgA in CRC development, we generated Iga- / - mice and performed CRC induction as described above. Throughout the entire CRC induction process, Iga- / - mice exhibited lower body weights than Iga+ / + mice (Fig. 5a). Consistently, the disease activity index of Iga- / - mice was higher than that of Iga+ / + mice (Fig. 5b). Two weeks after the completion of CRC induction, mice were sacrificed. Iga- / - mice developed more tumor nodules and their size was larger than that of Iga+ / + mice (Fig. 5c). 16S rRNA sequencing clearly revealed differences in the composition of the fecal microbiota between Iga+ / + and Iga- / - mice (Fig. 5d, e). Furthermore, the relative abundance of nine different bacterial genera changed significantly (Fig. 5f).

[0150] (2-8) Oral administration of IgA attenuates AOM / DSS-induced CRC in Mzb1- / - mice. IgA is the major Ab isotype in intestinal secretions and plays a crucial role in maintaining homeostasis of the intestinal microenvironment. Mzb1- / - mice exhibit reduced intestinal IgA levels, which may contribute to the accelerated progression of AOM / DSS-induced CRC. To further confirm that the worsening CRC observed in Mzb1- / - mice was due to reduced IgA production in the intestine, Mzb1- / - mice were given the monoclonal IgA antibody, W27, at a concentration of 2.5 μg / ml in their drinking water throughout the CRC induction process. Administration of W27 significantly ameliorated AOM / DSS-induced CRC in Mzb1- / - mice, as indicated by reduced weight loss (Fig. 6a), reduced disease activity index (Fig. 6b), and increased colon length (Fig. 7a). After sacrificing the mice 2 weeks after CRC induction, Mzb1- / - mice fed W27 were found to have fewer and smaller tumors than mice not supplemented with W27 (Fig. 6c, d). Furthermore, W27 administration attenuated intestinal inflammation in Mzb1- / - mice, as indicated by reduced macrophage percentage and cell number in the colonic lamina propria (Fig. 7b and Fig. 10d) and reduced expression of IL-17A, IL-6, and TNF-α in colonic tissue. Although W27 treatment did not alleviate the spleen enlargement observed in Mzb1- / - CRC mice (Fig. 15a), it did reduce the proportion of macrophages and neutrophils (Fig. 15b) and the total spleen cell count (Fig. 15a, right panel). W27 treatment did not affect serum Ig levels (day 82), including IgA, IgM, IgG3, IgG1, IgG2b, and IgG2c (Fig. 15c). Ten days after CRC induction was completed, distinct fecal microbiota compositions were evident between the Mzb1- / -Ctrl and Mzb1- / -W27 groups (Fig. 16a). W27 treatment restored the disrupted microbiota in Mzb1- / - CRC mice and promoted Verrucomicrobiae colonization (Fig. 16b). At the genus level, significant differences were observed in the mean proportions of 11 different bacterial genera (Fig. 16c). Oral administration of W27 to Mzb1- / - mice did not result in an increase in fecal IgA levels (Fig. 17a).

[0151] (2-9) Co-housed Mzb1+ / + and Mzb1- / - mice exhibit similar CRC onset and progression. On day 10 of life, Mzb1+ / + and Mzb1- / - mice were housed together in shared cages and breastfed by the same mother. At 8 weeks of age, these mice were treated with AOM / DSS. Notably, Mzb1+ / + and Mzb1- / - mice showed comparable changes in body weight and disease activity index (Fig. 8a, b). Two weeks after CRC induction was completed, the mice were sacrificed, but no significant differences were found in the number or size of tumor nodules between Mzb1+ / + and Mzb1- / - mice (Fig. 8c). Furthermore, the fecal microbiota of Mzb1+ / + and Mzb1- / - mice did not segregate into distinct groups (Fig. 8d). Consistently, the microbiota composition was comparable between the two groups (Fig. 8e, f). Fecal IgA levels were moderately reduced in Mzb1- / - mice compared with Mzb1+ / + mice (Fig. 17b).

[0152] (3) Discussion

[0153] Our previous study elucidated the role of MZB1 in promoting the secretion of J-chain-containing dimeric IgA, which is important for suppressing DSS-induced colitis (Xiong, E. et al. Proc Natl Acad Sci USA 116, 13480-13489 (2019)). In this study, we further demonstrated that the absence of MZB1 accelerates the development and progression of AOM / DSS-induced CRC. Specifically, MZB1 deficiency resulted in reduced intestinal IgA levels and exacerbated both intestinal and systemic inflammation during CRC formation. Notably, we found that oral administration of IgA attenuated AOM / DSS-induced CRC in Mzb1- / - mice, providing a potential therapeutic option for colitis-induced CRC.

