Pharmaceutical compositions for treating inflammatory bowel disease

Abstract

There are said to be approximately five million people worldwide who suffer from inflammatory bowel diseases including ulcerative colitis and Crohn's disease, and the development of therapeutic agents is hoped for. In particular, the development of a therapeutic agent that is effective for patients who have a recognized resistance to currently existing therapeutic agents is a problem of concern.　An enterocolitis amelioration effect was observed due to a neutralizing antibody that recognizes only active IL-18. Furthermore, a marked enterocolitis amelioration effect was observed due to using an anti-TNF-α antibody together therewith. A medicinal composition having an antibody that recognizes active IL-18, i.e. an antibody that recognizes a neoepitope, and a TNF-α inhibiting agent as active ingredients thereof has a marked enterocolitis amelioration effect, and thus is a therapeutic agent for inflammatory bowel disease.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for treating inflammatory bowel disease, which contains an antibody that recognizes active IL-18 as an active ingredient.

Background Art

[0002] Inflammatory bowel disease (IBD) includes ulcerative colitis and Crohn's disease as its main forms, and both are designated as intractable diseases. IBD is a disease in which inflammation occurs in the gastrointestinal mucosa mainly in the small intestine and large intestine, accompanied by symptoms such as diarrhea, bloody stools, abdominal pain, and fever. The main forms, ulcerative colitis and Crohn's disease, are diseases of unknown cause, and it is estimated that there are about 300,000 patients nationwide and about 5 million patients worldwide, and the number of patients is increasing rapidly, especially among the young.

[0003] For the treatment of IBD, 5-aminosalicylic acid preparations, immunosuppressive agents such as azathioprine, and adrenal cortical steroids are used, and when these show treatment resistance, biological agents are introduced. The emergence of an anti-TNF-α antibody preparation, which is one of the biological agents, has brought about a revolution in treatment (Non-Patent Document 1), but treatment resistance is recognized in about half of the patients (Non-Patent Documents 2 and 3), and the establishment of a new treatment method according to the disease state is an urgent task.

[0004] Regarding the pathological state of IBD, in addition to the disturbance of the intestinal flora (dysbiosis) and the breakdown of mucosal immunity, the importance of the inflammasome pathway related to both has been recognized. A plurality of inflammasome-related genes have been identified as disease susceptibility genes for IBD, and its dysfunction has been reported to induce dysbiosis, activate helper T cell type 1 (Th1) and Th2 cells, and exacerbate enteritis via the production of active IL-18.

[0005] The inventors of this invention have found that IL-18, an inflammasome-related cytokine, is highly expressed in the blood, intestinal epithelium, and stromal tissue of IBD patients resistant to anti-TNF-α antibodies and anti-IL-12 / 23 antibody preparations. Antiactive IL-18 neutralizing antibodies, which directly suppress active IL-18 produced by inflammasome activation, have a different mechanism of action from the existing TNF-α pathway and IL-12 / 23 pathway, and are expected to be effective as a novel therapeutic agent for refractory cases. Furthermore, since IL-18 gene-deficient mice do not show severe symptoms, it is considered unlikely that IL-18 suppression will cause serious side effects, making it a promising drug candidate. Therefore, we investigated the possibility of treating inflammatory bowel disease with anti-IL-18 antibodies.

[0006] IL-18 is a cytokine of the IL-1β family, primarily expressed in macrophages, but also in various other cells such as dendritic cells, epithelial cells, and keratinocytes. Furthermore, the IL-18 receptor is expressed in various cells including NK cells, NKT cells, CD4 T cells, as well as B cells, neutrophils, macrophages, vascular endothelial cells, and smooth muscle cells. In intestinal tissue, IL-18 is produced as a precursor by macrophages, dendritic cells, and intestinal epithelial cells, cleaved by caspases produced by the inflammasome, and secreted extracellularly as active IL-18. Anti-IL-18 neutralizing antibodies, which directly suppress active IL-18 produced by inflammasome activation, differ in mechanism from the existing TNF-α and IL-12 / 23 pathways, and are expected to be effective as novel therapeutic agents for refractory cases. However, it has already been disclosed that, although not using an active IL-18 neutralizing antibody, suppressing IL-18 can be a potential treatment for inflammatory bowel disease (Patent Documents 1-10). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2015 / 032932 [Patent Document 2] International Publication No. 2014 / 186728 [Patent Document 3] International Publication No. 2014 / 037899 [Patent Document 4] International Publication No. 2001 / 058956 [Patent Document 5] International Publication No. 2005 / 047307 [Patent Document 6] International Publication No. 2012 / 085015 [Patent Document 7] International Publication No. 2010 / 048183 [Patent Document 8] International Publication No. 2007 / 137984 [Patent Document 9] International Publication No. 2001 / 062285 [Patent Document 10] International Publication No. 2000 / 056771 [Patent Document 11] International Publication No. 2020 / 116423 [Patent Document 12] International Publication No. 2004 / 092219 [Non-patent literature]

[0008] [Non-Patent Document 1] Nielsenand Ainsworth, 2013, N Engl J Med, Vol.369(8), p.754-762 [Non-Patent Document 2] Gisbert et al., 2015, Aliment. Pharmacol. Ther. Vol.41(7), p.613-23 [Non-Patent Document 3] Singh et al., 2018,Aliment. Pharmacol. Ther. Vol. 47(2), p.162-175 [Non-Patent Document 4] Arimori, T., et al., Structure, 2017, Vol. 25, pp.1611-1622 [Non-Patent Document 5] Hezareh et al., J. Virol. 2001, Vol. 75, pp.12161-12168

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0009] Patent Documents 1 to 10 list Crohn's disease and ulcerative colitis as inflammatory diseases to be treated. However, there is not a single document showing the actual effect. Also, at present, several companies are conducting clinical trials or preparing for clinical trials, but there are no anti-IL-18 antibody pharmaceuticals that have reached practical application. Clinical trials of anti-IL-18 antibody pharmaceuticals are being conducted on sarcoidosis (NCT04064242, Phase II, Patent Document 3), atopic dermatitis (NCT04836858, Phase II, Patent Document 3), transplant rejection reaction (NCT02723786, Phase II, Patent Document 8), type 2 diabetes (NCT01648153, Phase II, Patent Document 8), Behçet's disease (NCT03522662, Phase II, Patent Document 8), Crohn's disease (NCT03681067, Phase II, Patent Document 8), inflammatory bowel disease (NCT01035645, Phase I, Patent Document 8), adult Still's disease (NCT0475237, Phase I, Patent Document 6), multiple myeloma (NCT04671251, Phase I, Patent Document 6) as target diseases. However, there are no tests reported as having no effect, nor are there any that have advanced to Phase III after the test ended. At present, no antibody pharmaceuticals close to being on the market are recognized.

