Inflammatory bowel disease testing method

Abstract

Provided is an inflammatory bowel disease testing method. This inflammatory bowel disease testing method comprises (1) a step for detecting biomarkers in a biological sample collected from a subject. The biomarkers include: proHp and LRG; or proHp and CRP.

Description

Method for examining inflammatory bowel disease

[0001] The present invention relates to a method for examining inflammatory bowel disease and the like.

[0002] Regarding inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, although the treatment results have been improved by the emergence of therapeutic drugs such as biological agents, the efficacy rate is still about 60%, and many cases follow a refractory course, resulting in irreversible damage, dysfunction, and inflammatory carcinogenesis in the intestinal tract.

[0003] For improving the long-term prognosis of patients, monitoring the activity of inflammation by gastrointestinal endoscopy is important, but it is an invasive examination, and a simple and minimally invasive examination that can substitute for endoscopy is desired.

[0004] There is no scientific criterion for how to select molecular target therapeutic drugs including biological agents, and in the guidelines, the selection of drugs is listed side by side, and optimizing and individualizing treatment have become major issues.

[0005] In addition to conventional biomarkers such as C-reactive protein (CRP) and erythrocyte sedimentation rate, several new biomarkers for monitoring the activity of inflammation using blood, feces, and urine have been developed and clinically applied (leucine-rich α2 glycoprotein (LRG) (Patent Document 1), fecal calprotectin, urinary prostaglandin E major metabolite), but these existing biomarkers are not sufficient in terms of accuracy, simplicity, disease specificity, etc., and no biomarker useful for drug selection has been found.

[0006] Patent No. 5246709

[0007] An object of the present invention is to provide a method for examining inflammatory bowel disease.

[0008] In view of the above problems, the inventors diligently conducted research and found that the above problems can be solved if the method for examining inflammatory bowel disease includes (1) a step of detecting a biomarker in a biological sample taken from a subject, wherein the biomarker includes proHp and LRG, or proHp and CRP. Based on this finding, the inventors furthered their research and completed the present invention. That is, the present invention encompasses the following aspects.

[0009] Item 1. (1) A method for testing for inflammatory bowel disease, comprising the step of detecting a biomarker in a biological sample taken from a subject, wherein the biomarker includes proHp and LRG, or proHp and CRP.

[0010] Item 2. The method according to item 1, wherein the biomarkers include proHp and LRG.

[0011] Item 3. The method according to item 1, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

[0012] Item 4. The method according to item 1, wherein the biological sample is at least one selected from the group consisting of blood, serum, and plasma.

[0013] Item 5. The method according to item 1, wherein the subject is a human.

[0014] Item 6. The method according to Item 1, wherein the examination for inflammatory bowel disease includes determining at least one of the following: the presence of inflammatory bowel disease, the severity of inflammatory bowel disease, and the therapeutic effect of drugs for treating inflammatory bowel disease.

[0015] Item 7. The method according to any one of items 1 to 6, further comprising (2) determining, based on the amount or concentration of the biomarker detected in step (1) or an index value calculated based on the amount or concentration, at least one selected from the group consisting of the prevalence of inflammatory bowel disease, the severity of inflammatory bowel disease, and the therapeutic effect of a drug for treating inflammatory bowel disease in the subject.

[0016] Item 8. Step (2) comprises: (2a) determining that the subject has inflammatory bowel disease and / or has a high severity of inflammatory bowel disease if the amount or concentration of the biomarker detected in step (1) or the index value calculated based on the amount or concentration is greater than or equal to a cutoff value; (2b) determining that the subject does not have inflammatory bowel disease and / or has a low severity of inflammatory bowel disease if the amount or concentration of the biomarker detected in step (1) or the index value calculated based on the amount or concentration is less than or equal to a cutoff value; (2c) determining that the subject has a high therapeutic effect of a Th17 inhibitor, a low therapeutic effect of a TNF-α inhibitor, a low therapeutic effect of an IL-12 / 23p40 inhibitor, a high therapeutic effect of a CN / JAK inhibitor, or a high therapeutic effect of a lymphocyte migration inhibitor (e.g., an anti-integrin inhibitor). (2d) A step in which, if the amount or concentration of proHp detected in step (1) or an index value calculated based on the amount or concentration is less than or equal to a cutoff value, the subject is determined to have a low therapeutic effect with Th17 inhibitors, a high therapeutic effect with TNF-α inhibitors, a high therapeutic effect with IL-12 / 23p40 inhibitors, a low therapeutic effect with CN / JAK inhibitors, or a low therapeutic effect with lymphocyte migration inhibitors (e.g., anti-integrin inhibitors); (2e) A step in which, if the amount or concentration of LRG and / or CRP detected in step (1) or an index value calculated based on the amount or concentration is greater than or equal to a cutoff value, the subject is determined to have a high therapeutic effect with TNF-α inhibitors or an IL-12 / 23p40 inhibitor; and The method according to claim 7, comprising at least one selected from the group consisting of (2f) determining that the subject has a low therapeutic effect on a TNF-α inhibitor or an IL-12 / 23p40 inhibitor when the amount or concentration of LRG and / or CRP detected in step (1) or an index value calculated based on the amount or concentration is less than or equal to a cutoff value.

