Composition for inhibiting fibrosis of intestinal tract

Abstract

Provided is a composition for inhibiting fibrosis of the intestinal tract, said composition comprising, as an active ingredient, a molecule that inhibits the function of the CCL19 gene.

Description

Composition for inhibiting intestinal fibrosis

[0001] The present invention relates to a composition for inhibiting intestinal fibrosis, and more particularly to a composition for inhibiting intestinal fibrosis, which comprises, as an active ingredient, a molecule that inhibits the function of the CCL19 gene.

[0002] Crohn's disease (CD) is an intractable disease of unknown etiology that occurs in young adults and is characterized by repeated chronic inflammation in the intestinal tract (predominantly the small intestine) (Non-Patent Document 1). The number of patients is increasing worldwide every year, reaching approximately 3 million worldwide by 2022 (Non-Patent Document 2). Although the causative antigen of CD is unknown, numerous studies suggest that it is a chronic inflammatory disease involving an autoimmune-like immune response (Non-Patent Document 3). Chronic inflammation is induced by various cytokines produced by activated immune cells such as macrophages, and in recent years, it has become possible to control the disease using various biological agents against these cytokines (Non-Patent Document 4).

[0003] Meanwhile, intestinal fibrosis is induced during the tissue repair process after inflammation is alleviated. Excessive fibrosis can cause intestinal stenosis, a serious problem in CD (Non-Patent Document 5). Intestinal stenosis is observed in more than half of CD patients, and many patients are forced to undergo surgery (intestinal resection), primarily due to small intestinal stenosis. Repeated inflammation, fibrosis, and intestinal resection (intestinal shortening) can lead to nutrient absorption disorders and the need for artificial anus placement, significantly reducing quality of life (Non-Patent Document 6). Therefore, controlling intestinal fibrosis is an urgent issue in CD treatment, but intestinal fibrosis has not been successfully suppressed.

[0004] Abraham, C. et al. N. Engl. J. Med. 361, 2066-2078 (2009). Ng, S. C. et al. Lancet. 390, 2769-2778 (2017). J,T. et al. Lancet. 389, 1741-1755 (2017). S.B., H. et al. Lancet. 359, 1541-1549 (2002). Di Sabatino, A. et al. Gut 58, 777-789 (2009). K.V., P. et al. Nat. Rev. Gastroenterol. Hepatol 13, 707-719 (2016). Murthy, S. K. et al. Gut 69, 274-282 (2020). Henderson, N. C. et al. Nature 587, 555-566 (2020). Denton CP. et al. Arthritis Rheum, 56, 323-333 (2007).

[0005] The present invention has been made in view of the problems associated with the prior art, and an object of the present invention is to provide a composition that can inhibit intestinal fibrosis.

[0006] The present inventors conducted extensive research to achieve the above-mentioned objectives. They noticed that the number and proportion of surgeries performed for CD have remained largely unchanged between before and now when various biological agents were approved (Non-Patent Document 7). This finding suggests that the mechanisms of inflammation and fibrosis in chronic inflammatory bowel diseases such as CD may differ, and suggests that the fibrosis mechanism involves not only the inflammatory immune response but also an immunosuppressive mechanism that controls the inflammatory immune response.

[0007] In fact, TGF-β, produced by macrophages and regulatory T cells (Tregs), is known to be a factor that causes fibrosis. Clinical trials focusing on TGF-β for fibrotic diseases using drugs that inhibit its action were conducted, but the trials were discontinued due to a significant number of serious adverse events, including progression of lesions and death (Non-Patent Document 8). Furthermore, TGF-β is a molecule that acts on many cellular functions, including intercellular adhesion, cell proliferation, cell differentiation, and cell migration, in addition to its immunosuppressive effect. Therefore, direct systemic inhibition of TGF-β action leads to a breakdown of biological homeostasis. In fact, when anti-TGF-β preparations were tested for fibrotic diseases, various complications occurred and the expected anti-fibrotic effects were not achieved (Non-Patent Document 9).

[0008] Based on the above results, in order to control intestinal fibrosis in conditions such as CD, it is necessary to develop an innovative treatment method that can prevent excessive intestinal fibrosis while maintaining homeostasis (avoiding inflammation recurrence). In other words, the inventors believe that it is crucial for next-generation treatments for CD and other conditions to develop a treatment method from a new perspective that combines not only conventional treatments aimed at improving inflammation, but also treatments aimed at preventing the subsequent excessive intestinal fibrosis and stenosis.

[0009] As shown in Figures 1A and 1B, fibrosis is a part of wound healing and an essential phenomenon in the recovery process from inflammation, but it can take a variety of different courses. In other words, tissues are normally repaired through moderate wound healing, but in CD and other diseases, there are many cases where excessive fibrosis occurs, causing intestinal stenosis. We believe that elucidating the mechanisms involved in these differences is essential for developing new treatments.

[0010] Therefore, the present inventors first compared molecular expression in areas of the small intestine of CD patients with excessive fibrosis and areas with mild fibrosis, attempting to identify factors associated with the differences between the two. As a result, they found that the chemokine CCL19 and its receptor CCR7 were highly expressed in fibrotic areas. Furthermore, they demonstrated that CCL19 expression increased over a certain period in areas of excessive fibrosis, accompanied by the accumulation of macrophages and Tregs positive for CCR7, the CCL19 receptor. Furthermore, they found that these cells survive for a long time and highly express TGF-β, promoting the transition from fibroblasts to myofibroblasts and causing excessive fibrosis.

[0011] The present inventors also succeeded in generating mice with human CD-like small intestinal fibrosis. Administration of an anti-CCL19 antibody to these CD model mice revealed a decrease in α-SMA, an indicator of intestinal fibrosis, in the small intestine. Furthermore, a decrease in CCL19 and CCR7-positive cells was observed in sync with this decrease in fibrosis, demonstrating that the anti-CCL19 antibody is effective against fibrosis by inhibiting tissue infiltration of CCR7-positive cells.

[0012] Furthermore, as a result of examining the timing of administration of such anti-CCL19 antibodies, it was found that a more effective administration time is from the middle stage of inflammation (the peak stage of inflammation) onwards, leading to the completion of the present invention.

[0013] That is, the present invention provides the following aspects.

[0014] [1] A composition for inhibiting intestinal fibrosis, comprising, as an active ingredient, a molecule that inhibits the function of the CCL19 gene.

[0015] [2] The composition according to [1], wherein the molecule is any one of the following molecules (a) to (f): (a) an anti-CCL19 protein antibody; (b) a nucleotide that binds to the CCL19 protein; (c) a nucleotide that binds to a transcription product of the CCL19 gene; (d) a peptide that has dominant-negative properties against the CCL19 protein; (e) an mRNA that encodes the anti-CCL19 protein antibody; or (f) a DNA that encodes the anti-CCL19 protein antibody.

[0016] [3] The composition according to [1] or [2], which is a pharmaceutical composition for treating or preventing chronic inflammatory bowel disease.

[0017] [4] The composition described in [3], wherein the chronic inflammatory bowel disease is Crohn's disease.

[0018] The present invention also provides the following aspects.

[0019] [5] Use of a molecule that suppresses the function of the CCL19 gene for producing a composition for suppressing intestinal fibrosis or a pharmaceutical composition for treating or preventing chronic inflammatory bowel disease.

[0020] [6] Use of a molecule that suppresses the function of the CCL19 gene for the suppression of intestinal fibrosis or the treatment or prevention of chronic inflammatory bowel disease.

[0021] [7] A molecule that suppresses the function of the CCL19 gene for use in suppressing intestinal fibrosis or treating or preventing chronic inflammatory bowel disease.

[0022] [8] A method for suppressing intestinal fibrosis or treating or preventing chronic inflammatory bowel disease, comprising administering to a subject an effective amount of a molecule that suppresses the function of the CCL19 gene.

[0023] According to the present invention, it is possible to suppress intestinal fibrosis. In particular, according to the present invention, by suppressing the function of the CCL19 gene, it is possible to suppress excessive fibrosis promoted by CCR7-positive cells that use CCL19 as a ligand, and thus it is possible to treat or prevent excessive intestinal fibrosis and further intestinal stenosis in chronic inflammatory bowel diseases such as Crohn's disease.

[0024] This figure shows an overview of the development of fibrosis at the cellular level. Myofibroblasts transitioning from fibroblasts vary in strength. This figure shows an overview of various fibrosis formations in the intestines of patients with Crohn's disease (CD) and other conditions. Fibrosis always occurs as a trigger of inflammation. It does not occur suddenly in normal areas. The pathological course of inflammation-triggered intestinal fibrosis progresses from normal (no inflammation) to mild inflammation to the peak inflammation stage, and ranges from mucosal healing to mild fibrosis to severe fibrosis. Severe fibrosis requires surgery. This heat map shows the results of comprehensive gene expression analysis of tissue samples taken from inflamed areas (tissue showing mild fibrosis before fibrotic stricture) and fibrotic areas (tissue from the fibrotic stricture area showing excessive (severe) fibrosis) of the small intestine of CD patients. RNA-seq results using small intestinal tissue showed high expression of CCL19 and CCR7 in areas of excessive fibrosis.

[0023] Figure 1 shows the results of an analysis (CIBERSORT) focusing on immune cells in inflammatory and fibrotic small intestinal tissues of CD patients. In this analysis, significant differences were observed between the two groups (mild fibrosis and severe fibrosis) in regulatory T cells (Tregs) and macrophages. This graph shows the results of flow cytometry (FACS) analysis of the level of CCR7-positive macrophage expression in normal and fibrotic small intestinal tissues. The vertical axis indicates the proportion (%) of CCR7-positive cells among CCD11b-positive cells (macrophages). Statistical analysis was performed using a t-test. A single asterisk indicates P<0.05, and two asterisks indicate P<0.01. Dots indicate individual samples, and bars represent the mean and standard deviation (statistical analysis and notations in the graphs are the same in Figures 3C, 5, 6A to 6D, 7A, 7C, 7E, 8, 9A, and 10A). These are fluorescence micrographs showing the results of analyzing the level of expression of CCR7-positive macrophages in normal sites and fibrotic sites of small intestinal tissue by multiple immunostaining. These are graphs showing the results of analyzing the level of expression of CCR7-positive Tregs in normal sites and fibrotic sites of small intestinal tissue by FACS. The vertical axis shows the proportion (%) of CCR7-positive cells among Foxp3-positive cells (Tregs).