[0154] Many studies have demonstrated that IBD patients are more likely to develop CRC (Olen, O. et al. Lancet 395, 123-131 (2020), Bopanna, S. et al. Lancet Gastroenterol Hepatol 2, 269-276 (2017)). The progression of IBD to CRC is correlated with the duration and severity of inflammation and genetic factors (Yashiro, M. World J Gastroenterol 20, 16389-16397 (2014), Rutter, M. et al. Gastroenterology 126, 451-459 (2004), Li, W. et al. Curr Oncol 29, 6091-6114 (2022)). The tumor microenvironment is a complex network of various cells and matrix components, including tumor cells, lymphocytes, macrophages, neutrophils, myofibroblasts, cytokines, and extracellular matrix, which contribute to tumor progression (Roma-Rodrigues, C., Mendes, R., Baptista, P.V. & Fernandez, A.R. Int J Mol Sci 20 (2019); Catalano, V. et al. Semin Cancer Biol 23, 522-532 (2013); Chiba, F. et al. Int J Oncol 44, 177-186 (2014)). In our study, we found that MZB1 deficiency exacerbates intestinal inflammation, characterized by increased macrophages, neutrophils, and proinflammatory cytokines. This altered intestinal microenvironment accelerated the transition from colitis to colon cancer and further exacerbated colon cancer progression. Furthermore, MZB1 deficiency also led to splenic enlargement accompanied by an increase in macrophages and neutrophils. Previous studies have shown that mice lacking activation-induced cytidine deaminase exhibited isolated lymphoid follicle (ILF) hyperplasia in the intestine and proliferation of GC B cells in secondary lymphoid tissues such as the spleen. Antibiotic treatment abolished both ILF hyperplasia and GC B proliferation, suggesting that local intestinal inflammation triggered systemic B cell activation (Fagarasan, S. et al. Science 298, 1424-1427 (2002)).Based on this observation, the spleen enlargement observed in Mzb1- / - mice is likely caused by exacerbated intestinal inflammation during CRC induction. Cortez-Retamozzo et al. also showed that the increased numbers of macrophages and neutrophils in Mzb1- / - mice may also contribute to the development of CRC. More recently, it has been shown that MZB1 may play a direct role in suppressing rectal adenocarcinoma (Wu, W. et al. Bioorg Chem 95, 103457 (2020)). However, MZB1 is primarily expressed in B cells (Flach, H. et al. Immunity 33, 723-735 (2010)) (see also BioGPS). Furthermore, when the Mzb1 gene was disrupted in plasma cells using CRISPR-Cas9-mediated genome editing, cell cycle progression was not affected (Figure S3 in Xiong, E. et al. Proc Natl Acad Sci USA 116, 13480-13489 (2019)).

[0155] MZB1 has been shown to regulate the differentiation of IgM-secreting PCs into innate-like B cells (Andreani, V. et al. Proc Natl Acad Sci USA 115, E9630-e9639 (2018); Flach, H. et al. Immunity 33, 723-735 (2010)). Consistent with this finding, we observed a decrease in the percentage and absolute number of IgA+ PCs in the lamina propria of Mzb1- / - mice compared to Mzb1+ / + mice after CRC induction. Therefore, MZB1 appears to be required for the differentiation / generation of IgA-secreting PCs in the intestine. We also observed a decrease in the percentage and absolute number of B cells in the lamina propria of Mzb1- / - mice compared to Mzb1+ / + mice after CRC induction. Secretory IgA has the ability to bind to gut microbiota and form a protective layer between the intestinal epithelium and the microbiota, maintaining the steady state of the gut microbiota and protecting against antigens (Wijburg, O.L. et al. J Exp Med 203, 21-26 (2006)). We observed that MZB1 deficiency causes a decrease in gut IgA levels during CRC formation, resulting in disruption of the fecal microbiota. Flow cytometry analysis of IgA + fecal bacteria showed that in the absence of MZB1, IgA binding to gut bacteria was reduced, especially after CRC induction. Wei et al. demonstrated that mice carrying the AIDG23S mutation exhibit germinal center (GC) hyperplasia in intestinal lymphoid tissues as a result of the lack of high-affinity IgA4 (Wei, M. et al. Nat Immunol 12, 264-270 (2011)). This observation suggests that affinity maturation of IgA is important for normal GC responses in gut-associated lymphoid tissues. Interestingly, unlike AIDG23S mice, which did not show increased susceptibility to DSS-induced colitis (unpublished data), Mzb1- / - mice did not exhibit GC B cell proliferation but showed a higher susceptibility to CRC development. Thus, both the quality (affinity) and quantity of IgA are important for orchestrating appropriate immune responses and reducing intestinal inflammation.