[0010] As described above, for Crohn's disease and inflammatory bowel disease, which are the disease targets of the present invention, although clinical trials have been conducted, they are Phase II trials that examined safety, and no effect on Crohn's disease and inflammatory bowel disease has been shown. Therefore, it is necessary to wait for future reports regarding confirmation of effectiveness. However, regarding the clinical trial of inflammatory bowel disease, although reports on safety and the like have been made after the trial ended in 2012, it has not advanced to Phase III thereafter and is presumed not to be put into practical use.

Means for Solving the Problems

[0011] The present invention relates to a therapeutic agent for inflammatory bowel disease. In particular, it relates to a therapeutic agent for refractory inflammatory bowel disease that shows resistance to existing drugs. (1) A pharmaceutical composition for treating inflammatory bowel disease, characterized by containing, as an active ingredient, an anti-active IL-18 neutralizing antibody, or a Fab, Fab', F(ab')2, single-chain antibody (scFv), disulfide-stabilized V-region fragment (dsFv), miniaturized antibody (Fv-clasp), or a peptide containing a CDR of the antibody. (2) The pharmaceutical composition for treating inflammatory bowel disease according to (1), further containing, as an active ingredient, a TNF-α inhibitor. (3) The pharmaceutical composition for treating inflammatory bowel disease according to (1), which is administered in combination with a pharmaceutical composition containing, as an active ingredient, a TNF-α inhibitor. (4) The TNF-α inhibitor is an anti-TNF-α antibody, or a Fab, Fab', F(ab')2, single-chain antibody (scFv), disulfide-stabilized V-region fragment (dsFv), miniaturized antibody (Fv-clasp), or a peptide containing a CDR of the antibody, in the pharmaceutical composition for treating inflammatory bowel disease according to (2) or (3). (5) A pharmaceutical composition for inflammatory bowel disease, containing, as an active ingredient, a bispecific antibody having a first antigen-binding site that binds to the N-terminal region of active IL-18 and a second antigen-binding site that binds to TNF-α, or a Fab, Fab', F(ab')2, single-chain antibody (scFv), disulfide-stabilized V-region fragment (dsFv), miniaturized antibody (Fv-clasp), or a peptide containing a CDR of the antibody. (6) The inflammatory bowel disease is ulcerative colitis or Crohn's disease, in the pharmaceutical composition for treating inflammatory bowel disease according to any one of (1) to (5). (7) The pharmaceutical composition for treating inflammatory bowel disease according to any one of (1) to (6), which is targeted at existing treatment-resistant inflammatory bowel disease. (8) The heavy chain variable domain of the anti-active IL-18 neutralizing antibody, or the first antigen-binding site that binds to the N-terminal region of active IL-18, includes a CDR1H region consisting of the amino acid sequence GFSLSSSGMG (SEQ ID NO: 1), a CDR2H region consisting of the amino acid sequence IWWDDDK (SEQ ID NO: 2), and a CDR3H region consisting of the amino acid sequence TRTRTYSNFGGGMAY (SEQ ID NO: 3), and the light chain variable domain includes a CDR1L region consisting of the amino acid sequence QSIAHSNGYTY (SEQ ID NO: 4), a CDR2L region consisting of the amino acid sequence KVS (SEQ ID NO: 5), and a CDR3L region consisting of the amino acid sequence VQGSHVPLT (SEQ ID NO: 6). A pharmaceutical composition for the treatment of inflammatory bowel disease according to any one of (1) to (7), characterized in that the heavy chain variable domain is a peptide, or the heavy chain variable domain comprises a CDR1H region consisting of the amino acid sequence of GFSLTSYG (SEQ ID NO: 7), a CDR2H region consisting of the amino acid sequence of IWAGGST (SEQ ID NO: 8), and a CDR3H region consisting of the amino acid sequence of ARESSYDAMDY (SEQ ID NO: 9), and the light chain variable domain is a peptide comprising a CDR1L region consisting of the amino acid sequence of ENVVTY (SEQ ID NO: 10), a CDR2L region consisting of the amino acid sequence of GAS (SEQ ID NO: 11), and a CDR3L region consisting of the amino acid sequence of GQGYSYPYT (SEQ ID NO: 12). (9) A pharmaceutical composition for treating inflammatory bowel disease according to any one of (1) to (7), characterized in that the amino acid sequence of the H chain variable region of the first antigen-binding site that binds to the N-terminal region of the anti-active IL-18 neutralizing antibody or active IL-18 is SEQ ID NO: 13, and the amino acid sequence of the L chain variable region is SEQ ID NO: 14, or the amino acid sequence of the H chain variable region is SEQ ID NO: 15, and the amino acid sequence of the L chain variable region is SEQ ID NO: 16. (10) A method for predetermining whether a target patient with inflammatory bowel disease has the therapeutic effect of any one of the pharmaceutical compositions for the treatment of inflammatory bowel disease described in (1) to (9), characterized in that the amount of active IL-18 expressed in the target patient is measured. (11) A method for treating inflammatory bowel disease, characterized by treating a patient with inflammatory bowel disease using the pharmaceutical composition described in (1), (2), or (5). (12) A method for treating inflammatory bowel disease, characterized by administering to a patient with inflammatory bowel disease the pharmaceutical composition described in (1) and a pharmaceutical composition containing an anti-TNF-α antibody, or a peptide containing Fab, Fab', F(ab')2, single-chain antibody (scFv), disulfide-stabilized V region fragment (dsFv), low molecular weight antibody (Fv-clasp), or CDR as an active ingredient. (13) A method for treating inflammatory bowel disease, characterized by first confirming the effectiveness of treatment with an anti-active IL-18 neutralizing antibody by the method described in (10), and then treating the disease by the method described in (11) or (12). [Brief explanation of the drawing]