[0033] Figure 1 is a fluorescence micrograph showing the results of analyzing the level of expression of CCR7-positive Tregs in normal and fibrotic areas of small intestinal tissue using multiple immunostaining. Figure 2 is a schematic diagram showing a hypothesis regarding the mechanism of excessive intestinal fibrosis caused by CCL19. In normal fibrotic areas, mild fibrosis occurs during wound healing triggered by inflammation. In contrast, in excessively fibrotic areas, CCL19 expression increases from a certain point in time, resulting in the accumulation of large numbers of CCR7-positive macrophages and CCR7-positive Tregs. Furthermore, it is speculated that these cells survive for a long time and highly express TGF-β, promoting the transition from fibroblasts to myofibroblasts and causing excessive fibrosis. Figure 3 is a graph showing the difference in TGF-β expression levels between CCR7-positive / negative macrophages and CCR7-positive / negative Tregs. Small intestinal tissue collected during surgery was sorted by CCR7, CD11b, and Tregs, and differences in TGF-β expression between CCR7-positive and -negative macrophages and CCR7-positive and -negative Tregs were examined by qPCR. Results indicated that CCR7-positive macrophages and CCR7-positive Tregs highly expressed TGF-β. This graph shows the difference in the expression level of Bcl-X between CCR7-positive and -negative macrophages at sites of intestinal fibrosis. CCR7-positive and -negative macrophages (CD11b) were sorted from small intestinal tissue collected during surgery, and differences in Bcl-X expression were examined by qPCR. Results indicated that CCR7-positive macrophages had significantly higher levels of the anti-apoptotic signal (Bcl-X). This graph shows the difference in the expression level of Bcl-X between CCR7-positive and -negative Tregs at sites of intestinal fibrosis. CCR7-positive and -negative Tregs (CD25) were sorted from small intestinal tissue collected during surgery, and differences in Bcl-X expression were examined by qPCR. The results showed that CCR7-positive Tregs had significantly higher levels of anti-apoptotic signals (Bcl-X). This is a graph showing the difference in the level of Bcl-2 expression in CCR7-positive / -negative macrophages at sites of intestinal fibrosis. CCR7-positive and -negative macrophages (CD11b) were sorted from small intestinal tissue collected during surgery, and differences in Bcl-2 expression were examined by qPCR.The results showed that CCR7-positive macrophages had significantly higher anti-apoptotic signals (Bcl-2). This graph shows the difference in the level of Bcl-2 expression between CCR7-positive and CCR7-negative Tregs at sites of intestinal fibrosis. CCR7-positive and -negative Tregs (CD25) were sorted from small intestinal tissue collected during surgery, and differences in Bcl-2 expression were examined by qPCR. The results showed that CCR7-positive Tregs had significantly higher anti-apoptotic signals (Bcl-2). This graph shows the results of administering an anti-CCL19 antibody to a mouse model of human CD-like small intestinal fibrosis, and examining its effect on reducing fibrosis in small intestinal tissue. The vertical axis shows the expression rate (%) of α-SMA, a fibrosis marker, in FACS. This is a fluorescence micrograph showing the results of administering an anti-CCL19 antibody to a mouse model of human CD-like small intestinal fibrosis, and examining its effect on reducing fibrosis in small intestinal tissue using multiple immunostaining.

[0023] Figure 1 is a graph showing the results of administering an anti-CCL19 antibody to a mouse model of human CD-like small intestinal fibrosis and analyzing the expression level of CCL19 in the small intestinal tissue by real-time PCR. Three asterisks indicate P<0.001.

[0024] Figure 2 is a micrograph showing the results of administering an anti-CCL19 antibody to a mouse model of human CD-like small intestinal fibrosis and analyzing the level of expression of CCR7-positive cells in the small intestinal tissue by immunostaining.

[0025] Figure 3 is a graph showing the results of administering an anti-CCL19 antibody to a mouse model of human CD-like small intestinal fibrosis and analyzing the level of expression of CCR7-positive macrophages in the small intestinal tissue by FACS. The vertical axis indicates the proportion (%) of CCR7-positive cells among CCD11b-positive cells (macrophages).

[0026] Figure 4 is a fluorescent micrograph showing the results of administering an anti-CCL19 antibody to a mouse model of human CD-like small intestinal fibrosis and analyzing the small intestinal tissue by multiplex immunostaining. 1 is a graph showing the transition of CCL19 expression from the normal phase to the inflammatory phase to the fibrotic phase. Changes in CCL19 depending on the disease stage were examined by qPCR. It was revealed that CCL19 levels increase in the fibrotic phase (later inflammatory phase and beyond). 1 is a graph showing the results of analyzing the expression level of TGF-b in small intestinal tissue by real-time PCR after administering an anti-CCL19 antibody to a mouse model of human CD-like small intestinal fibrosis at the early stage of inflammation, the intermediate stage of inflammation, or both the early and intermediate stages (early-to-mid stage of inflammation).In the figure, "fibrotic mice" indicates the results of analysis of human CD-like small intestinal fibrosis model mice administered with anti-CCL19 antibody at the same time as analysis of mice administered with anti-CCL19 antibody without administration of anti-CCL19 antibody. "Control" indicates the results of analysis of wild-type mice (mice without fibrosis). All mice (7 weeks old) were first administered TNBS for the first time to induce fibrosis. TNBS and anti-CCL19 antibody were then administered sequentially at the same time, and all mice were recovered at 10 weeks (within 1 week after administration in the late inflammatory phase) and analyzed. (The terms "fibrotic mice" and "control" in the figure are the same in Figures 9B to 10C. The timing of administration of TNBS and anti-CCL19 antibody and the time of analysis are also the same in these figures.) 1 shows a dot plot diagram and fluorescent micrographs showing the results of analyzing the level of CCR7 expression in small intestinal tissue by FACS and immunostaining after administering an anti-CCL19 antibody to a mouse model of human CD-like small intestinal fibrosis at the early stage, the intermediate stage, or both the early and intermediate stages (early to mid-stage inflammation). In the figures, the numbers in the dot plots indicate the percentage of α-SMA-positive cells. This is a graph showing the results of analyzing the level of CCL19 expression in small intestinal tissue by real-time PCR after administering an anti-CCL19 antibody to a mouse model of human CD-like small intestinal fibrosis at the early stage, the intermediate stage, or both the early and intermediate stages (early to mid-stage inflammation). This is a dot plot diagram showing the results of analyzing the level of CCR7 expression in small intestinal tissue by FACS after administering an anti-CCL19 antibody to a mouse model of human CD-like small intestinal fibrosis at the early stage, the intermediate stage, or both the early and mid-stages (early to mid-stage inflammation). In the figure, the numbers in the dot plots indicate the percentage of CCR7-positive cells. Figure 1 shows fluorescence micrographs showing the results of analyzing the expression of CCR7-positive macrophages in small intestinal tissue by multiple immunostaining after administering an anti-CCL19 antibody to a mouse model of human CD-like small intestinal fibrosis at the early stage of inflammation, the middle stage of inflammation, or both the early and middle stages of inflammation (early to middle stage of inflammation).

[0025] As shown in the Examples below, the present inventors have discovered a pathogenic mechanism in which increased expression of CCL19 in the intestine leads to the infiltration and accumulation of CCR7-positive cells (CCR7-positive macrophages and CCR7-positive Tregs) that use CCL19 as a ligand, and these cells survive for a long period of time and highly express TGF-β, promoting the transition from fibroblasts to myofibroblasts and causing excessive fibrosis. The present inventors have also demonstrated that administration of an anti-CCL19 antibody to a human CD-like small intestinal fibrosis model suppresses this pathogenic mechanism and intestinal fibrosis. Thus, the present invention provides a composition for suppressing intestinal fibrosis, which comprises as an active ingredient a molecule that suppresses the function of the CCL19 gene.

[0026] In the present invention, the term "intestinal tract" refers to the organ extending from the part of the intestine that continues to the pharynx to the anus, and examples thereof include the small intestine (duodenum, jejunum, ileum, etc.) and the large intestine (cecum, colon, rectum, etc.). "Intestinal fibrosis" refers to the activation of fibroblasts in the intestinal tract, the massive production and deposition of extracellular matrix proteins (mainly collagen), and the abnormal proliferation of connective tissue.

[0027] "Excessive intestinal fibrosis" and "severe intestinal fibrosis" generally refer to the same condition. However, "excessive intestinal fibrosis" refers to one of the fibrosis processes in intestinal wound healing triggered by inflammation, and refers to a state in which excessive intestinal fibrosis has occurred, such as a state of intestinal fibrotic stenosis. More specifically, this refers to a state in which intestinal passage obstruction has occurred due to fibrotic stenosis (a state in which uncontrollable abdominal pain or intestinal obstruction due to obstruction of food and intestinal fluid passage has occurred (history) or is likely to occur, such as a state in which endoscopic passage obstruction is observed in clinical and laboratory findings), requiring surgical treatment. "Severe intestinal fibrosis" refers to a phenomenon in which the intestine (mainly the submucosal tissue) is severely fibrosed due to the accumulation of extracellular matrix and the proliferation of collagen fibers and elastic fibers. Furthermore, the intestine undergoing the "excessive fibrosis" process has undergone "severe fibrosis."

[0028] On the other hand, "normal intestinal fibrosis" and "mild intestinal fibrosis" generally refer to the same condition, but "normal intestinal fibrosis" refers to one of the fibrosis processes in intestinal wound healing triggered by inflammation, and includes, for example, a state in which intestinal fibrosis stenosis does not occur. More specifically, it includes a state in which there is no intestinal passage obstruction (for example, clinical and laboratory findings show that endoscope passage is possible) and surgery is not required. "Mild intestinal fibrosis" refers to a phenomenon in which the accumulation of extracellular matrix and proliferation of connective tissue, etc. are relatively mild, and fibrosis itself of the intestinal tract (mainly the tissue under the mucosa) is mild. Furthermore, the intestine in the "normal fibrosis" process often undergoes "mild fibrosis."

[0029] In the present invention, "suppression of gene function" means both suppression of gene expression (suppression of transcription, suppression of translation) and suppression of the activity of the translation product (protein) of the gene.