[0156] To further verify that reduced IgA production worsened CRC progression, we conducted a two-stage study. First, we generated Iga- / - mice and induced CRC. Our findings demonstrated for the first time that IgA deficiency worsens the onset and progression of AOM / DSS-induced CRC. Next, we added a high-affinity monoclonal IgA, W27, to the drinking water throughout the CRC induction process. W27 is derived from plasma cells in the mouse intestinal lamina propria and has been reported to bind to and inhibit the growth of Escherichia coli and several other bacteria (Okai, S. et al. Nat Microbiol 1, 16103 (2016)). Our previous study demonstrated that oral administration of W27 attenuated DSS-induced colitis in Mzb1- / - mice (Xiong, E. et al. Proc Natl Acad Sci USA 116, 13480-13489 (2019)). In this study, we further demonstrated that oral administration of W27 significantly attenuated AOM / DSS-induced CRC. Thus, MZB1 deficiency leads to reduced IgA production, exacerbating the development and progression of AOM / DSS-induced CRC, but this condition can be reversed by IgA supplementation.

[0157] 16S rRNA sequencing of fecal microbiota revealed that the abundance of Akkermansia, a member of the Verrucomicrobiae family, was significantly reduced and nearly eliminated in Mzb1- / - CRC mice. Akkermansia is considered a beneficial bacterium. Recent studies have demonstrated that oral administration of Akkermansia muciniphila reduces colitis and colorectal cancer through the modulation of CTLs (Wang, L. et al. Gut 69, 1988-1997 (2020)). Previous studies have shown that cohousing mice with different genotypes can affect the composition of the gut microbiota and determine phenotypic expression (Tschurtschenthaler, M. et al. Gut 63, 1921-1931 (2014); Hu, B. et al. Proc Natl Acad Sci USA 110, 9862-9867 (2013); J Immunol 209, 2227-2238 (2022)). To investigate the influence of the gut microbiota on CRC development, we cohousing Mzb1+ / + and Mzb1- / - mice from day 10 of age and found that the differences in microbiota abundance between the two types of mice disappeared. As a result, the onset and progression of CRC were similar in both Mzb1+ / + and Mzb1- / - mice. Extending cohousing time and initiating cohousing earlier have been shown to improve microbiota transmission and intermediate tumor burden phenotypes (Yu, A.I. et al. Cell Rep 31, 107471 (2020)). In our study, mice were cohoused from day 10 of age and shared with their nursing mothers. These factors were essential for gut microbiota transmission and colorectal tumor burden phenotypes. These findings suggest that the composition of the microbiota, shaped by intestinal IgA levels, ultimately determines the severity of colitis and the development of CRC. However, when Mzb1+ / + and Mzb1- / - mice were cohoused, the intermediate phenotype was not obtained; rather, the WT phenotype became similar to that of Mzb1- / - mice. In the current experiment, it is difficult to establish a direct correlation between the severity of colitis and subsequent colon cancer development and the presence or absence of a specific microbiota.

[0158] This study revealed that MZB1 deficiency accelerates the development and progression of AOM / DSS-induced colorectal cancer by exacerbating intestinal inflammation through reduced intestinal IgA production. Supplementation with monoclonal IgA, W27, was shown to alleviate the exacerbated CRC.

Claims

1. A composition for preventing or treating colorectal cancer, comprising an IgA antibody or an antigen-binding fragment thereof.

2. A composition comprising the IgA antibody or its antigen-binding fragment described in claim 1, wherein the IgA antibody is an IgA antibody present in the intestinal lumen.

3. A composition comprising the IgA antibody or antigen-binding fragment thereof of claim 1, wherein the IgA antibody or antigen-binding fragment thereof binds to and / or inhibits the growth of Fusobacterium and / or Clostridium difficile bacterial organisms.