[0017] Item 9. A diagnostic reagent for use in any of the methods described in items 1 to 6, comprising at least one selected from the group consisting of proHp-binding molecules, LRG-binding molecules, and CRP-binding molecules.

[0018] Item 10. The diagnostic agent described in Item 9, which is at least one companion diagnostic agent selected from the group consisting of Th17 inhibitors, TNF-α inhibitors, IL-12 / 23p40 inhibitors, CN / JAK inhibitors, and lymphocyte migration inhibitors (e.g., anti-integrin inhibitors).

[0019] Item 11. (X) A method for determining the therapeutic effect of a therapeutic agent for inflammatory bowel disease, comprising the step of detecting a biomarker in a biological sample taken from a subject, wherein the biomarker includes proHp and / or LRG.

[0020] Item 12. A diagnostic reagent for use in the method described in Item 11, comprising at least one selected from the group consisting of proHp-binding molecules and LRG-binding molecules.

[0021] Item 13. The diagnostic agent according to item 12, which is at least one companion diagnostic agent selected from the group consisting of Th17 inhibitors, TNF-α inhibitors, IL-12 / 23p40 inhibitors, CN / JAK inhibitors, and lymphocyte migration inhibitors (e.g., anti-integrin inhibitors).

[0022] According to the present invention, it is possible to provide a method for examining inflammatory bowel disease, a diagnostic agent for inflammatory bowel disease, a companion diagnostic agent for inflammatory bowel disease, and the like.

[0023] The measurement results for each inflammatory marker are shown (Test Example 1-1). The disease of the patient is shown on the left side of the graph, and the inflammatory markers are shown at the top of the graph. The horizontal axis shows the severity, and the vertical axis shows the median of the distribution when the secretion amount distribution is transformed using the fourth root. The results of principal component analysis of the correlation between the three inflammatory markers are shown (Test Example 1-3). The disease of the patient is shown in the upper left of the graph. The black arrows show biplots of factor loadings for each serum biomarker. The relationship between index values ​​(horizontal axis) for the secretion amount of LRG (left graph) and proHp (right graph) and the efficacy (vertical axis) of each drug (legend) is shown (Test Example 1-4). HP gene expression graphs quantified by Real-time RT-PCR after stimulating Caco-2 and THP-1 with each cytokine (20 ng / mL) for 24 hours are shown. (N=3 each, Tukey-krumer HSD test, MEAN+SD, ***P<0.001). The results of Western blot analysis of the culture supernatants of Caco-2 and THP-1 cells stimulated with each cytokine (20 ng / mL) for 24 hours are shown. The 10⁻⁷G values ​​of the culture supernatants of Caco-2 and THP-1 cells stimulated with each cytokine (20 ng / mL) for 24 hours are shown (N=3 each, Tukey-Krumer HSD test, MEAN+SD, *P<0.05). The results of Western blot analysis of phosphorylated STAT3 antibodies on cell lysates of Caco-2 cells stimulated with each cytokine (20 ng / mL) for 24 hours are shown. The results of Western blot analysis of phosphorylated STAT3 antibodies on cell lysates of Caco-2 cells stimulated with IL-22 (20 ng / mL) in the presence of a STAT3 inhibitor (10 ng / mL) for 24 hours are shown. Linear prediction of treatment efficacy by logistic regression analysis is shown (Study Example 3). Black circles indicate non-remission patients, and white circles indicate remission patients. The integrated mapping of the linear predictions in Figure 9 is shown (Study Example 3). Based on the most effective treatment predicted by LOOCV, patients were classified into those who received the most effective treatment (matched patients) and those who did not (unmatched patients). The results for each category of patients are plotted on the integrated mapping in Figure 10. Black circles indicate patients in non-remission, and white circles indicate patients in remission.Figure 11 shows the percentage of patients who achieved remission with each drug, for both matched and unmatched patients.