[0030] In the present invention, the "CCL19 (C-C motif chemokine ligand 19) gene" the function of which is to be inhibited is a gene also known as CKb11, ELC, MIP-3b, MIP3B, or SCYA19. If derived from a human, the gene is typically a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 (a gene comprising the DNA sequence set forth in SEQ ID NO: 1). More specific examples include a gene encoding a protein specified by UniProt ID: Q99731 and a gene encoding a protein consisting of the amino acid sequence set forth in NCBI Reference Sequence: NP_006265 (a gene comprising the DNA sequence set forth in NCBI Reference Sequence: NM_006274). If derived from a mouse, the gene is typically a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 (a gene comprising the DNA sequence set forth in SEQ ID NO: 3). More specifically, examples of such genes include a gene encoding a protein identified by UniProt ID: Q548P0 and a gene encoding a protein consisting of the amino acid sequence set forth in NCBI Reference Sequence: NP_036018 (a gene containing a DNA sequence identified by NCBI Reference Sequence: NM_011888). However, the DNA sequence of a gene can mutate in nature (i.e., non-artificially) due to mutations or the like. Therefore, in the present invention, such naturally occurring mutants can also be targets for functional inhibition.

[0031] (Molecules that suppress the function of the CCL19 gene) Examples of "molecules that suppress the function of the CCL19 gene" include, for example, anti-CCL19 protein antibodies, nucleotides that bind to the CCL19 protein, nucleotides that bind to transcription products of the CCL19 gene, peptides that have dominant-negative traits against the CCL19 protein, and genome editing systems that target the CCL19 gene.

[0032] (a) Anti-CCL19 Protein Antibody In the present invention, the term "anti-CCL19 protein antibody" refers to an antibody that recognizes the CCL19 protein, i.e., an antibody that has binding activity to the CCL19 protein. The antibody of the present invention may be any antibody that recognizes the CCL19 protein, but from the viewpoint of being able to suppress intestinal fibrosis by inhibiting the binding of the CCL19 protein to its receptor, CCR7 protein (so-called, having neutralizing activity), the antibody is preferably an antibody that has binding activity to a peptide containing the binding site or binding groove for the CCR7 protein. As such an antibody, as long as it recognizes human-derived CCL19 protein, it is preferably an antibody having binding activity to a peptide consisting of the amino acid sequence of positions 22 to 98 in the amino acid sequence of SEQ ID NO: 2, more preferably an antibody having binding activity to a peptide consisting of the amino acid sequence of positions 29 to 87, even more preferably an antibody having binding activity to a binding site with the CCR7 protein (at least one of amino acid sequences of positions 29 to 33, 35 to 42, 54 to 55, and 77 to 87), or even more preferably an antibody having binding activity to a binding groove with the CCR7 protein (at least one of amino acids of positions 31, 61, and 70). Note that the "position" in the amino acid sequence of SEQ ID NO: 2 refers to the order in which the first methionine is placed, with position 1 being the first.

[0033] As used herein, "antibodies" include all classes and subclasses of immunoglobulins. "Antibodies" include polyclonal and monoclonal antibodies. A "polyclonal antibody" is an antibody preparation containing different antibodies directed against different epitopes. A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. In contrast to polyclonal antibodies, monoclonal antibodies recognize a single determinant on an antigen. Antibodies according to the present invention are preferably monoclonal antibodies. Antibodies according to the present invention are antibodies that have been separated and / or recovered (i.e., isolated) from components of their natural environment.

[0034] The antibody of the present invention is not particularly limited in terms of origin, type, shape, etc., as long as it can recognize the CCL19 protein. Specific examples include antibodies derived from non-human animals (e.g., mouse antibodies, rabbit antibodies, rat antibodies, and camel antibodies), antibodies derived from humans, chimeric antibodies, and humanized antibodies. When the antibody of the present invention is administered to humans as a therapeutic or prophylactic agent, chimeric antibodies, humanized antibodies, or human antibodies are preferred from the viewpoint of reducing side effects.

[0035] In the present invention, a "chimeric antibody" refers to an antibody in which the variable region of one antibody is linked to the constant region of a heterologous antibody. Chimeric antibodies can be obtained, for example, by immunizing a mouse with an antigen, excising the antibody variable region (variable region) that binds to the antigen from the mouse monoclonal antibody gene, ligating it to an antibody constant region (constant region) gene derived from human bone marrow, incorporating it into an expression vector, and introducing it into a host for production (e.g., JP-A-8-280387, U.S. Pat. No. 4,816,397, U.S. Pat. No. 4,816,567, U.S. Pat. No. 5,807,715).

[0036] The constant region of a chimeric antibody is usually derived from a human antibody. For example, Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, and Cε can be used as the heavy chain constant region. Furthermore, Cκ and Cλ can be used as the light chain constant region. The amino acid sequences of these constant regions and the nucleotide sequences encoding them are known. Furthermore, to improve the stability of the antibody itself or the stability of antibody production, one or several amino acids in the human antibody constant region can be substituted, deleted, added, and / or inserted.

[0037] In the present invention, a "humanized antibody" refers to an antibody in which the gene sequence of the antigen-binding region (CDR) of a non-human antibody has been grafted onto a human antibody gene (CDR grafting). Methods for producing such antibodies include known overlap extension PCR methods (see, for example, EP 239400, EP 125023, WO 90 / 07861, and WO 96 / 02576). The variable region of an antibody typically consists of three CDRs sandwiched between four FRs. CDRs essentially determine the binding specificity of an antibody. While the amino acid sequences of CDRs are highly diverse, the amino acid sequences constituting FRs often show high homology or identity even between antibodies with different binding specificities. Therefore, it is generally believed that CDR grafting can transfer the binding specificity of one antibody to another. Furthermore, from the viewpoint of maintaining the function of the CDR, when grafting a non-human CDR onto a human FR, a human FR having high homology or identity to the non-human animal-derived FR is selected. That is, since the amino acids in the CDR not only recognize the antigen but also coordinate with the amino acids in the FR adjacent to the CDR and are involved in maintaining the loop structure of the CDR, it is preferable to use a human FR having an amino acid sequence having high homology or identity to the amino acid sequence of the FR adjacent to the CDR to be grafted.

[0038] A search for known human FRs that are highly homologous or identical to non-human animal-derived FRs can be performed, for example, using an antibody-specific search system available on the Internet (http: / / www.bioinfo.org.uk / abysis / ). Mutations can be introduced into sequences other than the CDRs of the non-human antibody so that they match the sequence of the human FR obtained in this manner. Alternatively, if a gene (cDNA) encoding the amino acid sequence of the human FR obtained by the search is available, the non-human CDRs can be introduced into that sequence. Introduction of mutations, etc., can be performed using techniques known in the art, such as nucleic acid synthesis and site-directed mutagenesis.

[0039] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody thus prepared, it is possible to suitably select FRs of a human-derived antibody that form a good antigen-binding site when linked via the CDRs. Furthermore, if necessary, amino acid residues in the FRs can be substituted so that the CDRs of the humanized antibody form a suitable antigen-binding site, according to the method described in Sato, K. et al., Cancer Res, 1993, 53, 851-856, etc., and mutant FR sequences with desired properties can be selected by measuring and evaluating the antigen-binding activity of mutant antibodies with the amino acid substitutions.

[0040] In the present invention, a "human antibody" is an antibody in which all regions are derived from humans. In producing human antibodies, it is possible to use transgenic animals (e.g., mice) that are capable of producing a repertoire of human antibodies upon immunization. Techniques for producing human antibodies are known (e.g., Nature, 1993, vol. 362, pp. 255-258; Intern. Rev. Immunol, 1995, vol. 13, pp. 65-93; J. Mol. Biol, 1991, vol. 222, pp. 581-597; Nature Genetics, 1997, Vol. 15, pp. 146-156; Proc. Natl. Acad. Sci. USA, 2000, Vol. 97, pp. 722-727; JP-A Nos. 10-146194, 10-155492, Japanese Patent Nos. 2938569, 11-206387, JP-A Nos. 8-509612 and 11-505107).

[0041] Anti-CCL19 protein antibodies also include antibodies whose amino acid sequences have been modified without reducing desired activities (such as binding activity to CCL19 protein, activity to inhibit intestinal fibrosis, and / or other biological properties). Amino acid sequence variants can be produced by introducing mutations into DNA encoding the antibody chain or by peptide synthesis. The site at which the antibody amino acid sequence is modified may be the constant region of the heavy or light chain of the antibody, or may be the variable region (framework region and CDRs), as long as the antibody has activity equivalent to that of the antibody before modification. Although modification of amino acids other than those in the CDRs is thought to have a relatively small effect on the binding affinity to the antigen, currently, techniques are known in which amino acids in the CDRs are modified to screen for antibodies with increased affinity to the antigen (PNAS, 2005, Vol. 102, pp. 8466-8471; Protein Engineering, Design & Selection, 2008, Vol. 21, pp. 485-493; WO2002 / 051870; J. Biol. Chem., 2005, Vol. 280, pp. 24880-24887; Protein Engineering, Design & Selection, 2008, Vol. 21, pp. 345-351).

[0042] The number of amino acids to be modified is preferably 10 or less, more preferably 5 or less, and most preferably 3 or less (e.g., 2 or less, 1 or less). The amino acid modification is preferably a conservative substitution. In the present invention, "conservative substitution" refers to substitution with another amino acid residue having a chemically similar side chain. Groups of amino acid residues having chemically similar side chains are well known in the art to which the present invention pertains. For example, acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), and neutral amino acids can be classified into amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), amino acids with hydroxyl groups (serine, threonine), sulfur-containing amino acids (cysteine, methionine), amino acids with amide groups (asparagine, glutamine), amino acids with imino groups (proline), and amino acids with aromatic groups (phenylalanine, tyrosine, tryptophan). It is preferable that the antigen-binding activity of the amino acid sequence variant is equivalent to that of the target antibody. The antigen-binding activity can be evaluated by analysis using, for example, a flow cytometer, ELISA, Western blotting, immunoprecipitation, or the like.

[0043] Furthermore, in the present invention, for the purpose of increasing antibody stability, deamidation may be suppressed by substituting the deamidated amino acid or the amino acid adjacent to the deamidated amino acid with another amino acid. Furthermore, antibody stability can also be increased by substituting glutamic acid with another amino acid. The present invention also provides antibodies stabilized in this manner.

[0044] In the present invention, anti-CCL19 protein antibodies include not only the above-mentioned antibodies themselves but also functional fragments thereof. A "functional fragment" of an antibody refers to a portion (partial fragment) of the antibody that recognizes the CCL19 protein. Specific examples include Fab, Fab', F(ab')2, variable region fragments (Fv), disulfide-linked Fv, single-chain variable region fragments (single-chain Fv, scFv), sc(Fv)2, and polymers thereof.