4. The IgA antibody is: a) an antibody comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:7, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of the light chain variable region comprising the amino acid sequence represented by SEQ ID NO:8; b) an antibody comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:17, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of the light chain variable region comprising the amino acid sequence represented by SEQ ID NO:18; c) an antibody comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:27, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:28; d) a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:81, and e) an antibody comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:93, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:94; f) an antibody comprising a heavy chain variable region comprising the amino acid sequence of heavy chain CDR1, the amino acid sequence of heavy chain CDR2, and the amino acid sequence of heavy chain CDR3 of the heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:37, and a light chain variable region comprising the amino acid sequence of light chain CDR1, the amino acid sequence of light chain CDR2, and the amino acid sequence of light chain CDR3 of the light chain variable region comprising the amino acid sequence represented by SEQ ID NO:38;or g) a composition comprising the IgA antibody or antigen-binding fragment thereof according to claim 1, which has at least one amino acid mutation in at least one region selected from heavy chain CDR1-3, light chain CDR1-3, and light chain FR1, relative to a reference antibody comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:37, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:38, and which binds to the amino acid sequence RQEEHIELIAS in Escherichia coli SHMT protein and the amino acid sequence VLDMMMKLEKPE in C. difficile iPGM protein.

5. The IgA antibody has a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:1, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:2, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:3, and a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:4, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:5, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

6.

6. A composition comprising the IgA antibody or its antigen-binding fragment described in claim 1, wherein the IgA antibody is an antibody comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:11, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:12, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:13, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:14, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:15, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

16.

7. A composition comprising the IgA antibody or its antigen-binding fragment described in claim 1, wherein the IgA antibody is an antibody comprising a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO:21, a heavy chain CDR2 having the amino acid sequence represented by SEQ ID NO:22, and a heavy chain CDR3 having the amino acid sequence represented by SEQ ID NO:23, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence represented by SEQ ID NO:24, a light chain CDR2 having the amino acid sequence represented by SEQ ID NO:25, and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:

26.

8. A composition comprising the IgA antibody or its antigen-binding fragment described in claim 1, wherein the IgA antibody is an antibody comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 83, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 84, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 85, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 86, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 87, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

88.

9. A composition comprising the IgA antibody or its antigen-binding fragment described in claim 1, wherein the IgA antibody is an antibody comprising a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 95, a heavy chain CDR2 having the amino acid sequence represented by SEQ ID NO: 96, and a heavy chain CDR3 having the amino acid sequence represented by SEQ ID NO: 97, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 98, a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 99, and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO:

100.

10. A composition comprising the IgA antibody or its antigen-binding fragment described in claim 1, wherein the IgA antibody is an antibody comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 32, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:

36.

11. The IgA antibody binds to Clostridium difficile bacteria, and has at least one amino acid mutation in at least one region selected from heavy chain CDR1-3, light chain CDR1-3, and light chain FR1, relative to a reference antibody comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:37 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:38, and binds to the amino acid sequence RQEEHIELIAS in Escherichia coli SHMT protein and the amino acid sequence VLDMMMKLEKPE in C. difficile iPGM protein, or an antigen-binding fragment thereof, comprising: X １ Heavy chain CDR1 comprising the amino acid sequence of YYIH, RIDPENX ２ X ３ a heavy chain CDR2 comprising the amino acid sequence of TTYAPKFQ; a heavy chain CDR3 comprising the amino acid sequence of YCARSTTVL; and a heavy chain variable region comprising: ４ a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SQSIVHTNG; a light chain CDR2 comprising the amino acid sequence of KLLIYKV; a light chain CDR3 comprising the amino acid sequence of GVYYCFQGS; and a light chain FR1 comprising the amino acid sequence of TPLSLPVSLGDQA or SPASX. ５ SVSLGDRX ６ X １ , X ２ , X ３ are each independently a neutral polar amino acid or an acidic polar amino acid; ４ is a non-polar amino acid or a neutral polar amino acid; ５ , X ６ The composition comprising the IgA antibody or antigen-binding fragment thereof of claim 1 , wherein each of the amino acids is independently a non-polar amino acid.

12. The IgA antibody comprises: (1) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:32, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:33; a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO:53; (2) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:31, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:32, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:33; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:52, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO:53, (3) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO: 53; (4) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO: 54, a light chain variable region comprising:(5) A heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO: 55; or (6) A heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 33; a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 52, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 35, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 36; and a light chain FR1 comprising the amino acid sequence represented by SEQ ID NO: 53, A composition comprising the IgA antibody or antigen-binding fragment thereof of claim 1.

13. The composition according to claim 1, for administration to the gastrointestinal tract.

14. The composition according to claim 1, provided in the form of a pharmaceutical composition, a food composition or a feed composition.

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