[0024] In this specification, the terms “contains” and “includes” include the concepts of “contains,” “includes,” “substantially consist of,” and “consist solely of.”

[0025] 1. Method for Testing Inflammatory Bowel Disease In one aspect of the present invention, the present invention relates to a method for testing inflammatory bowel disease (sometimes referred to as "the testing method of the present invention") comprising the step of (1) detecting a biomarker (the biomarker of the present invention) in a biological sample taken from a subject, wherein the biomarker includes proHp and LRG, or proHp and CRP. The present invention will be described below.

[0026] 1-1. Process (1) The inflammatory bowel disease to be examined is not particularly limited, and examples include ulcerative colitis and Crohn's disease. In one embodiment of the present invention, the inflammatory bowel disease is ulcerative colitis and / or Crohn's disease. Inflammatory bowel disease encompasses all classes, grades, and stages of inflammatory bowel disease in various classification criteria regarding severity, progression, and condition. Severity is preferably endoscopic activity, and in the case of ulcerative colitis, for example, the severity is indicated by the MES (Mayo Endoscopic Subscore) endoscopic score (4 levels (0-3) from normal to severe), and in the case of Crohn's disease, for example, the severity is indicated by the CDEIS (Crohn's Disease Endoscopic Index of Severity) endoscopic score (less than 3 is Inactive (normal), 3 to less than 7 is Mild (mild), 7 to less than 16 is Moderate (moderate), and 16 or more is Severe (severe)).

[0027] The subject is the organism targeted by the testing method of the present invention, and the species of the organism is not particularly limited. Examples of subject species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, with humans being preferred.

[0028] The condition of the subject is not particularly restricted. Examples of subjects include those whose status as having inflammatory bowel disease is unknown, those who have already been determined to have inflammatory bowel disease by another method, those who have already been determined to not have inflammatory bowel disease by another method, and those who are currently undergoing treatment for inflammatory bowel disease.

[0029] The biological sample is not particularly limited as long as it can contain the biomarker of the present invention. Examples of biological samples include body fluids and tissues (e.g., digestive tract tissues such as small intestine tissue and large intestine tissue). Among these, body fluids are preferred from the viewpoint of ease of collection and minimal invasiveness.

[0030] Body fluids are not particularly limited. Examples of body fluids include blood, ascites, cerebrospinal fluid, saliva, synovial fluid, urine, tissue fluid, sweat, tears, sputum, nasal secretions, and exhaled breath, with blood being preferred.

[0031] A biological sample may be a body fluid or tissue itself taken from a living organism, or it may be derived from these (a sample prepared from these). Examples of such samples include those obtained through processes such as the removal of certain components from body fluids, or the concentration and purification of proteins contained in body fluids. Examples of samples derived from body fluids include serum and plasma.

[0032] Serum is the portion of blood from which blood cells and certain blood clotting factors have been removed, and can be obtained, for example, as the supernatant after blood has been allowed to clot. Plasma is the portion of blood from which blood cells have been removed, and can be obtained, for example, as the supernatant after centrifugation under conditions that do not allow blood to clot.

[0033] Examples of biological samples include blood, serum, and plasma.

[0034] Biological samples may be used individually or in combination of two or more types.

[0035] In step (1), a biological sample is used as the test sample, and the biomarker of the present invention contained in the test sample, namely a biomarker containing both proHp and LRG, or both proHp and CRP, is detected.

[0036] proHp (prohaptoglobin) is a known protein, and in humans, for example, it is a precursor to the protein (haptoglobin) with UniProt Number: 3240. The amino acid sequences of haptoglobin and prohaptoglobin can be determined based on known genetic information (NCBI Gen ID: P00738). The protein to be detected in step (1) includes those with variations observed between individuals.

[0037] Human prohaptoglobin is a heterodimer in which two subunits, an α-chain and a β-chain, are linked, and is a precursor of human haptoglobin. Human prohaptoglobin is cleaved into α-chain and β-chain by the serine protease activity of complement C1r subcomponent-like protein (C1RL), and the two chains are linked via disulfide bonds to form human haptoglobin. There are two types of α-chains in human haptoglobin (Hpt): α1-1 ((α1β)2) and Hpt2-1 ((α1β)). m (α2β) n ), and HPT2-2 type ((α2β) n They are classified into three types. m and n are integers of 1 or more, and may be the same or different. The β-chains of the three types are the same. In this invention, the HPT2-2 type is preferred.