[0045] "Fab" refers to a monovalent antigen-binding fragment of an immunoglobulin consisting of one light chain and part of a heavy chain. It can be obtained by papain digestion of an antibody or by recombinant methods. "Fab'" differs from Fab by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines in the antibody hinge region. "F(ab')2" refers to a bivalent antigen-binding fragment of an immunoglobulin consisting of both light chains and part of both heavy chains.

[0046] A "variable region fragment (Fv)" is the smallest antibody fragment that has a complete antigen recognition and binding site. Fv is a dimer in which a heavy chain variable region and a light chain variable region are tightly linked by non-covalent bonds. A "single-chain variable region fragment (single-chain Fv, scFv)" contains an antibody's heavy chain variable region and light chain variable region, and these regions are present in a single polypeptide chain. An "sc(Fv)2" is a single chain formed by linking two heavy chain variable regions and two light chain variable regions with a linker or the like.

[0047] The antibodies of the present invention can be produced by the hybridoma method or by recombinant DNA technology. A representative example of the hybridoma method is the method of Kohler and Milstein (Nature, 256:495 (1975)). The antibody-producing cells used in the cell fusion step in this method are spleen cells, lymph node cells, peripheral blood leukocytes, etc. of animals (e.g., mice, rats, hamsters, rabbits, monkeys, goats) immunized with an antigen (CCL19 protein, its partial peptides, proteins in which CCL19 protein or its partial peptides are fused with Fc protein or the like, or cells expressing these). Antibody-producing cells obtained by reacting the antigen in a culture medium with the above-mentioned cells or lymphocytes previously isolated from an unimmunized animal can also be used. Various known cell lines can be used as myeloma cells. The antibody-producing cells and myeloma cells may be derived from different animal species, as long as they are fusible, but are preferably derived from the same animal species. Hybridomas can be produced, for example, by cell fusion between spleen cells obtained from a mouse immunized with an antigen and mouse myeloma cells, and then screening can be performed to obtain hybridomas that produce monoclonal antibodies that recognize the CCL19 protein. Monoclonal antibodies that recognize the CCL19 protein can be obtained by culturing the hybridomas or from the ascites fluid of a mammal to which the hybridoma has been administered.

[0048] The recombinant DNA method involves cloning DNA encoding the antibody of the present invention from hybridomas, B cells, or the like, incorporating it into an appropriate vector, and then introducing it into host cells (e.g., mammalian cell lines such as HEK cells, E. coli, yeast cells, insect cells, plant cells, etc.) to produce the antibody of the present invention as a recombinant antibody (e.g., P. J. Delves, Antibody Production: Essential Techniques, 1997 Wiley, P. Shepherd and C. Dean, Monoclonal Antibodies, 2000 Oxford University Press; Vandamme A. M. et al., Eur. J. Biochem. 192:767-775 (1990)). In expressing DNA encoding an antibody of the present invention, DNA encoding the heavy chain and DNA encoding the light chain may be separately incorporated into an expression vector and used to transform host cells, or DNA encoding the heavy chain and DNA encoding the light chain may be incorporated into a single expression vector and used to transform host cells (see WO 94 / 11523). The antibody of the present invention can be obtained in a substantially pure and homogeneous form by culturing the host cells and isolating and purifying it from within the host cells or from the culture medium. Antibody isolation and purification can be performed using methods commonly used for purifying polypeptides. By using transgenic animal production techniques to produce transgenic animals (such as cows, goats, sheep, and pigs) incorporating antibody genes, it is possible to obtain large quantities of monoclonal antibodies derived from the antibody genes from the milk of the transgenic animals.

[0049] Furthermore, as described above, antibodies according to the present invention can also be expressed from nucleotides encoding them. Thus, antibodies according to the present invention can also take the form of nucleotides (DNA, RNA (mRNA), etc.) encoding the anti-CCL19 protein antibodies according to the present invention. Note that, like the "nucleotides binding to the transcription product of the CCL19 gene" described below, such nucleotides can be appropriately prepared by those skilled in the art, and can also take the form of a nucleotide construct (expression cassette).

[0050] (b) Nucleotides that bind to CCL19 protein The "nucleotides that bind to CCL19 protein" contained as an active ingredient in the composition of the present invention are typically nucleic acid aptamers. The "nucleic acid aptamer" of the present invention may be DNA or RNA, but DNA is preferred from the viewpoint of high in vivo stability. Furthermore, from the same viewpoint, the nucleic acid aptamer may include a nucleotide molecule in which the phosphate backbone in the nucleotide molecular structure has been modified to increase in vivo stability. For details of such modifications and nucleotide molecules, see "Nucleotides that bind to the transcription product of the CCL19 gene" below.

[0051] In the present invention, the "nucleic acid aptamer" may have, as structural characteristics, at least one loop structure, a primary structure containing many deoxyguanosines (including guanosine and guanosine analogs), or a tetrameric cluster structure of deoxyguanosines (so-called "G-quartet structure"). Furthermore, the nucleic acid aptamer of the present invention may be either a double-stranded nucleotide or a single-stranded nucleotide, but a single-stranded nucleotide is preferred.

[0052] In the present invention, the "length of the nucleic acid aptamer" is not particularly limited as long as it has the number of bases that enables it to specifically bind to the CCL19 protein, but is, for example, 10 to 200 bases, preferably 20 to 150 bases, more preferably 30 to 150 bases, and even more preferably 50 to 150 bases, and specific examples include aptamers of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 bases.

[0053] Furthermore, the "nucleic acid aptamer" of the present invention may be one that specifically binds to the CCL19 protein. However, from the viewpoint that intestinal fibrosis can be suppressed by inhibiting the binding of the CCL19 protein to its receptor, CCR7 protein, it is desirable that the nucleic acid aptamer has binding activity to a peptide that contains the binding site or binding groove for the CCR7 protein. As such an antibody, so long as it recognizes human-derived CCL19 protein, a nucleic acid aptamer having binding activity to a peptide consisting of the amino acid sequence of positions 22 to 98 in the amino acid sequence of SEQ ID NO: 2 is preferred, a nucleic acid aptamer having binding activity to a peptide consisting of the amino acid sequence of positions 29 to 87 is more preferred, a nucleic acid aptamer having binding activity to a binding site with CCR7 protein (at least one of amino acid sequences of positions 29 to 33, 35 to 42, 54 to 55, and 77 to 87) is even more preferred, or a nucleic acid aptamer having binding activity to a binding groove with CCR7 protein (at least one of amino acids of positions 31, 61, and 70).

[0054] Those skilled in the art can appropriately select and produce nucleic acid aptamers using known techniques, such as the in vitro selection method (SELEX) (Tuerk, C. et al., Science, 1990, vol. 249, pp. 505-510; Green, L. et al., Meths. Enzymol., 1991, vol. 2, pp. 75-86; Gold, L. et al., Annu. Rev. Biochem., 1995, vol. 64, pp. 763-797; Uphoff, K. W. et al., Curr. Opin. Struct. Biol., 1996, vol. 6, pp. 281-288).

[0055] The "in vitro selection method" refers to a method of selecting nucleotides with affinity for a desired protein from a pool of nucleic acids containing random sequences and eliminating nucleotides without affinity. Specifically, when producing a nucleic acid aptamer using the in vitro selection method, nucleotides containing a random base sequence of approximately 20 to 300 bases, preferably 30 to 150 bases, and more preferably 30 to 100 bases, are first prepared. The nucleic acid may be synthesized directly, or in the case of an RNA molecule, it may be prepared by first synthesizing a DNA molecule and then performing a transcription reaction. When the nucleotide is DNA, it is preferable that both ends of the nucleotide have base sequences that serve as primers for enabling PCR amplification. The primer binding sequence portion may be prepared to have an appropriate restriction enzyme recognition sequence for excising the primer portion with a restriction enzyme after PCR amplification. The length of the primer binding sequence portion is not particularly limited, but is usually 20 to 50 bases, preferably 20 to 30 bases. Furthermore, the 5' end of the primer may be labeled with a radioactive substance, fluorescent substance, or the like to separate the PCR-amplified DNA by electrophoresis or the like. Furthermore, when preparing RNA, it may be prepared by transcribing DNA molecules into RNA molecules using a suitable promoter, such as DNA having a T7 promoter sequence, in the 5'-terminal primer. Next, the random nucleotides obtained by PCR amplification are mixed with the CCL19 protein or a partial peptide thereof and incubated. After incubation, the mixture is subjected to electrophoresis to separate the complex of the nucleotides and the CCL19 protein from the free nucleotides. Then, the nucleic acid forming the complex with the CCL19 protein is extracted according to a known method. If the recovered nucleotides are DNA, they are further amplified by PCR, and the amplified DNA is recovered as single-stranded DNA by heat denaturation or the like. If the recovered nucleotides are RNA, the RNA is reverse transcribed to cDNA, which is then amplified by PCR, and the amplified DNA is transcribed to prepare RNA.Alternatively, the procedure of mixing the nucleic acid with the CCL19 protein, isolating the nucleic acid bound to the CCL19 protein, PCR amplification (in the case of RNA, amplification after reverse transcription), and using the amplified nucleic acid again to bind to the CCL19 protein may be repeated several times. The resulting nucleic acid may be sequenced according to a known method.

[0056] Furthermore, in the present invention, nucleic acid aptamers may be produced based on the nucleotide sequence obtained by the above-described operations by chemical synthesis, in vitro transcription using DNA-dependent RNA polymerase or DNA-dependent DNA polymerase, or amplification by PCR.

[0057] (c) Nucleotide that binds to the transcription product of the CCL19 gene More specifically, the "nucleotide that binds to the transcription product of the CCL19 gene" contained as an active ingredient in the composition of the present invention includes the following embodiments: (c1) siRNA against the transcription product of the CCL19 gene (c2) miRNA against the transcription product of the CCL19 gene (c3) antisense oligonucleotide against the transcription product of the CCL19 gene (c4) a nucleotide having ribozyme activity against the transcription product of the CCL19 gene.

[0058] (c1) siRNA Against a CCL19 Gene Transcription Product In the present invention, the "siRNA against a CCL19 gene transcription product" is not particularly limited as long as it is a double-stranded RNA molecule (dsRNA) that specifically suppresses the expression of the CCL19 gene. The double-stranded RNA portion in which RNAs in a dsRNA pair with each other is not limited to perfect pairing, and may contain unpaired portions due to mismatches (corresponding bases are not complementary), bulges (there is no corresponding base in one strand), etc. Furthermore, the double-stranded RNA region in which RNAs pair with each other in a dsRNA may contain both bulges and mismatches.