[0012] [Figure 1A] This figure shows elevated blood IL-18 levels in Crohn's disease patients who are resistant to existing treatments. [Figure 1B] This image shows the immunohistochemical staining of endoscopic specimens of Crohn's disease patients resistant to existing treatments, specifically focusing on activated IL-18. [Figure 2] A diagram showing the sequence homology between human and mouse IL-18. [Figure 3A] This figure shows the results of examining the epitopes of 5-4.1 antibodies that recognize mouse active IL-18 using the alanine scanning method. [Figure 3B] This figure shows the results of examining the epitopes of the 9-3.1 antibody that recognizes mouse active IL-18 using the alanine scanning method. [Figure 4A] This diagram shows the experimental schedule for inducing colitis with dextran sulfate sodium (DSS) and administering antibodies. [Figure 4B] This figure shows the effect of the 5-4.1 antibody, an anti-active IL-18 neutralizing antibody, on DSS-induced colitis, analyzed by weight change. [Figure 4C] This figure shows the effect of an anti-active IL-18 neutralizing antibody on DSS-induced colitis, analyzed using a colitis activity score. [Figure 5A] This figure shows the results of an investigation into the mechanism of action of the inhibitory effect of administering an anti-IL-18 neutralizing antibody on enteritis, based on the serum concentration of orally administered FITC-dextran. [Figure 5B] This figure shows the results of an analysis of the mechanism of action of the inhibitory effect of administering an anti-active IL-18 neutralizing antibody on enteritis, based on tissue staining of the constituent molecules of tight junctions. [Figure 5C] This figure shows the results of analyzing the mechanism of action of the inhibitory effect of administering an anti-active IL-18 neutralizing antibody on colitis, using PAS staining of goblet cells that secrete intestinal mucus. The number of goblet cells measured is shown on the right. [Figure 6A] This figure, based on weight loss percentage, shows that combined administration of an anti-active IL-18 neutralizing antibody and an anti-TNF-α antibody significantly improves DSS-induced colitis. [Figure 6B] This figure, using an enteritis activity score, shows that co-administration of an anti-active IL-18 neutralizing antibody and an anti-TNF-α antibody significantly improves DSS-induced colitis. [Figure 6C] This figure shows, based on intestinal length, that combined administration of an anti-active IL-18 neutralizing antibody and an anti-TNF-α antibody significantly improves DSS-induced colitis. [Figure 6D] This figure, based on HE staining of colon specimens, shows that combined administration of an anti-active IL-18 neutralizing antibody and an anti-TNF-α antibody significantly improves DSS-induced colitis. [Figure 7A] This figure shows the experimental schedule for inducing enteritis with 2,4,6-trinitrobenzenesulfonic acid (TNBS) and administering antibodies. [Figure 7B] This figure shows the effect of an anti-active IL-18 neutralizing antibody on TNBS-induced colitis, analyzed by weight loss rate. [Figure 7C] This figure shows the effect of an anti-active IL-18 neutralizing antibody on TNBS-induced colitis, analyzed using a colitis activity score. [Figure 7D] This figure shows the effect of an anti-active IL-18 neutralizing antibody on TNBS-induced colitis, analyzed by intestinal length. [Figure 8A] This figure, based on weight loss percentage, shows that combined administration of an anti-active IL-18 neutralizing antibody and an anti-TNF-α antibody significantly improves TNBS-induced colitis. [Figure 8B]This figure, using an enteritis activity score, shows that co-administration of an anti-active IL-18 neutralizing antibody and an anti-TNF-α antibody significantly improves TNBS-induced colitis. [Figure 8C] This figure shows, based on intestinal length, that combined administration of an anti-active IL-18 neutralizing antibody and an anti-TNF-α antibody significantly improves TNBS-induced colitis. [Figure 9A] This figure shows the α-diversity (left) and β-diversity (right) of the gut microbiota in the non-inducible enteritis group, the IgG-administered group, and the 5-4.1 antibody-administered group 9 days after DSS administration. [Figure 9B] Relative abundance at the phylum level of the gut microbiota in the non-inducible enteritis group, the IgG administration group, and the 5-4.1 antibody administration group. [Figure 9C] The relative abundance of gut microbiota at the genus level in the IgG administration group and the 5-4.1 antibody administration group is shown in the bar graph. The bacteria shown represent those that significantly increased in each group. [Figure 9D] The relative abundances of Enterococcus, Staphylococcus, and Lactococcus species, which increased in the enteritis-induced group, are shown. [Figure 10A] Figure showing the epitopes of anti-IL-18 antibodies analyzed by Western blotting. [Figure 10B] Figure showing the results of ELISA analysis of the epitopes of anti-IL-18 antibodies. [Figure 10C] This figure shows the results of an analysis of the effects of 5-4.1 antibodies and anti-IL-18 antibodies on DSS-induced colitis using a colitis activity score. [Figure 10D] This figure shows the results of an analysis of the effects of 5-4.1 antibodies and anti-IL-18 antibodies on DSS-induced colitis, measured by the rate of weight loss. [Figure 11A] This figure shows the results of an analysis of the effects of 5-4.1 antibodies and anti-IL-18 antibodies on TNBS-induced colitis using a colitis activity score. [Figure 11B] This figure shows the results of an analysis of the effects of 5-4.1 antibodies and anti-IL-18 antibodies on TNBS-induced colitis, measured by the rate of weight loss. [Modes for carrying out the invention]

[0013] The inventors of this invention hypothesized that one reason why anti-IL-18 antibody drugs have not been clinically applied is that the antibodies used do not recognize only active IL-18, but also the abundant precursor. Therefore, they conducted an investigation using an antibody that recognizes only active IL-18. Unlike cytokines such as TNF, IL-18 is not regulated at the mRNA level and is abundant in cells as an inactive precursor (pro-IL-18). pro-IL-18 is cleaved by caspase 1 or caspase 4, becoming active and released outside the cell. Therefore, even if an antibody that binds to the precursor protein is used, it does not necessarily lead to the suppression of active IL-18. Furthermore, even if an antibody were to bind to both the active and precursor forms, it would not efficiently suppress the activity of active IL-18 because a large amount of IL-18 precursor is present in cells.

[0014] The IL-18 antibody of the present invention is an antibody that recognizes only active IL-18, that is, an antibody whose epitope is a neoepitope newly formed when the precursor pro-IL-18 is cleaved by caspase 1 / 4. "Epitope" refers to a part of the antigen recognized by the antibody, and means the site on the antigen to which the domain containing the antibody variable region disclosed herein binds. For example, when the antigen is a polypeptide such as IL-18, the antibody may recognize a linear amino acid sequence or a three-dimensional structure, so the epitope can be defined by the amino acid sequence or the structure of the antigen. The neoepitope of IL-18 is a sequence that exists only in active IL-18 cleaved by caspase 1 / 4, and an antibody that recognizes this can be said to be an antibody that recognizes only active IL-18.