[0038] LRG (Leucine-Rich Alpha-2 Glycoprotein) is a known protein, and in humans, for example, it is the protein with UniProt Number: P02750. The amino acid sequence of this protein can be determined based on known genetic information (NCBI Gen ID: 116844). The protein to be detected in step (1) includes those with variations found between individuals.

[0039] CRP (C-reactive protein) is a known protein, and in humans, for example, it is the protein with UniProt Number: P02741. The amino acid sequence of this protein can be determined based on known genetic information (NCBI Gen ID: 1401). The protein targeted for detection in step (1) includes those with variations observed between individuals.

[0040] In the step of detecting the biomarker of the present invention, the method for detecting the target protein (preferably a method for measuring the amount or concentration of the target protein) is not particularly limited as long as it is a method capable of detecting the target protein. Examples of such methods include immunoassays. Immunoassays can be widely employed, regardless of whether they are direct, indirect, homogeneous, heterogeneous, competitive, or non-competitive methods. More specifically, examples of immunoassays include ELISA (e.g., direct, indirect, sandwich, competitive methods), radioimmunoassay (RIA), immunoradiometric assay (IRMA), enzyme immunoassay (EIA), sandwich EIA, immunochromatography, Western blotting, immunoprecipitation, slot or dot blotting assays, immunohistochemistry, fluorescence immunoassays, immunoassays using avidin-biotin or streptavidin-biotin systems, and immunoassays using surface plasmon resonance (SPR). By immunoassay, specifically, a target protein can be detected by directly or indirectly contacting a target protein-binding molecule bound to the target protein with a labeled antibody, and quantifying the signal derived from the label of the bound labeled antibody. The labeled antibody used in this process, or the antibody mediating the connection between the labeled antibody and the target protein-binding molecule or target protein, is not particularly limited; for example, antibodies against the constant region of an antibody, anti-idiotype antibodies, etc., can be used.

[0041] The detection method may be employed as a single method or as a combination of two or more methods.

[0042] The type of label in the labeling agent (such as a labeled antibody, etc.) used when detecting the target protein is not particularly limited. Examples of labels include fluorescent substances, luminescent substances, dyes, enzymes, gold colloids, radioisotopes, and the like. Among these, enzyme labels such as peroxidase and alkaline phosphatase are preferable from the viewpoints of safety, economy, detection sensitivity, and the like.

[0043] In one aspect, step (1) preferably includes (1a) a step of contacting the target protein-binding molecule with the biological sample, and (1b) a step of measuring the amount or concentration of the target protein bound to the target protein-binding molecule.

[0044] The target protein-binding molecule is not particularly limited as long as it can selectively (specifically) recognize the target protein. Here, "selectively (specifically) recognize" means, for example, that the target protein can be specifically detected in Western blotting or ELISA, but is not limited thereto, and as long as those skilled in the art can determine that the detected substance is derived from the target protein.

[0045] The target protein-binding molecule includes, for example, molecules containing a part of the above antibodies having antigen-binding properties, such as polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, or fragments generated by Fab fragments or Fab expression libraries. The target protein-binding molecule having antigen-binding properties against a polypeptide consisting of at least continuously, usually 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids among the amino acid sequence of the target protein is also included in the target protein-binding molecule of the present invention.

[0046] Methods for producing these target protein-binding molecules are already well-known, and the target protein-binding molecules of the present invention can also be produced according to these conventional methods (Current protocols in Molecular Biology, Chapter 11.12 - 11.13 (2000)). Specifically, when the target protein-binding molecule of the present invention is a polyclonal antibody, it can be obtained from the serum of an immunized animal according to a conventional method by using a target protein expressed and purified in Escherichia coli or the like according to a conventional method, or by synthesizing an oligopeptide having a partial amino acid sequence of the target protein according to a conventional method and immunizing a non-human animal such as a rabbit. On the other hand, in the case of a monoclonal antibody, it can be obtained from hybridoma cells prepared by cell fusion of spleen cells and myeloma cells obtained by immunizing a non-human animal such as a mouse with a target protein expressed and purified in Escherichia coli or the like according to a conventional method, or an oligopeptide having a partial amino acid sequence of the target protein (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4 - 11.11).