[0059] In the present invention, the "chain length of siRNA" is not particularly limited as long as it can suppress the expression of the CCL19 gene and is not toxic, but is, for example, 15 to 49 nucleotides, preferably 15 to 35 nucleotides, more preferably 20 to 30 nucleotides, and more preferably 21 to 23 nucleotides.

[0060] The "siRNA" according to the present invention may be shRNA (small hairpin RNA, short hairpin RNA). The shRNA can be designed so that a portion thereof forms a stem-loop structure. For example, the shRNA can be designed to have a spacer sequence (e.g., 5 to 25 nucleotides) between the target sequence and the sequence complementary to the target sequence, with these sequences present in a single RNA strand, and to have a total length of, for example, 45 to 70 nucleotides.

[0061] The "siRNA" according to the present invention may have additional nucleotides at the 5' or 3' end. The length of the additional nucleotides is usually 2 to 4 nucleotides. The additional nucleotides may be DNA or RNA, but using DNA may improve the stability of the nucleic acid. Examples of such additional nucleotide sequences include, but are not limited to, the sequences ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3', and uuuuu-3'.

[0062] The "siRNA" according to the present invention may have a protruding sequence (overhang) at the 3' end, specifically, one to which dTdT (dT: deoxythymidine) has been added. Alternatively, it may be blunt-ended with no additional end. The sense strand and the antisense strand of the siRNA may have different numbers of bases, for example, the antisense strand may be an "asymmetrical interfering RNA (aiRNA)" having overhangs at the 3' end and the 5' end.

[0063] The target sequence of the "siRNA" according to the present invention is not particularly limited as long as it can specifically suppress the expression of the CCL19 gene, and candidates can be appropriately selected from sequences encoding the CCL19 protein (e.g., the sequence set forth in SEQ ID NO: 1) in accordance with the rules described in, for example, Sayda M. Elbashir et al., Genes Dev., 2001, vol. 15, pp. 188-200. Furthermore, it is preferable to check the selected target sequence candidates for homology to a consecutive sequence of 16 to 17 nucleotides in mRNAs other than CCL19 using homology search software such as BLAST, thereby confirming the specificity of the selected target sequence.

[0064] (c2) miRNA Targeting a Transcription Product of the CCL19 Gene The "miRNA targeting a transcription product of the CCL19 gene" according to the present invention is not particularly limited, as long as it inhibits translation of the gene encoding the CCL19 protein. For example, it may inhibit its translation by pairing with the 3' untranslated region (UTR) of the target. Furthermore, the "miRNA" according to the present invention may take the form of any of primary miRNA, precursor miRNA, and mature miRNA, but is preferably a pre-miRNA or mature miRNA, and more preferably a mature miRNA. Furthermore, the length of the miRNA is not particularly limited, but typically, primary miRNA is 100 to 3,000 bases long, pre-miRNA is 50 to 80 bases long, and mature miRNA is 18 to 30 bases long.

[0065] Those skilled in the art can also select miRNA candidates using target prediction software on websites such as TargetScan (http: / / www.targetscan.org / vert_72 / ) and DIANA-micro-T-CDS (http: / / diana.imis.athena-innovation.gr / DianaTools / index.php?r=microT_CDS / index). Furthermore, miRNA candidates according to the present invention can be obtained by searching a database related to miRNAs (TarBase (http: / / carolina.imis.athena-innovation.gr / diana_tools / web / index.php?r=tarbasev8 / index)).

[0066] (c3) Antisense oligonucleotide against a transcription product of the CCL19 gene In the present invention, an "antisense oligonucleotide against a transcription product of the CCL19 gene" is a nucleotide containing a base sequence complementary or substantially complementary to the sequence of the mRNA of the gene encoding CCL19, or a part thereof, and having the function of inhibiting CCL19 protein synthesis by binding to the mRNA to form a specific and stable double strand.

[0067] The "antisense oligonucleotide" of the present invention may be DNA, RNA, or a DNA / RNA chimera. When the antisense oligonucleotide is DNA, the RNA-DNA hybrid formed by the target RNA and the antisense oligonucleotide is recognized by endogenous ribonuclease H (RNase H) and causes selective degradation of the target RNA. Therefore, in the case of an antisense oligoDNA directed to degradation by RNase H, the target sequence may be not only a sequence in mRNA but also a sequence of an intron region in the initial translation product of the CCL19 gene. The intron sequence can be determined by comparing the genomic sequence with the cDNA sequence of the CCL19 gene using a homology search program such as BLAST or FASTA.

[0068] The target region of the "antisense oligonucleotide" of the present invention is not limited in length as long as hybridization of the antisense oligonucleotide results in inhibition of translation into CCL19 protein, and may be the entire sequence of mRNA encoding the CCL19 protein or a portion thereof. However, from the viewpoints of ease of synthesis, antigenicity, intracellular internalization, etc., the target region is preferably 10 to 40 bases, and more preferably 15 to 30 bases. More specifically, the 5'-terminal hairpin loop, 5'-terminal untranslated region, translation initiation codon, protein coding region, ORF translation termination codon, 3'-terminal untranslated region, 3'-terminal palindrome region, and 3'-terminal hairpin loop of the CCL19 gene can be selected as preferred target regions for the "antisense oligonucleotide" of the present invention, but are not limited thereto.

[0069] Furthermore, the antisense oligonucleotide of the present invention may not only hybridize with the mRNA or initial transcription product of the CCL19 gene to suppress translation into protein, but may also bind to these genes, which are double-stranded DNA, to form a triplex and inhibit transcription into RNA (antigene).

[0070] (c4) Nucleotides Having Ribozyme Activity Against Transcription Products of the CCL19 Gene In the present invention, "nucleotides having ribozyme activity against transcription products of the CCL19 gene" encompass not only RNAs having enzymatic activity for cleaving nucleotides, which are ribozymes in the narrow sense, but also DNAs as long as they have sequence-specific nucleic acid cleavage activity. The most versatile ribozymes are self-splicing RNAs found in infectious RNAs such as viroids and virusoids, and hammerhead and hairpin types are known. Hammerhead types exert their enzymatic activity with approximately 40 bases (e.g., 30 to 60 bases), and by arranging several bases (totaling approximately 10 bases) on both ends adjacent to the hammerhead structure to have sequences complementary to the desired cleavage site in the mRNA, they can specifically cleave only the target mRNA.

[0071] Furthermore, when the mRNA of the CCL19 gene itself has a double-stranded structure, the target sequence can be made single-stranded by using a hybrid ribozyme linked to an RNA motif derived from viral nucleic acid that can specifically bind to RNA helicase (see, e.g., Warashina M. et al., PNAS, 2001, Vol. 98, No. 10, pp. 5572-5577). Furthermore, when a ribozyme is used in the form of an expression vector containing DNA encoding it, a hybrid ribozyme can also be formed by further linking a sequence of modified tRNA to promote translocation of the transcript into the cytoplasm (see, e.g., Kuwabara T. et al., Nucleic Acids Res., 2001, Vol. 29, No. 13, pp. 2780-2788).

[0072] Specific embodiments of the "nucleotide that binds to the transcription product of the CCL19 gene" according to the present invention have been described above, but such nucleotides can be prepared appropriately by those skilled in the art using known techniques. For example, as described above, such nucleotides can be prepared by determining the target sequence of the transcription product of the CCL19 gene (such as the initial transcription product or mRNA) and synthesizing a complementary sequence thereto using a commercially available automated DNA / RNA synthesizer.

[0073] siRNA can be prepared by synthesizing the sense and antisense strands of the target sequence on mRNA using an automated DNA / RNA synthesizer, denaturing them, and then annealing them. Alternatively, shRNA can be synthesized and then cleaved using the RNA-cleaving protein dicer.

[0074] Furthermore, the "nucleotides bound to the transcription product of the CCL19 gene" according to the present invention may be not only natural RNA or DNA, but also those containing various chemical modifications to improve stability and specific activity (affinity with RNA). For example, in order to prevent degradation by hydrolases such as nucleases, the phosphate residues of each constituent nucleotide can be substituted with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, phosphorodithioate, etc. Furthermore, the hydroxyl group at the 2'-position of the sugar (ribose) of each nucleotide can be substituted with -OR (R=CH ３ (2'-O-Me), CH ２ CH ２ OCH ３ (2'-O-MOE), CH ２ CH ２ NHC (NH) NH ２ , C.H. ２ CONHCH ３ , C.H. ２ CH ２ Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a carbonyl group at the 2-position with a thiocarbonyl group.

[0075] The sugar moiety of RNA predominantly has two conformations, C2'-endo (S-type) and C3'-endo (N-type), and in single-stranded RNA, these two conformations exist in equilibrium, but when double-stranded RNA is formed, it is fixed to the N-type. Therefore, in order to impart strong binding ability to target RNA, BNA (LNA), an RNA derivative in which the sugar moiety conformation is fixed to the N-type by bridging the 2' oxygen and 4' carbon, is also preferably used. Furthermore, in addition to BNA, other non-natural nucleotides (artificial nucleotides, nucleotide analogs) such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), peptide nucleic acid (PNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), morpholino nucleic acid, tricyclo-DNA (tcDNA), 2'-O-methylated nucleic acid, 2'-MOE (2'-O-methoxyethyl) modified nucleic acid, 2'-AP (2'-O-aminopropyl) modified nucleic acid, 2'-fluorinated nucleic acid, and 2'F-arabino nucleic acid (2'-F-ANA) can also be suitably used. Note that nucleotides containing such various modifications can also be chemically synthesized by known techniques.

[0076] Furthermore, the "nucleotides binding to the transcription product of the CCL19 gene" of the present invention may take the form of a nucleotide construct (expression cassette) that can express the nucleotide in vivo. The expression cassette of the present invention is not particularly limited as long as it is a polynucleotide into which the above-mentioned nucleotides have been incorporated in an expressible state, and examples thereof include a polynucleotide containing a promoter, a sequence encoding the nucleotides, and, if necessary, a transcription termination signal.

[0077] The promoter is not particularly limited as long as it can induce the expression of the nucleotide, but from the viewpoint of accurately transcribing short RNAs such as siRNA, Pol III promoters are preferred, and more specific examples include mouse and human U6-snRNA promoters, human H1-RNase P RNA promoters, and human valine-tRNA promoters. Furthermore, a sequence of four or more consecutive Ts is used as a transcription termination signal.

[0078] The expression cassette constructed in this manner may be inserted into a plasmid vector or a viral vector, such as a retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, or Sendai virus, or an animal cell expression plasmid.