[0015] In this invention, the term "antibody" also includes functional fragments of the antibody that exhibit substantially the same antigen specificity as the original antibody, i.e., recognize the same epitope. Functional fragments of antibodies include Fab, Fab', F(ab')2, single-chain antibodies (scFv), disulfide-stabilized V-region fragments (dsFv), low-molecular-weight antibodies (Fv-clasp, Non-Patent Literature 4), or functional fragments of antibodies such as peptides containing CDRs.

[0016] Furthermore, in the embodiments of the present invention, since the N-terminal sequence of active IL-18 differs between humans and mice (see Figure 2), an antibody that recognizes mouse active IL-18 is used. However, it goes without saying that when applying this to humans, it is necessary to use an antibody that recognizes human active IL-18. Examples of antibodies that recognize such human active IL-18 include the 9-10.2 antibody and the 8-4.1 antibody (Patent Document 11).

[0017] Specifically, the heavy chain variable domain includes a CDR1H region consisting of the amino acid sequence GFSLSSSGMG (SEQ ID NO: 1), a CDR2H region consisting of the amino acid sequence IWWDDDK (SEQ ID NO: 2), and a CDR3H region consisting of the amino acid sequence TRTRTYSNFGGGMAY (SEQ ID NO: 3), while the light chain variable domain includes a CDR1L region consisting of the amino acid sequence QSIAHSNGYTY (SEQ ID NO: 4), a CDR2L region consisting of the amino acid sequence KVS (SEQ ID NO: 5), and a CDR3L region consisting of the amino acid sequence VQGSHVPLT (SEQ ID NO: 6). (9-10.2 antibody) Examples include antibodies (8-4.1 antibodies) in which the heavy chain variable domain includes a CDR1H region consisting of the amino acid sequence of GFSLTSYG (SEQ ID NO: 7), a CDR2H region consisting of the amino acid sequence of IWAGGST (SEQ ID NO: 8), and a CDR3H region consisting of the amino acid sequence of ARESSYDAMDY (SEQ ID NO: 9), and the light chain variable domain includes a CDR1L region consisting of the amino acid sequence of ENVVTY (SEQ ID NO: 10), a CDR2L region consisting of the amino acid sequence of GAS (SEQ ID NO: 11), and a CDR3L region consisting of the amino acid sequence of GQGYSYPYT (SEQ ID NO: 12). In addition, antibodies (9-10.2 antibodies) in which the amino acid sequence of the H chain variable region is SEQ ID NO: 13 and the amino acid sequence of the L chain variable region is SEQ ID NO: 14, or antibodies (8-4.1 antibodies) in which the amino acid sequence of the H chain variable region is SEQ ID NO: 15 and the amino acid sequence of the L chain variable region is SEQ ID NO: 16, or antibodies having 80% or more, preferably 90% or more, homology to these antibodies can be used.

[0018] Furthermore, while the 9-10.2 and 8-4.1 antibodies that recognize human active IL-18 are mouse monoclonal antibodies, for clinical application in humans, these monoclonal antibodies can be transformed into human-type chimeric antibodies, humanized CDR-transplanted antibodies, and other humanized antibodies using genetically modified mice, as well as human antibodies produced using genetically modified mice, are also included in the antibodies of this invention. When administered to humans, humanized antibodies and human antibodies have fewer side effects and their therapeutic effects last longer compared to antibodies from non-human animals. In addition, by introducing mutations into the Fc region of IgG1, the binding affinity to C1q and Fc receptors can be reduced, preventing the antibody from being taken up by immune cells such as macrophages, and resulting in an antibody with stronger neutralizing activity. Examples of such mutations include K322A, L234A, L235A (Non-Patent Literature 5), the triple mutation L234F / L235E / P331S (Non-Patent Literature 6), and N297Q (Non-Patent Literature 7, 8). Based on these findings, for example, an antibody (Patent Document 12) may be developed in which an LALA mutation (L234A, L235A) is introduced to suppress its uptake by immune cells such as macrophages.

[0019] Furthermore, as detailed below, studies using a mouse model revealed that combining this drug with an anti-TNF-α antibody can more effectively suppress colitis. For human application, existing antibody drugs such as infliximab, adalimumab, golimumab, and certolizumab pegol can be used as anti-TNF-α antibodies, but the drug is not limited to these. Future anti-TNF-α antibody drugs can also be used.

[0020] Furthermore, since a significant inhibitory effect has been observed when antibodies against IL-18 neoepitopes are used in combination with anti-TNF-α antibodies, any TNF inhibitor may be usable. Examples include soluble TNF-α receptors that bind to TNF-α and inhibit its action, and small molecule compounds that inhibit the action of TNF-α. In addition, bispecific antibodies that bind to active IL-18 and TNF-α, as well as functional antibody fragments that maintain the antigen specificity of bispecific antibodies, can also be used as pharmaceutical compositions for the treatment of inflammatory bowel disease.

[0021] The following data will be presented in detail. In a group of patients with inflammatory bowel disease, we investigated the differences between the treatment-responsive and treatment-resistant groups in response to anti-TNF-α antibody and anti-IL-12 / 23 antibody preparations. The results revealed that IL-18 expression was significantly higher in the treatment-resistant group (Figure 1A, B). Blood IL-18 concentrations were measured using the human IL-18 ELISA kit (MBL) in patients who responded to anti-TNF-α antibody and anti-IL-12 / 23 antibody treatment and in patients who showed treatment resistance (Figure 1A). Blood IL-18 concentrations were significantly higher in the treatment-resistant group compared to the treatment-responsive group (*** indicates p<0.001; the same applies below).

[0022] Colonoscopy specimens from Crohn's disease patients were immunostained using an antibody that recognizes only active IL-18 (9-10.2) (Figure 1B). Specimens obtained from patients resistant to existing treatments showed higher expression of active IL-18 in the intestinal epithelium and stromal tissue compared to the treatment-responding group.

[0023] Given that IL-18 concentrations in the blood were high in the treatment-resistant group, and that active IL-18 was present in colonic samples, we investigated whether inflammatory bowel disease could be suppressed using antibodies that recognize and neutralize active IL-18.