[0047] The target protein used as an immunizing antigen for producing the target protein-binding molecule can be obtained by operations such as DNA cloning, construction of each plasmid, transfection into a host, culturing of the transformant, and recovery of the protein from the culture, based on known gene sequence information. These operations can be carried out according to methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, D.M. Glover, IRL PRESS (1985), etc.).

[0048] Specifically, a recombinant DNA (expression vector) is created that allows the gene encoding the target protein to be expressed in a desired host cell. This is then introduced into the host cell to transform it, and the transformed cells are cultured. The target protein is then recovered from the resulting culture, thereby obtaining a protein that serves as an immune antigen for the production of the target protein-binding molecule of the present invention. Alternatively, a partial peptide of the target protein can be produced by general chemical synthesis methods (peptide synthesis) according to known gene sequence information.

[0049] Furthermore, the target protein-binding molecule of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of the target protein. The oligo(poly)peptide used for the production of such a target protein-binding molecule does not need to have functional biological activity, but it is desirable that it has immunogenic properties similar to those of the target protein. Preferably, an example of an oligo(poly)peptide having these immunogenic properties and consisting of at least 8 consecutive amino acids, preferably 15 amino acids, and more preferably 20 amino acids, in the amino acid sequence of the target protein can be given.

[0050] The production of target protein-binding molecules for such oligo(poly)peptides can also be carried out by enhancing the immunological response using various adjuvants depending on the host. Such adjuvants include, but are not limited to, Freund's adjuvants, mineral gels such as aluminum hydroxide, as well as surfactants such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol, and human adjuvants such as BCG (Bacillus calmette-Guéran) and Corynebacterium parvum.

[0051] The mode of contact in step (1a) is not particularly limited, and an appropriate mode can be selected depending on the type of detection method for the target protein described above (e.g., various immunoassays). Examples of contact modes include a mode in which only the protein (antigen) in the biological sample and the target protein-binding molecule are immobilized on the solid phase and brought into contact, and a mode in which neither of these are immobilized on the solid phase and brought into contact. Among these, from the viewpoint of efficiency, a mode in which at least one is immobilized on the solid phase and brought into contact is preferred. In the mode of contact in step (1a), when at least one is immobilized on the solid phase, it is preferable to wash the solid phase after immobilization.

[0052] The solid phase is not particularly limited as long as it can immobilize the target protein or the target protein-binding molecule. Examples of such solid phases include plates, slides, and membranes mainly composed of polystyrene, glass, or nitrocellulose. The solid phase may also be coated with a component that facilitates the immobilization of the target protein or the target protein-binding molecule, such as a highly reactive compound (e.g., a compound having a highly reactive group, or a gold colloid). Compounds having easily reactive groups include compounds having groups that can form covalent bonds with proteins, such as (1H-imidazole-1-yl)carbonyl groups, succinimidyloxycarbonyl groups, epoxy groups, aldehyde groups, amino groups, thiol groups, carboxyl groups, azide groups, cyano groups, active ester groups (1H-benzotriazole-1-yloxycarbonyl groups, pentafluorophenyloxycarbonyl groups, p-nitrophenyloxycarbonyl groups, etc.), or halogenated carbonyl groups (carbonyl chloride groups, carbonyl fluoride groups, carbonyl bromide groups, carbonyl iodide groups, etc.).

[0053] Examples of compounds having easily reactive groups include epoxysilanes and polylysine.

[0054] The solid phase is preferably blocked using bovine serum albumin (BSA) buffer or the like.

[0055] The method of measuring the amount or concentration of the target protein in step (1b) is not particularly limited, and an appropriate method can be selected depending on the type of detection method for the target protein described above (e.g., various immunoassays). The measurement can be carried out, for example, by quantifying the signal derived from the label of the label used. More specifically, in this method, the measurement can be carried out by contacting a labeled antibody with a complex of the target protein and a target protein-binding molecule, and quantifying the signal derived from the label of the bound labeled antibody.