[0079] (d) Peptides Having Dominant-Negative Traits Against CCL19 Protein Examples of "peptides having dominant-negative traits against CCL19 protein" contained as an active ingredient in the composition of the present invention include peptides that competitively inhibit the activation of CCR7 protein by CCL19 protein. More specifically, these include partial peptides containing the binding site or binding groove of CCL19 protein with CCR7 protein, and decoy peptides that mimic the binding site or binding groove. Examples of such partial peptides include peptides containing the amino acid sequence of positions 29 to 87 of SEQ ID NO: 2, peptides containing at least one of the amino acid sequences of positions 29 to 33, 35 to 42, 54 to 55, and 77 to 87 of SEQ ID NO: 2, and peptides containing at least one of the amino acid sequences of positions 31, 61, and 70 of SEQ ID NO: 2. Furthermore, in the present invention, the length of a peptide having a dominant-negative trait against the CCL19 protein is not particularly limited as long as it has the trait, and examples include 70 amino acids or less, 60 amino acids or less, 50 amino acids or less, 40 amino acids or less, 30 amino acids or less (e.g., 29 amino acids, 28 amino acids, 27 amino acids, 26 amino acids), 25 amino acids or less (e.g., 24 amino acids, 23 amino acids, 22 amino acids, 21 amino acids), 20 amino acids or less (e.g., 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids), 15 amino acids or less (e.g., 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids), 10 amino acids or less (e.g., 9 amino acids, 8 amino acids, 7 amino acids, 6 amino acids), and 5 amino acids or less (e.g., 4 amino acids, 3 amino acids, 2 amino acids).

[0080] (Compositions) The compositions of the present invention may be in the form of, for example, pharmaceutical compositions or reagents used for research purposes (eg, in vitro or in vivo experiments).

[0081] More specifically, the pharmaceutical composition of the present invention is a pharmaceutical composition for treating or preventing chronic inflammatory bowel disease. In the present invention, "chronic inflammatory bowel disease" refers to a disease that causes chronic inflammation or ulcers in the mucosa of the large intestine or small intestine, and examples thereof include Crohn's disease (CD) and ulcerative colitis (UC). CD is a disease in which inflammatory lesions such as ulcers and swelling occur in any part of the intestinal tract (gastrointestinal tract) (the small intestine is the most common site). UC is a disease in which chronic inflammation occurs in the large intestine, causing ulcers. In such chronic inflammatory bowel disease, stenosis due to intestinal fibrosis may occur, necessitating surgery.

[0082] In the present invention, "treatment" includes suppression of intestinal fibrosis in chronic inflammatory bowel disease, and more specifically includes elimination of intestinal fibrotic state, reduction of intestinal fibrotic tissue, suppression (prevention or delay) of the progression of intestinal fibrosis, etc. Furthermore, "prevention" includes prevention, delay or reduction of the risk of recurrence of intestinal fibrosis in chronic inflammatory bowel disease, maintenance of improved intestinal fibrotic tissue, etc.

[0083] The "composition" of the present invention may contain other pharmacologically acceptable components in addition to the molecule that inhibits the function of the CCL19 gene. Examples of such other components include carriers, emulsifiers, humectants, pH buffers, culture media, excipients, disintegrants, buffers, isotonicity agents, suspending agents, solubilizers, soothing agents, stabilizers, preservatives, and antiseptics. More specifically, in the case of liquid preparations such as injections, examples of pharmacologically acceptable other components include aqueous solutions (e.g., physiological saline, water for injection, phosphate buffer, aqueous glucose solution, aqueous glycerol solution), and aluminum hydroxide. In the case of lyophilized preparations, examples include, but are not limited to, sugars (e.g., mannitol, lactose, sucrose), albumin, and the like. Furthermore, the pharmaceutical composition of the present invention may be in the form of a kit in which the above components can be mixed prior to administration. Furthermore, when used as an injection, the composition may be in the form of a syringe.

[0084] Furthermore, in the present invention, when the substance that inhibits the function of the CCL19 gene is in the form of a nucleotide, it can be incorporated into a delivery system such as a liposome or microsphere, or other molecules can be attached. Examples of such additives include polycations such as polylysine, which act to neutralize the charge of the phosphate backbone, and hydrophobic substances such as lipids (phospholipids, cholesterol, etc.) that enhance interaction with cell membranes or increase nucleotide uptake. Examples of lipids that can be attached include cholesterol or its derivatives (cholesteryl chloroformate, cholic acid, etc.). Such substances can be attached to the 3' or 5' end of the nucleotide, or via a nucleotide, sugar, or intramolecular nucleoside bond. Other examples of groups include capping groups specifically placed at the 3' or 5' end of the nucleotide to prevent degradation by nucleases such as exonucleases and RNases. Examples of such capping groups include hydroxyl-protecting groups known in the art (e.g., glycols such as polyethylene glycol and tetraethylene glycol).

[0085] Furthermore, particularly when the composition of the present invention is intended for oral administration, it may be combined with a composition that enables efficient delivery into the intestine. The composition that enables delivery into the intestine is not particularly limited, and known compositions can be appropriately used, for example, pH-sensitive compositions, compositions that suppress release into the intestine (cellulose-based polymers, acrylic acid polymers and copolymers, vinyl acid polymers and copolymers, etc.), bioadhesive compositions that specifically adhere to the intestinal mucosa, compositions containing protease inhibitors, and compositions that are specifically degraded by intestinal enzymes.

[0086] The pharmaceutical composition of the present invention may contain, as an active ingredient, only the molecule that suppresses the function of the CCL19 gene, or may contain the molecule and at least one other therapeutic or preventive agent for chronic inflammatory bowel disease (hereinafter also simply referred to as "other therapeutic agent, etc."). Alternatively, the molecule that suppresses the function of the CCL19 gene and the other therapeutic agent, etc. may be administered separately. Such a combination can enhance the therapeutic or preventive effect for chronic inflammatory bowel disease. Examples of the "other therapeutic agent, etc." to be used in combination include, but are not limited to, anti-inflammatory agents, immunomodulators, and corticosteroids. More specifically, examples of anti-inflammatory drugs include anti-TNFα antibodies (adalimumab (trade name: Humira), infliximab (trade name: Remicade), certolizumab or certolizumab pegol (both trade names: Cimzia), golimumab (Simponi)), anti-IL-12 / 23p40 antibodies (ustekinumab (trade name: Stelara)), anti-IL-23p19 antibodies (risankizumab (trade name: Skyrizi)), anti-α4β7 antibodies (vedolizumab (trade name: Entyvio)), JAK inhibitors (Upa Examples of immunomodulatory agents include biological agents such as dacitinib (trade name: RINVOQ), sulfasalazine (SASP), and 5-aminosalicylic acid (5-ASA). Examples of immunomodulatory agents include azathioprine, 6-mercaptopurine (6-MP), cyclosporine, and tacrolimus (Prograf). Examples of corticosteroids include prednisone (PSL), prednisolone (methylprednisolone, etc.), dexamethasone, triamcinolone, and betamethasone.

[0087] The product (drug, reagent, etc.) of the composition of the present invention or its instruction manual may bear a label indicating that the composition is used for inhibiting intestinal fibrosis or for treating or preventing chronic inflammatory bowel disease. Here, "bearing a label on the product or instruction manual" means that the label is affixed to the product itself, container, packaging, etc., or to an instruction manual, package insert, promotional material, other printed matter, etc. that discloses information about the product.

[0088] The composition of the present invention may also be in the form of a kit. For example, the molecule that suppresses the function of the CCL19 gene and the other pharmacologically acceptable components, etc., typically exist as two or more substances, but can be mixed or otherwise prepared into a single composition before administration to a subject. When used as an injection, the kit may be in the form of a syringe.

[0089] <Treatment and prevention methods> The present invention also provides a method for suppressing intestinal fibrosis or treating or preventing chronic inflammatory bowel disease, which comprises administering to a subject an effective amount of a molecule that suppresses the function of the CCL19 gene.

[0090] The "subject" of treatment, etc., in the present invention is not particularly limited and may be not only humans but also non-human animals. Non-human animals are not particularly limited and may include various livestock, poultry, pets, laboratory animals, etc. More specifically, vertebrates, preferably mammals, more preferably primates (humans, monkeys, chimpanzees, orangutans, gorillas, etc.), ungulates (cattle, horses, sheep, goats, etc.), and rodents (mice, rats, guinea pigs, etc.), are included. However, the subject of the present invention is usually humans, and is not limited by gender, age, race, etc. More specifically, examples of the subject include humans suffering from chronic inflammatory bowel disease, humans at risk of suffering from chronic inflammatory bowel disease, humans at risk of recurrence of chronic inflammatory bowel disease, and humans with recurrence of chronic inflammatory bowel disease.

[0091] The method of administration of the composition of the present invention is not particularly limited and varies depending on the form of the molecule or composition, the age, weight, sex, health condition, etc. of the subject, but can be administered by any of the following administration routes: parenteral administration (e.g., topical administration, intravenous administration, intra-arterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration, intramuscular administration, administration by infusion), and oral administration. Furthermore, when the molecule that suppresses the function of the CCL19 gene is in the form of a nucleotide, it can be administered locally using a gene gun, a catheter, or the like. An example of local administration is direct administration to the target tissue (intestinal tract).

[0092] When administering the compositions of the present invention, the dosage can be appropriately selected by those skilled in the art depending on the subject's age, body weight, degree of progression of symptoms, health condition, the type of molecule that inhibits the function of the CCL19 gene, the dosage form of the composition, the method of administration, etc. For example, the dosage of the pharmaceutical composition of the present invention (equivalent to the active ingredient) varies depending on the type of active ingredient. For example, when the active ingredient is RNA or DNA, it is typically 0.1 μg to 10 mg / kg body weight, and when the active ingredient is an antibody, it is typically 1 to 10 mg / kg body weight. Furthermore, when the active ingredient is DNA encoding RNA that binds to the transcription product of the CCL19 gene, an expression cassette may be prepared and administered so that an appropriate amount of RNA is expressed. Furthermore, the number of administrations per day is not particularly limited and can be appropriately selected taking into consideration the various factors described above. For example, the composition may be administered once or multiple times (e.g., twice) per day.