[0024] [Production of antibodies that recognize mouse IL-18 neoepitope] The human inflammatory cytokine IL-18 protein (uniprot:Q14116, SEQ ID NO: 17) has a polypeptide at positions 37-193 that is cleaved by caspases, and this polypeptide functions as active IL-18. Although mouse IL-18 (NP_001344150, SEQ ID NO: 18) and human IL-18 have high homology, the N-terminal sequence that forms the neoepitope of active IL-18 is different (Figure 2, the C-terminal side indicated by the arrow functions as active IL-18. The cleaved end becomes the neoepitope). The mouse IL-18 neoepitope sequence (NFGRLHCTTC, SEQ ID NO: 19) was synthesized by conventional methods by adding cysteine ​​(C) to the C-terminus of the peptide from positions 36-44 of mouse IL-18, which is the cleaved end by caspases.

[0025] As a sensitizing antigen, keyhole-limpet hemocyanin (hereinafter referred to as KLH) was crosslinked to the peptide of SEQ ID NO: 19 using the Imject Maleimide-Activated mcKLH spin Kit (Thermo Scientific), and then mice were immunized according to a standard procedure.

[0026] The same peptide was crosslinked to bovine serum albumin (BSA) using the Imject Maleimide-Activated BSA spin Kit (Thermo Scientific), and screening was performed by ELISA to select and establish hybridomas 5-4.1 and 9-3.1. Antibodies produced by hybridoma 5-4.1 are referred to as 5-4.1 antibodies, and antibodies produced by hybridoma 9-3.1 are referred to as 9-3.1 antibodies.

[0027] Using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Sigma), the isotypes of monoclonal antibodies 5-4.1 and 9-3.1 that recognize active IL-18 produced by hybridomas 5-4.1 and 9-3.1 were identified as IgG1,κ.

[0028] [Epitope analysis of 5-4.1 antibodies and 9-3.1 antibodies] The 5-4.1 and 9-3.1 antibodies were confirmed to recognize and neutralize only mouse active IL-18 by ELISA, immunoprecipitation, and mouse IL-18 function inhibition activity testing. Only the results of the alanine scanning method by ELISA are shown below. As mentioned above, when proteins are cleaved by proteolytic enzymes such as caspases, protein cleavage sites, or neoepitopes, which are not present in normal proteins, are formed. Epitope analysis was performed on the neoepitope peptides used in antibody production by the alanine scanning method. Here, quantitative analysis was performed by ELISA as shown below.

[0029] The N-terminal peptide of the active mouse IL-18 protein (positions 36-44, NGFRLHCTT, SEQ ID NO: 20) and peptides obtained by adding biocitin to the C-terminus of alanine variants of the N-terminal four amino acids (SEQ ID NOs: 21-24) were synthesized according to conventional methods. Each peptide was immobilized on a NeuroAvidin plate via biotin, and recognition sites of 5-4.1 and 9-3.1 antibodies were analyzed by ELISA (Figure 3A, B). To the right of the ELISA plate photograph in Figure 3, the absorbance obtained with a plate reader is shown along with the standard deviation, with the wild-type peptide set to 100.

[0030] Both the 5-4.1 antibody and the 9-3.1 antibody showed less than 10% recognition of the peptide (N36A) and F37A peptide, which are modified peptides in which the first amino acid (asparagine (N) at position 36) of the N-terminal sequence of the active mouse IL-18 protein is replaced with alanine (A). They also showed slightly stronger reactivity to the R39A mutant than to the wild type. On the other hand, for the G38A mutant, the 5-4.1 antibody showed weak binding of about 10% to the wild type peptide, while the 9-3.1 antibody showed reactivity of about 30%.

[0031] Results from the alanine scanning method described above revealed that the peptides from position 36 to 38 are particularly important for recognition by the 5-4.1 antibody and the 9-3.1 antibody. Specifically, it was revealed that both the 5-4.1 antibody and the 9-3.1 antibody recognize NGFRLHCTT (SEQ ID NO: 20) as an epitope and NFG (SEQ ID NO: 25) as a minimum epitope. Next, we investigated whether the 5-4.1 antibody has a therapeutic effect on two types of inflammatory bowel disease model mice (DSS-induced colitis and TNBS-induced colitis model mice).

[0032] [Example 1] [Therapeutic effect of 5-4.1 antibody on a mouse model of DSS-induced colitis] DSS (dextran sulfate sodium)-induced colitis is an acute colitis model that induces damage to intestinal epithelial cells and activation of macrophages, Th1 cells, and Th17 cells by DSS. Eight-week-old C57BL / 6J male mice were divided into two groups: a colitis induction group that was given drinking water mixed with 2.5% DSS, and a non-induction group that was given distilled water. The colitis induction group was further divided into a 5-4.1 antibody administration group and an isotype IgG (InVivoMab mouse IgG1 isotype control BioXCell) administration group (control group), and administered for 7 days (Figure 4A). Six mice were used in each group for the experiment. 5-4.1 antibody or isotype IgG was administered intraperitoneally at a dose of 200 μg each on days 3, 5, and 7 of DSS administration, and dissection was performed on day 9. Colitis activity scores (Non-Patent Literature 9, Figure 4C), measured from body weight (Figure 4B), stool shape, degree of bloody stool, and weight loss rate, were measured daily. In the 5-4.1 antibody-treated group, compared to the control group, significant suppression of weight loss and improvement in the enteritis activity score were observed (** indicates p<0.01, * indicates p<0.05; the same applies below). Autopsy performed on day 9 of DSS administration revealed suppression of intestinal length shortening in the 5-4.1 antibody-treated group, and improvement was also observed in the pathological score (Non-Patent Literature 9), which scores inflammatory cell infiltration into the submucosa and epithelial damage. Based on these results, it was identified that the 5-4.1 antibody improves enteritis in DSS-induced enteritis model mice.

[0033] Possible mechanisms by which DSS induces colitis include disruption of the defense mechanisms of intestinal epithelial cells, excessive activation of immune cells, and disruption of the gut microbiota (dysbiosis). To investigate the effect of 5-4.1 antibodies on intestinal epithelium, mice were given DSS for 7 days, and FITC-labeled 4kDa dextran was orally administered to mice 9 days after the start of colitis induction. Blood was collected 4 hours later, and the concentration of FITC-dextran in the serum was measured (Figure 5A). On 9 days after colitis induction, dextran permeability was increased, and elevated blood dextran concentrations were observed. However, in the 5-4.1 antibody-administered group, blood FITC-dextran concentrations were lower compared to the control group, indicating an improvement in increased permeability.