[0056] One example of this method is the ELISA method. The ELISA method allows for the quantification of the concentration of a target protein by creating a standard curve using a standard antibody. Standard antibodies can be prepared from commercially available antibodies, for example, by affinity purification using biotin-labeled antigen and streptavidin-agarose resin. First, the wells of a suitable ELISA plate are coated with antigen or antibody, and then blocked with BSA buffer. Next, standard antibodies or samples of each concentration are added to the wells and allowed to stand for a certain period. After washing the wells, peroxidase-labeled secondary antibodies are added to the wells and allowed to stand for a certain period. Then, the wells are washed again, peroxidase substrate is added to the wells and allowed to stand for a certain period to develop color, and after adding a reaction stop solution such as sulfuric acid, the absorbance is measured using a plate reader. A standard curve is created based on the concentrations of the standard antibodies and their absorbances. Based on this standard curve, the concentration of the target protein in the sample is quantified.

[0057] Based on the obtained signal intensity, the amount of the target protein can be calculated. For example, in the case of a non-competitive method, the obtained signal intensity can be used directly as the amount of the target protein. Alternatively, in the case of a competitive method, the obtained signal intensity and the amount of the target protein are inversely proportional, so the amount of the target protein can be calculated from the obtained signal intensity based on this relationship.

[0058] Furthermore, the concentration of the target protein can be calculated by dividing the amount of the target protein by the amount of the biological sample or the amount of components within the biological sample (for example, the total amount of protein).

[0059] The testing method of the present invention, including step (1), can provide the amount and / or concentration of the biomarker of the present invention, which is a testing indicator for inflammatory bowel disease, thereby assisting in the testing of inflammatory bowel disease and the like.

[0060] The examination for inflammatory bowel disease may include at least one determination selected from the group consisting of the presence of inflammatory bowel disease, the severity of inflammatory bowel disease, and the therapeutic effect of inflammatory bowel disease medications. In one embodiment, information for such determination may be provided.

[0061] In determining whether or not a subject has inflammatory bowel disease, it is possible to determine whether or not the subject has inflammatory bowel disease, and whether or not the subject is likely to have inflammatory bowel disease.

[0062] In determining the severity of inflammatory bowel disease, it is possible to determine whether the subject has a high or low severity of inflammatory bowel disease, or whether the subject is likely to have a high or low severity of inflammatory bowel disease.

[0063] In evaluating the therapeutic effects of drugs for inflammatory bowel disease, it is possible to predict the therapeutic effect when a subject is administered a particular drug. For example, it is possible to determine whether a subject has a high or low therapeutic effect from a particular drug, or whether there is a high probability that a particular drug will have a high or low therapeutic effect.

[0064] The severity of inflammatory bowel disease is preferably measured by endoscopic activity. For example, in the case of ulcerative colitis, the severity is indicated by the MES (Mayo Endoscopic Subscore) endoscopic score (4 levels from normal to severe (0-3)), and in the case of Crohn's disease, the severity is indicated by the CDEIS (Crohn's Disease Endoscopic Index of Severity) endoscopic score (less than 3 is Inactive (normal), 3 to less than 7 is Mild (mild), 7 to less than 16 is Moderate (moderate), and 16 or more is Severe (severe)).

[0065] Therapies for treating inflammatory bowel disease are particularly molecularly targeted drugs. Examples of such therapies include Th17 inhibitors, TNF-α inhibitors, IL-12 / 23p40 inhibitors, CN / JAK inhibitors, and lymphocyte migration inhibitors (e.g., anti-integrin inhibitors). CN inhibitors include calcineurin inhibitors (e.g., cyclosporine, tacrolimus, etc.).

[0066] Th17 inhibitors can be, for example, inhibitors of differentiation into Th17 cells or inhibitors of Th17-producing cytokines. Examples of Th17 differentiation inhibitors include IL-6 inhibitors, IL-23 inhibitors, and TNF inhibitors, while examples of Th17-producing cytokine inhibitors include IL-22 inhibitors, IL-21 inhibitors, and IL-17 inhibitors.

[0067] Therapeutic agents for inflammatory bowel disease can include, for example, antibodies (especially neutralizing antibodies), (poly)peptides, and small molecule compounds (e.g., molecular weights of 50-2000 or 100-1000).

[0068] More specifically, examples of medications used to treat inflammatory bowel disease include 5-ASA preparations, steroids (prednisolone, budesonide), thiopurine preparations (azathioprine), calcineurin inhibitors (tacrolimus, cyclosporine), anti-TNF-α preparations (infliximab, adalimumab, golimumab), anti-integrin inhibitors (vedolizumab, carotegrast), anti-IL-12 / 23 inhibitors (ustekinumab), anti-IL-23 inhibitors (mirikizumab, risankizumab), and JAK inhibitors (tofacitinib, filgotinib, upadacitinib).