[0093] The administration schedule of the composition of the present invention can be adjusted appropriately depending on the various factors described above. However, as shown in the examples below, it is desirable to administer the composition after the mid-inflammatory stage (peak inflammation stage). Here, "mid-inflammatory stage" does not refer to the inflammation exacerbation stage from the early stage to the peak inflammation stage, but rather refers to the stage after the inflammation has started to converge (the inflammation resolution stage). More specifically, it refers to the stage after the disease activity score (CDAI, etc.) has started to decline.

[0094] From this viewpoint, the pharmaceutical composition of the present invention may also be in the form of a pharmaceutical composition for treating or preventing chronic inflammatory bowel disease, comprising as an active ingredient a molecule that suppresses the function of the CCL19 gene, wherein the molecule is administered to a subject at or after the middle stage of inflammation in the intestinal tract.

[0095] The composition of the present invention may be administered in a single dose or multiple times continuously or periodically. Furthermore, the degree of fibrosis in the intestinal tract may be monitored after administration, and the timing of administration may be determined based on the results. Administration may be discontinued depending on the degree of recovery, but from the viewpoint of preventing recurrence, administration may be continued without discontinuation. Note that "continuous" may mean daily or at intervals.

[0096] Furthermore, the treatment methods of the present invention may be used in combination with other known treatment methods for chronic inflammatory bowel disease. Examples of such treatment methods include the administration of the other therapeutic agents (anti-inflammatory drugs, immunomodulators, corticosteroids, etc.) mentioned above, granulocyte apheresis, endoscopic treatment (balloon dilation), surgical treatment, etc. The "surgical treatment" is not particularly limited, and examples include partial resection of the intestinal tract. Furthermore, the other therapeutic agent may be administered to a subject simultaneously, at different times, or consecutively with the molecule that suppresses the function of the CCL19 gene, or the two may be administered at different administration intervals. For example, when used in combination with an anti-inflammatory drug, the molecule that suppresses the function of the CCL19 gene may be administered after a period (e.g., at least 7 days, preferably 10 to 15 days) following administration of the anti-inflammatory drug. Such a combined use can have the effect of further suppressing not only inflammation but also excessive fibrosis.

[0097] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. The examples were carried out using the materials and methods shown below.

[0098] [Materials and Methods] (Sample Collection) For CD patients who consented to participate in a clinical study (Research on the immune status of patients with chronic inflammatory bowel disease, approved by the Nagoya University School of Medicine Genome Analysis Research Expert Review Committee: 2020-0599), tissue samples were collected using a small intestine endoscope (Fujifilm, EN-580T) and biopsy forceps, while distinguishing between areas of severe fibrous stenosis (areas of excessive stenosis), areas of inflammation (mild stenosis), and normal areas of the small intestine. The collected tissue samples were placed in RNA Later tissue preservation reagent (Invitrogen, USA), transported to the laboratory on ice as quickly as possible, and subjected to the RNA extraction described below.

[0099] (RNA extraction from specimen) The collected tissue specimen was pulverized using a microtissue disrupter (Biomasher (registered trademark) II, manufactured by Kanto Kagaku Co., Ltd.). Then, RNA was extracted using NucleoSpin (registered trademark) RNA Plus (manufactured by Macherey-Nagel). After that, RNA was extracted using a nucleic acid concentration measuring instrument, Multiska ＴＭ The RNA concentration was measured using GO (Thermo Scientific).

[0100] (Synthesis of cDNA from Sample) 2,000 μg of the extracted RNA was used for RT-PCR using RT-PCR reagents (Invitrogen, SuperScript IV VILO Master Mix) and Veriti ＴＭ cDNA synthesis was carried out using a 96-well thermal cycler (manufactured by Applied Biosystems (USA)).

[0101] (Bulk RNA seq) The collected RNA was subjected to next-generation sequencing (NGS) analysis (contracted to the National Cancer Center). The obtained sequence data was subjected to QC (FastP) and mapping (HISAT2) using FastQfile software. Furthermore, gene expression levels were quantified using featureCounts. The obtained count data was used for clustering using the statistical software R. For the purpose of additional immune cell analysis, cell subset analysis was performed using Cybersort.

[0102] (Tissue Processing) Collected tissue specimens (endoscopic specimens, surgical specimens) were pulverized, suspended in an enzyme reaction solution, and treated at 37°C for 20 minutes. The tissue was then passed through a 100 μm filter and centrifuged (4°C / 300 g / 5 minutes) to collect cells. The enzyme reaction solution was prepared by adding 1% collagenase 1 (Fujifilm Wako (JPN)), DNAase (Roche, catalog number: 10104159001), and Dyspase (Roche, catalog number: 4942078001)) to HBSS.

[0103] (Flow Cytometry (FACS)) The cells obtained by the tissue processing were washed with FACS buffer, the supernatant was discarded, and then the cell surface was stained. Specifically, APC-cy7 Fixable viability dye, PerCP5.5 anti-CD3 mAb, BV510 anti-CD4 mAb, BUV737 anti-CD11b mAb, PEcy7 anti-CCR7 mAb were mixed with FACS buffer at a ratio of 1:100 according to the number of specimens, and staining was performed (4°C, 15 minutes). Antibodies were appropriately selected depending on the origin (human or mouse) of the tissue to be analyzed. All antibodies used were manufactured by Biolegend (USA).

[0104] Next, the cells were washed with FACS buffer, and after fixation treatment with Fix / Perm buffer, staining was performed with PE, anti-FOXP3 mAb (diluted 1:100 with Perm-wash buffer) (4°C, 15 minutes). After washing with Perm-wash buffer, the buffer was replaced with FACS buffer and the cells were analyzed using a flow cytometer (BD FACSSymphony). ＴＭ The cells were subjected to FACS analysis using a BD Biosciences (USA) FACSDiva software (version 8.0.1, BD Biosciences), FlowJo software (version 10, BD Biosciences), and Excel for Mac 16 (Microsoft).

[0105] (Cell sorting) Cells obtained by tissue processing were subjected to hemolysis treatment using RBC Lysis Buffer (Biolegend) at room temperature for 10 minutes. The collected cells were suspended in 40 μl of Dead Cell Removal Beads (Biolegend) and 200 μl of MACS buffer, and reacted at 4°C for 10 minutes. 2,000 μl of MACS buffer was added, and the cells were incubated with a dedicated magnet at room temperature for 15 minutes to adsorb dead cells, and the supernatant was collected. Subsequently, staining was performed in the same manner as described above for FACS. After staining and washing, the cells were replaced with 200 μl of FACS buffer and analyzed using a BD FACSSymphony. ＴＭ CCR7 was sorted using a S6 cell sorter (BD bioscience, USA). ＋ CD11b ＋ , CCR7 － CD11b ＋ , CCR7 ＋ CD25 ＋ or CCR7 － CD25 ＋ The cells were isolated and harvested.

[0106] (RNA extraction from isolated cells and cDNA synthesis) The cells isolated above (CCR7 ＋ CD11b ＋ , CCR7 － CD11b ＋ , CCR7 ＋ CD25+ or CCR7-CD25+) using SuperScript ＴＭ RNA was extracted and cDNA synthesis was carried out using the IV Single Cell / Low Input cDNA PreAmp Kit (Invitrogen).

[0107] (Real-time PCR) The cDNA synthesized above was used to ＴＭReal-time PCR was performed using a 7 real-time PCR system (Applied biosystems (USA)). The PCR reaction solution (per sample) consisted of 1 μl of DNA sample (diluted 1 / 4 with MilliQ water), 10 μl of SYBR Green qPCR Mix (Thermo Fisher), 0.5 μl of forward primer and reverse primer, and 8 μl of MilliQ water, totaling 20 μl, and was subjected to PCR under the following conditions. PCR conditions: 95°C for 1 minute, 40 cycles of (95°C for 15 seconds, 60°C for 40 seconds), 60°C for 40 seconds. Human / mouse actin was also amplified as a correction intrinsic factor. The sequences of the primers used are shown below.

[0108]

[0109] (Preparation of specimens) The surgical specimens were fixed in formalin for 24 hours, then replaced with ethanol, embedded in paraffin, and the resulting blocks were sliced ​​thinly.

[0110] (Fluorescent Immunostaining) The sliced ​​specimens were deparaffinized and subjected to an activating solution (Epitope Retrieval Solution pH 9, manufactured by Leica biosystems), and then treated in a microwave oven for 2 minutes and in an electric kettle for 15 minutes. The specimens were then cooled at room temperature for 15 minutes to activate the antigens. The specimens were then blocked, and multiple immunostaining was performed using an Opal 6-Plex Manual Detection Kit (manufactured by AKOYA biosciences). Specifically, the specimens were stained (at room temperature in the dark for 1 hour) with the following appropriately diluted primary antibodies. The primary antibodies were diluted with a dedicated diluent (Antibody Diluent), with the anti-α-SMA antibody being used at a 1:1000 dilution, and the other antibodies being used at a 1:100 dilution. After staining with the primary antibody, the sections were washed and stained with a secondary antibody (Polymer HRP secondary antibody) (10 minutes at room temperature in the dark). Then, the sections were washed and stained with Pulus Amplification Diluent (each of the desired colors diluted 150-fold) (10 minutes at room temperature in the dark), followed by washing. If the colors overlapped, the sections were returned to the activation step and stained one factor at a time with the primary antibody, HRP-labeled secondary antibody, and desired dye. After staining all factors, the sections were stained with DAPI (manufactured by Dojin Kagaku (JPN)). After washing, the sections were mounted and placed in a dark room at 4°C for 24 hours, and then photographed using a tissue quantitative analysis imaging system (The Vectra (registered trademark) 3).Human primary antibody CCR7 (EPR23192-57: ab253187: Abcam USA), CD11b (EP45: AC-0043RUO: Epitomics USA), a-SMA (A14: ab7817: Abcam USA), CD4 (4B12: PA0427 Leica USA), FOXP3 (236A / E7: ab20034: Abcam USA), DAPI (D523 Cellstain (registered trademark) - DAPI solution), Mouse primary antibody CCR7 (EPR23192-57:ab253187:Abcam USA), CD11b (EP45:AC-0043RUO:Epitomics USA), a-SMA (A14:ab7817:Abcam USA), CD4 (GHH4:DIA-404 dianova USA), Foxp3 (FJK-16s: eBioscience USA), DAPI (D523 Cellstain®-DAPI solution).