[0034] Immunohistochemical staining was performed for claudin 1, occludin, and ZO-1, molecules that constitute tight junctions controlling permeability (Figure 5B). Colon tissue specimens obtained from the DSS-free group (control group), the IgG-administered group (where colitis was induced by DSS and then administered isotype IgG), and the 5-4.1 antibody-administered group were immunostained using conventional methods. The primary antibodies used were anti-claudin 1 antibody (Invitrogen, #71-7800), anti-occludin antibody (Invitrogen, #40-4700), and anti-ZO-1 antibody (#61-7300, Thermo Scientific). DSS administration resulted in decreased expression of all of the above molecules constituting tight junctions (DSS + Isotype IgG), but the decreased expression of claudin 1 and occludin was improved by 5-4.1 antibody administration (DSS + 5-4.1 Ab). In addition, PAS staining was performed on colon tissue specimens to observe goblet cells that secrete intestinal mucus. 5-4.1 In the antibody-administered group, the number of goblet cells was significantly increased compared to the isotype IgG-administered group (Figure 5C, right graph).

[0035] These results indicate that the 5-4.1 antibody protects intestinal epithelial function by restoring the reduced expression of molecules constituting tight junctions caused by DSS administration, suppressing increased permeability, and restoring the number of goblet cells. Although data are not shown here, the 5-4.1 antibody-treated group showed a decrease in the expression of inflammatory cytokines such as IFN-γ and TNF-α, and the chemokine CXCL2, compared to the control group.

[0036] [Example 2] [5-4.1 Therapeutic effects of combined use of antibodies and anti-TNF-α antibodies on DSS-induced colitis] Anti-TNF-α antibodies are clinically used as a treatment for IBD. To investigate the combined use of anti-TNF-α antibodies and the effective anti-active IL-18 antibodies, we investigated the therapeutic effect of combining anti-mouse TNF-α antibodies (Invivo Mab anti-mouse TNF-α, BioXCell) with 5-4.1 antibodies on DSS-induced colitis. Participants were divided into four groups: an isotype IgG administration group (control group), a 5-4.1 antibody administration group, an anti-TNF-α antibody administration group, and a group receiving a combination of 5-4.1 and anti-TNF-α antibodies. Colitis was induced using the same protocol for each group, and the therapeutic effect was investigated (Figures 6A-D). Enteritis was induced by DSS in the same manner as in Example 1, and 100 μg each of 5-4.1 antibody and anti-TNF-α antibody were administered intraperitoneally. In the combination therapy group, 100 μg of 5-4.1 antibody and 100 μg of anti-TNF-α antibody were administered intraperitoneally on days 3, 5, and 7 from the start of DSS administration, along with 100 μg of isotype IgG as a control.

[0037] In the 5-4.1 antibody administration group, the rate of weight loss and the enteritis activity score showed a similar level of improvement in enteritis compared to the anti-TNF-α antibody administration group. The combination of 5-4.1 antibody and anti-TNF-α antibody showed a marked improvement in both the rate of weight loss and the enteritis activity score (Figure 6A, B). Regarding shortening of the intestinal tract due to inflammatory bowel disease, the 5-4.1 antibody administration group showed a similar level of improvement as the anti-TNF-α antibody administration group, while the combination group showed a stronger improvement compared to monotherapy (Figure 6C). Pathological examinations also showed a similar level of improvement as the anti-TNF-α antibody administration group, while the combination group showed a stronger improvement compared to monotherapy (Figure 6D). Although not shown here, the combination group significantly suppressed anti-TNF-α production more than the 5-4.1 antibody administration group.

[0038] [Example 3] [Therapeutic effects of 5-4.1 antibodies on a mouse model of TNBS-induced colitis] TNBS (2,4,6-trinitrobenzenesulfonic acid)-induced colitis is an acute colitis model in which Th1 cell activation is induced by rectal administration of TNBS, and is considered a Crohn's disease model. As shown in Figure 7A, TNBS was administered rectally (10 mg / ml, 100 μl) on day 1, and 100 μg each of 5-4.1 antibody or control IgG were administered intraperitoneally on days 1, 2, 3, and 4, and autopsy was performed on day 5. The 5-4.1 antibody-administered group showed a significantly reduced rate of weight loss (Figure 7B) and an improvement in the colitis activity score (Figure 7C) compared to the control group. Upon autopsy, the 5-4.1 antibody-administered group showed suppression of intestinal length shortening (Figure 7D), and pathological improvement was also observed. Based on these results, it was identified that the 5-4.1 antibody improves colitis in TNBS-induced colitis model mice.

[0039] [Example 4] [5-4.1 Therapeutic effects of combined use of antibodies and anti-TNF-α antibodies on TNBS-induced colitis] The therapeutic effect of combining anti-TNF-α antibody and 5-4.1 antibody on TNBS-induced colitis was investigated (Figure 8). Participants were divided into four groups: an IgG administration group (control group), a 5-4.1 antibody administration group, an anti-TNF-α antibody administration group, and a group receiving both 5-4.1 antibody and anti-TNF-α antibody. Colitis was induced using the same protocol as in Example 3, and the therapeutic effect was investigated. Specifically, TNBS was administered on day 1, and 100 μg each of 5-4.1 antibody and anti-TNF-α antibody were administered intraperitoneally on days 1, 2, 3, and 4, either alone or in combination. In the 5-4.1 antibody administration group, a similar level of improvement in colitis was observed in weight loss rate (Figure 8A) and colitis activity score (Figure 8B) compared to the anti-TNF-α antibody administration group. The combination of 5-4.1 antibody and anti-TNF-α antibody showed a significant improvement in both weight loss rate and colitis activity score. Furthermore, regarding intestinal length, the 5-4.1 antibody administration group showed a similar level of suppression of intestinal length shortening compared to the anti-TNF-α antibody administration group, and the combination therapy group showed a remarkable effect, with intestinal length being similar to that of mice that had not undergone TNBS treatment (Figure 8C).

[0040] [Example 5] [5.4.1 Effects of antibodies on the gut microbiota] As shown above, the 5-4.1 antibody administration group showed therapeutic effects against both DSS-induced and TNBS-induced colitis. It is known that the diversity of the gut microbiota is lost in inflammatory bowel disease, so we investigated whether administration of anti-activated IL-18 antibody could improve the gut environment.