[0111] (DAB staining) The sliced ​​specimens were deparaffinized and washed, and then subjected to antigen retrieval in an activating solution (Epitope Retrieval Solution pH 9, manufactured by Leica biosystems) (MW method: heated in a microwave oven for 3 minutes, then further heated in a pressure cooker). Then, the specimens were subjected to blocking treatment (Dako protein block serum-free for 30 minutes at room temperature) (H ２ O ２ After 10 minutes of incubation with the primary antibody (100-fold dilution) at room temperature, the sections were stained and washed with TBS-T. ２ O ２After incubation with a POD-labeled secondary antibody at room temperature for 30 minutes, the sections were washed with TBS-T. Then, the sections were stained with DAB and washed with TBS-T. After HE staining, the sections were washed and further dehydrated, mounted, and photographed using a tissue quantitative analysis imaging system (The Vectra® 3). Primary antibody / Human CD45 (UCHL1:ab23 Abcam USA), CCL19 (HPA067758 Anti-CCL19 Sigma), CCR7 (EPR23192-57:ab253187: Abcam USA), a-SMA (A14:ab7817: Abcam USA), Primary antibody / Mouse CD45 (30-F11:550539 BD Pharmingen), CCR7 (EPR23192-57:ab253187: Abcam USA), a-SMA (A14:ab7817:Abcam USA), (primary antibody dilution solution PBS in 1% BSA + 0.1% NAN3), secondary antibody / Dako Envision system HRP labeled polymer (Anti-Rabbit) K4003, H2O2 0.5 ml + methanol 50 ml (for peroxidase blocking), DAB Dako Liquid DAB + substrate chromogen K3488.

[0112] (Elastica Masson Staining) The sliced ​​specimens were deparaffinized and washed in running water for 10 minutes. They were then stained with Bouin's solution (60°C, 60 minutes), washed, and then stained with resorcinol fuchsin solution (room temperature, 90 minutes). They were washed with 3% hydrochloric acid alcohol (room temperature, 1 minute), and stained with iron hematoxylin solution (room temperature, 1 minute). After washing with acetic acid, they were stained with Masson's solution (room temperature, 5 minutes). They were then washed with acetic acid, stained with phosphomolybdic acid orange solution (room temperature, 3 minutes), and further washed with acetic acid. They were stained with phosphotungsten solution (room temperature, 7 minutes), washed with acetic acid, and stained with 0.8% light green solution (room temperature, 15 minutes). They were then treated with ethanol and xylol, washed, mounted, and photographed using a tissue quantitative analysis imaging system (The Vectra® 3).

[0113] (Preparation of mice with small intestinal fibrosis) As an enteritis inducer, 2,4,6-trinitrobenzenesulfonic acid sodium hydrate (TNBS, manufactured by Wako (JPN)) was diluted with 50% ethanol to a concentration of 1 mg / 100 μl and used.

[0114] The mice were anesthetized and underwent abdominal section, and the enteritis-inducing agent was directly administered to multiple sites from the ileum to the small intestine (200 μl per mouse). This administration was repeated three times at weekly intervals.

[0115] The mice with small intestinal fibrosis prepared in this manner were then sacrificed, and the small intestines were collected and fixed using the Swiss-roll method to prepare pathological sections. The small intestines isolated from the mice with small intestinal fibrosis were crushed, washed, and filtered through a 100 μm filter. After centrifugation, the supernatant was discarded and the cells were collected.

[0116] (Treatment with anti-CCL19 antibody) In the above-described process for producing mice with small intestinal fibrosis, two days after the third TNBS administration, an anti-mouse CCL19 antibody (R&D, clone: ​​87102, Mouse CCL19 / MIP-3 beta Antibody) was directly administered from the ileum to the small intestine (dosage: 20 μg / administration / mouse) in the same manner as above.

[0117] The results obtained using the above materials and methods are shown below.

[0118] Example 1 Identification of Factors Causing Excessive Intestinal Fibrosis To prevent intestinal fibrosis, it is necessary to identify and intervene against factors that cause excessive fibrosis. Therefore, we compared molecular expression in areas with excessive fibrosis and areas with mild fibrosis in an attempt to identify factors associated with the differences between the two. Specifically, we collected tissue from the inflamed area (before fibrosis) and tissue from the fibrotic area (after fibrosis) of the small intestine of CD patients using an enteroscope, which allows direct visual confirmation of the differences between the two, and comprehensively analyzed gene and molecular expression.

[0119] As shown in Figure 2A, comprehensive gene expression analysis (bulk RNA sequencing analysis) was performed using collected small intestinal tissue, revealing that the chemokine CCL19 and its receptor CCR7 were highly expressed in fibrotic sites compared to inflammatory sites. Furthermore, as shown in Figure 2B, analysis of immune cells (CIBERSORT) at both sites revealed differences in the expression of Treg- and macrophage-associated molecules.

[0120] Focusing on CCR7, the receptor for CCL19, we analyzed immune cells infiltrating into tissues and found that the CCR7-positive cell fraction increased in fibrotic sites, revealing the presence of many CCR7-positive macrophages and CCR7-positive Tregs, as shown in Figures 3A to 3D. These data suggest that these CCR7-positive cells may induce intestinal fibrosis.

[0121] Based on the above results, we hypothesized that "at sites of excessive fibrosis, CCL19 expression increases from a certain point in time, resulting in the accumulation of CCR7-positive macrophages and CCR7-positive Tregs. Furthermore, these cells survive for a long period of time and highly express TGF-β, promoting the transition from fibroblasts to myofibroblasts and causing excessive fibrosis" (see Figure 4), and proceeded with the following investigation.

[0122] Example 2 Analysis of CCR7-Positive Macrophages and CCR7-Positive Tregs in Severely Fibrotic Sites in the Intestinal Tract Inflammation was progressing toward resolution in fibrotic sites, and as shown in Figure 2B, immune cell infiltration, particularly macrophage and Treg infiltration, was reduced compared to inflamed and normal conditions. However, as shown in Figures 3A to 3D, although the overall number was reduced, the CCR7-positive cell fraction was increased.

[0123] Therefore, when CCR7-positive macrophages and CCR7-positive Tregs were examined by cell sorting, as shown in Figure 5, CCR7-positive cells at sites of severe fibrosis expressed significantly higher levels of TGF-β than CCR7-negative cells. Furthermore, as shown in Figures 6A-D, it was revealed that CCR7-positive macrophages and CCR7-positive Tregs highly expressed anti-apoptotic signals (factors associated with long-term survival), such as Bcl-XL and Bcl-2. Furthermore, although not shown in the figures, analysis using a public database of intestinal scRNA-seq (GSE134809) also confirmed that the expression of Bcl-XL and Bcl-2 was high in CCR7-positive macrophages and CCR7-positive Tregs.

[0124] Example 3: Administration of anti-CCL19 antibody to mice with human CD-like small intestinal fibrosis One of the difficulties in developing a drug to prevent intestinal fibrosis is the lack of an animal model of small intestinal fibrosis similar to human CD. Conventional CD model mice only exhibit acute inflammation in the large intestine. However, the present inventors used a unique method to successfully create a mouse model with inflammation, fibrosis, and stenosis in the terminal ileum, a common site of CD. In this mouse model, fibrosis worsened with each drug administration, and irreversible fibrosis accompanied by collagen fiber proliferation was observed after three drug administrations. This mouse model reflected the human pathology, making it a novel mouse model of human CD-like small intestinal fibrosis. Furthermore, macrophages and Tregs were reduced in fibrotic sites compared with normal sites. Meanwhile, fibrotic sites exhibited high CCL19 expression and extensive infiltration of CCR7-positive cells. In particular, extensive infiltration of CCR7-positive macrophages and CCR7-positive Tregs was observed.

[0125] We then investigated the potential of anti-mouse CCL19 antibody as a fibrosis preventive agent using mice with human CD-like small intestinal fibrosis. When anti-CCL19 antibody was administered to mice with small intestinal fibrosis, a decrease in α-SMA, an indicator of intestinal fibrosis, was observed in the small intestine, as shown in Figures 7A and 7B. Furthermore, as shown in Figures 7C to 7F, a decrease in CCL19 and CCR7-positive cells was observed in sync with the decrease in fibrosis. Therefore, it was demonstrated that anti-CCL19 antibody is effective against fibrosis by inhibiting tissue infiltration of CCR7-positive cells.

[0126] Example 4: Study on the Timing of Administration of Anti-CCL19 Antibody The timing of administration of anti-CCL19 antibody was studied. First, human analysis revealed that CCL19 expression increased and was highly expressed from the late inflammatory to fibrotic stages, as shown in Figure 8. Based on these results, anti-CCL19 antibody was administered at the early, middle, and late inflammatory stages to study its therapeutic effect. As a result, as shown in Figures 9A and 9B, the most effective way to reduce fibrosis was observed when anti-CCL19 antibody was administered after the middle inflammatory stage (peak inflammatory stage). Consistent with this, as shown in Figures 10A to 10C, it was found that the reduction in CCL19 and CCR7-positive cells was significantly reduced when anti-CCL19 antibody was administered after the middle inflammatory stage (peak inflammatory stage). Furthermore, a significant reduction in CCR7-positive macrophages was observed, and an anti-fibrotic effect was also observed.

[0127] The above results revealed the following: (1) CCR7-positive macrophages and Tregs accumulated by CCL19 significantly contribute to excessive intestinal fibrosis. (2) Anti-CCL19 antibodies reduce tissue-infiltrating CCR7-positive cells in the intestinal tract and reverse fibrosis. (3) The best time to use anti-CCL19 antibodies is after the mid-inflammatory stage (peak inflammatory stage).

[0128] As described above, according to the present invention, it is possible to inhibit intestinal fibrosis by suppressing the function of the CCL19 gene. That is, it is possible to treat or prevent intestinal fibrosis in CD and the like. Therefore, the present invention is useful in the medical field related to chronic inflammatory bowel diseases such as CD, and can greatly contribute to improving the QOL of many patients with chronic inflammatory bowel diseases.

Claims

1. A composition for inhibiting intestinal fibrosis, comprising as an active ingredient a molecule that inhibits the function of the CCL19 gene.

2. The composition according to claim 1, wherein the molecule is any one of the following molecules (a) to (f): (a) an anti-CCL19 protein antibody; (b) a nucleotide that binds to the CCL19 protein; (c) a nucleotide that binds to a transcription product of the CCL19 gene; (d) a peptide that has dominant-negative properties against the CCL19 protein; (e) an mRNA that encodes the anti-CCL19 protein antibody; or (f) a DNA that encodes the anti-CCL19 protein antibody.

3. The composition according to claim 1 or 2, which is a pharmaceutical composition for treating or preventing chronic inflammatory bowel disease.

4. The composition of claim 3, wherein the chronic inflammatory bowel disease is Crohn's disease.

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