[0041] In the same manner as in Example 1, 8-week-old C57BL / 6J male mice were given drinking water contaminated with DSS for 7 days to induce enteritis. 5-4.1 The mice were divided into two groups: an antibody administration group and an isotype IgG administration group (6 mice per group), with a DSS-free group used as a control. Stool samples were collected 9 days after the first DSS administration, and DNA was extracted from the stool samples using the DNeasy PowerSoil Kit (Qiagen). The V3-4 region of the 16S rRNA gene was amplified by 1st and 2nd PCR to create a DNA library, and nucleotide sequence information was obtained using the Illumina next-generation sequencer Miseq. SILVA (Version 38) and QIIME2 were used to analyze the obtained nucleotide sequences. Linear discriminant analysis was used to compare the composition of the gut microbiota between the groups. α-diversity and β-diversity were evaluated using Microbiome Analyst software 36.

[0042] When comparing the control group (non-inducible enteritis), the IgG-administered group (inducible enteritis), and the 5-4.1 antibody-administered group, the Chao-1 index, which indicates α diversity, was significantly lower in the enteritis-inducible group, but no significant difference was observed between the IgG-administered group and the 5-4.1 antibody-administered group (Figure 9A left). In addition, the UniFrac distance, which indicates β diversity, formed different clusters depending on the induction of enteritis, but no significant difference was observed between the IgG-administered group and the 5-4.1 antibody-administered group (Figure 9A right).

[0043] While no significant differences were observed at the phylum level in the composition of the gut microbiota, the enteritis induction group showed a decrease in the relative abundance of Bacteroides and an increase in Firmicutes, while the 5-4.1 antibody administration group tended to suppress these changes in the gut microbiota (Figure 9B). Furthermore, analysis was performed at the genus level (Figure 9C). The relative abundance of gut microbiota at the genus level was analyzed in the IgG administration group (enteritis induction group) and the 5-4.1 antibody administration group, and the bacteria that significantly increased at the genus level in each group are shown. In addition, when comparing the three groups, the relative abundance of Enterococcus, Staphylococcus, and Lactococcus increased in the IgG administration group (enteritis induction group), and this increase was significantly suppressed by the administration of the 5-4.1 antibody (Figure 9D). The Tukey method was used for the comparison of the three groups, and *P<0.05 is indicated.

[0044] [Example 6] [Comparison of the inhibitory effects of 5-4.1 antibodies and anti-mouse IL-18 antibodies on colitis in a mouse model of DSS-induced colitis] We analyzed whether the effect of the antibody against inflammatory bowel disease shown in the examples is specific to the 5-4.1 antibody, which recognizes neoepitopes, or whether the effect can be obtained if there is IL-18 neutralizing activity. The anti-mouse IL-18 antibody (Bio X cell, BE0237) (hereinafter referred to as the anti-IL-18 antibody) is known to have neutralizing activity (Non-patent Literature 10). First, we confirmed whether the anti-IL-18 antibody is a neutralizing antibody that recognizes epitopes other than the IL-18 activation stump (neoepitope).

[0045] Full-length mouse IL-18 or active mouse IL-18 (positions 36-192) protein was prepared and analyzed by Western blot. Active mouse IL-18 was purified as follows: Full-length mouse IL-18 expressed in E. coli was purified, followed by the expression of active caspase-4 in E. coli. 105-377 (This refers to the peptides at positions 105 to 377 of caspase-4.) is mixed with activated mouse IL-18 protein (IL-18 36-192The purified protein was obtained by purification. The N-terminal sequence of the purified protein was determined by the Edman degradation method and, as expected, was NFGR. This corresponds to the transition from asparagine at position 36, which is the N-terminus of active IL-18, to arginine at position 39. Western blot analysis using these proteins showed that the anti-IL-18 antibody recognized both full-length mouse IL-18 and active IL-18 (Figure 10A).

[0046] Furthermore, the region recognized by the anti-IL-18 antibody was confirmed by ELISA. A peptide (NFGRLHCTTC, SEQ ID NO: 19) was created by adding cysteine ​​to the C-terminus of the N-terminal sequence peptide (positions 36-44) of the active mouse IL-18 protein for crosslinking (NFGRLHCTTC). BSA was crosslinked to this peptide using the Imject Maleimide-Activated BSA spin Kit, and the recognition sites (epitopes) of the anti-IL-18 antibody and the 5-4.1 antibody were analyzed by ELISA on a plate (Figure 10B). The graph on the right shows the absorbance values ​​obtained with a plate reader, along with their mean ± standard deviation. ** indicates a P value < 0.005, and ns indicates no significant difference (P = 0.810). The anti-IL-18 antibody did not recognize the N-terminal sequence peptide, which is a neoepitope due to activation, at all. As the above results show, the 5-4.1 antibody recognizes the mouse IL-18 active fragment peptide, but the anti-IL-18 antibody does not recognize the mouse IL-18 active fragment peptide. In other words, the anti-IL-18 antibody is a neutralizing antibody that recognizes regions other than the neoepitope.

[0047] Eight-week-old C57BL / 6J male mice were divided into two groups: a 5-4.1 antibody group and an anti-mouse IL-18 antibody group (5 mice per group). They were administered drinking water mixed with 2.5% DSS for 5 days. 200 μg of either 5-4.1 antibody or anti-mouse IL-18 antibody was administered intraperitoneally on days 3, 5, and 7, and dissection was performed on day 9. Enteritis activity score (Figure 10C), measured from stool shape, degree of bloody stool, and weight loss rate, and body weight (Figure 10D) were measured daily. The 5-4.1 antibody group showed significantly reduced weight loss and improved enteritis activity score compared to the anti-mouse IL-18 antibody group (** indicates P<0.01, * indicates P<0.05). These results indicate that the 5-4.1 antibody significantly improves enteritis in DSS-induced enteritis model mice compared to the anti-mouse IL-18 antibody.

[0048] [Example 7] [Comparison of the inhibitory effects of 5-4.1 antibodies and anti-mouse IL-18 antibodies on enteritis in a mouse model of TNBS-induced enteritis] In a TNBS-induced colitis mouse model, we analyzed whether there was a difference in the therapeutic effects of 5-4.1 antibody and anti-mouse IL-18 antibody. Using 8-week-old C57BL / 6J male mice, TNBS was administered rectally on Day 1, and 100 μg each of 5-4.1 antibody or anti-mouse IL-18 antibody was administered intraperitoneally on Days 1, 2, 3, and 4 (5 mice in each group). Colitis activity score (Figure 11A), measured from stool shape, degree of bloody stool, and weight loss rate, and body weight (Figure 11B) were measured daily (* indicates P<0.05). The 5-4.1 antibody group showed significantly suppressed weight loss and improved colitis activity score compared to the anti-mouse IL-18 antibody group. These results indicate that 5-4.1 antibody significantly improves colitis in TNBS-induced colitis model mice compared to anti-mouse IL-18 antibody.

[0049] As demonstrated in the above examples, administration of the 5-4.1 antibody improved colitis in two types of IBD model mice. Furthermore, the effect of the 5-4.1 antibody was significantly superior to that of anti-IL-18 antibodies that bind to and neutralize substances other than neoepitopes. Currently, there are no IL-18 suppression therapies that have reached clinical application, and antibodies specific to active IL-18 could become novel therapeutic agents for refractory cases, which are a major clinical problem. Moreover, since a more synergistic effect in improving colitis was observed when used in combination with existing anti-TNF-α antibodies, combination therapy with anti-active IL-18 antibodies and anti-TNF-α antibodies can be expected to be a novel treatment for refractory cases.

[0050] IBD presents with diverse pathologies involving multiple factors, requiring treatment selection tailored to each individual's condition. Identifying patient groups with high IL-18 expression will lead to the identification of patient groups where IL-18 plays a central role in the disease's pathology, and to the application of treatments appropriate to those conditions. Specifically, when resistance to existing treatments is observed, checking for IL-18 expression, particularly active IL-18 expression, and confirming high IL-18 expression may yield therapeutic effects through treatment with an anti-active IL-18 antibody drug, or a combination of an anti-active IL-18 antibody drug and an anti-TNF-α antibody drug. Furthermore, when resistance to 5-aminosalicylic acid preparations, immunomodulators such as azathioprine, and corticosteroids is observed, checking for IL-18 expression and confirming high expression may yield even greater efficacy through the administration of an anti-active IL-18 antibody drug.

Claims

1. For inflammatory bowel disease resistant to anti-TNF-α antibody and / or anti-IL-12 / 23 antibody, Antiactive IL-18 neutralizing antibody, or Fab, Fab', F(ab') of said antibody ２ A pharmaceutical composition for the treatment of inflammatory bowel disease, characterized by containing a single-chain antibody (scFv), a disulfide-stabilized V region fragment (dsFv), or a low-molecular-weight antibody (Fv-clasp) as an active ingredient.

2. Furthermore, the pharmaceutical composition for treating inflammatory bowel disease according to claim 1 further comprises a TNF-α inhibitor as an active ingredient.

3. The pharmaceutical composition for treating inflammatory bowel disease according to claim 1, which is administered in combination with a pharmaceutical composition containing a TNF-α inhibitor as an active ingredient.

4. The aforementioned TNF-α inhibitor Anti-TNF-α antibody, or Fab, Fab', F(ab') of said antibody ２ A pharmaceutical composition for treating inflammatory bowel disease according to claim 2 or 3, wherein the antibody is a single-chain antibody (scFv), a disulfide-stabilized V region fragment (dsFv), or a low-molecular-weight antibody (Fv-clasp).

5. For inflammatory bowel disease resistant to anti-TNF-α antibody and / or anti-IL-12 / 23 antibody, A bispecific antibody having a first antigen-binding site that binds to the N-terminal region of active IL-18 and a second antigen-binding site that binds to TNF-α, or Fab, Fab', F(ab') of said antibody ２ A pharmaceutical composition for inflammatory bowel disease comprising a single-chain antibody (scFv), a disulfide-stabilized V-region fragment (dsFv), or a low-molecular-weight antibody (Fv-clasp) as an active ingredient.

6. A pharmaceutical composition for treating inflammatory bowel disease according to any one of claims 1 to 5, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

7. The heavy chain variable domain of the anti-active IL-18 neutralizing antibody, or the first antigen-binding site that binds to the N-terminal region of active IL-18, includes a CDR1H region consisting of the amino acid sequence GFSLSSSGMG (SEQ ID NO: 1), a CDR2H region consisting of the amino acid sequence IWWDDK (SEQ ID NO: 2), and a CDR3H region consisting of the amino acid sequence TRTRTYSNFGGGGMAY (SEQ ID NO: 3). The light chain variable domain is a peptide comprising a CDR1L region consisting of the amino acid sequence QSIAHSNGYTY (SEQ ID NO: 4), a CDR2L region consisting of the amino acid sequence KVS (SEQ ID NO: 5), and a CDR3L region consisting of the amino acid sequence VQGSHVPLT (SEQ ID NO: 6), or The heavy chain variable domain includes a CDR1H region consisting of the amino acid sequence of GFSLTSYG (SEQ ID NO: 7), a CDR2H region consisting of the amino acid sequence of IWAGGST (SEQ ID NO: 8), and a CDR3H region consisting of the amino acid sequence of ARESSYDAMDY (SEQ ID NO: 9). A pharmaceutical composition for treating inflammatory bowel disease according to any one of claims 1 to 6, characterized in that the light chain variable domain is a peptide comprising a CDR1L region consisting of the amino acid sequence of ENVVTY (SEQ ID NO: 10), a CDR2L region consisting of the amino acid sequence of GAS (SEQ ID NO: 11), and a CDR3L region consisting of the amino acid sequence of GQGYSYPYT (SEQ ID NO: 12).

8. A pharmaceutical composition for treating inflammatory bowel disease according to any one of claims 1 to 6, characterized in that the amino acid sequence of the H chain variable region of the antigen-binding site that binds to the N-terminal region of the anti-active IL-18 neutralizing antibody or active IL-18 is SEQ ID NO: 13, and the amino acid sequence of the L chain variable region is SEQ ID NO: 14, or the amino acid sequence of the H chain variable region is SEQ ID NO: 15, and the amino acid sequence of the L chain variable region is SEQ ID NO:

16.

9. A method for determining in advance whether a target patient with inflammatory bowel disease will have the therapeutic effect of the pharmaceutical composition for treating inflammatory bowel disease described in any one of claims 1 to 8, A method for predicting therapeutic effect, characterized by measuring the expression level of the active IL-18 of the target subject.

Images