[0069] 1-2. Step (2) In one embodiment, the inspection method of the present invention preferably further includes (2) determining at least one selected from the group consisting of the presence or concentration of inflammatory bowel disease in the subject, the severity of inflammatory bowel disease, and the therapeutic effect of a drug for treating inflammatory bowel disease, based on the amount or concentration of the biomarker detected in step (1) or an index value calculated based on the amount or concentration.

[0070] Furthermore, the present invention relates, in one embodiment, to a determination method comprising steps (1) and (2).

[0071] In a more specific embodiment, step (2) is: (2a) determining that the subject has inflammatory bowel disease and / or has a high severity of inflammatory bowel disease if the amount or concentration of the biomarker detected in step (1) or the index value calculated based on the amount or concentration is greater than or equal to a cutoff value; (2b) determining that the subject does not have inflammatory bowel disease and / or has a low severity of inflammatory bowel disease if the amount or concentration of the biomarker detected in step (1) or the index value calculated based on the amount or concentration is less than or equal to a cutoff value; (2c) determining that the subject has a high therapeutic effect of a Th17 inhibitor, a low therapeutic effect of a TNF-α inhibitor, a low therapeutic effect of an IL-12 / 23p40 inhibitor, a high therapeutic effect of a CN / JAK inhibitor, or a high therapeutic effect of a lymphocyte migration inhibitor (e.g., an anti-integrin inhibitor) if the amount or concentration of proHp detected in step (1) or the index value calculated based on the amount or concentration is greater than or equal to a cutoff value; (2d) A step in which, if the amount or concentration of proHp detected in step (1) or an index value calculated based on the amount or concentration is less than or equal to a cutoff value, the subject is determined to have a low therapeutic effect with a Th17 inhibitor, a high therapeutic effect with a TNF-α inhibitor, a high therapeutic effect with an IL-12 / 23p40 inhibitor, a low therapeutic effect with a CN / JAK inhibitor, or a low therapeutic effect with a lymphocyte migration inhibitor (e.g., an anti-integrin inhibitor); (2e) A step in which, if the amount or concentration of LRG and / or CRP (preferably LRG) detected in step (1) or an index value calculated based on the amount or concentration is greater than or equal to a cutoff value, the subject is determined to have a high therapeutic effect with a TNF-α inhibitor or an IL-12 / 23p40 inhibitor; and (2f) A step in which the subject determines that the therapeutic effect of a TNF-α inhibitor is low or that the therapeutic effect of an IL-12 / 23p40 inhibitor is low, when the amount or concentration of LRG and / or CRP (preferably LRG) detected in step (1) or an index value calculated based on the amount or concentration is less than or equal to a cutoff value. Preferably, the step includes at least one selected from the group consisting of the above.

[0072] "The index value calculated based on the aforementioned quantity or concentration" is a value calculated by substituting "the aforementioned quantity or concentration" into an arithmetic formula. This arithmetic formula can be set from the standpoint of the accuracy of the judgment, and can be determined, for example, by regression analysis.

[0073] The cutoff value is predetermined based on statistical analysis or ROC analysis, using a database that tracks the amount or concentration of the target protein in the evaluation population, along with the incidence of inflammatory bowel disease, the severity of inflammatory bowel disease, and the therapeutic effect of inflammatory bowel disease medications. Alternatively, the cutoff value can be set on a case-by-case basis. When determining the cutoff value by statistical analysis, for example, the median, arithmetic mean, or other mean values ​​of the data on the amount or concentration of the target protein in the evaluation population can be used. When determining the cutoff value by ROC analysis, for example, the cutoff value based on ROC analysis can be the amount or concentration of the target protein at the point on the ROC curve where the distance to the point where the vertical axis (sensitivity or true positivity) is 1.0 and the horizontal axis (1 - specificity) is 0.0 is minimized, or it can be a cutoff value derived from the Youden index of the ROC curve (Cancer 1950;3:32-35.). The database of the evaluation population may be used to set the cutoff value in the testing method of the present invention without any changes once it has been established. Alternatively, new subjects may be incorporated into the evaluation population, and the database of the evaluation population may be updated as appropriate while being used to set the cutoff value. The cutoff value can be, for example, a percentile value of the amount or concentration of the target protein in the body fluid sample in the reference group of subjects, for example, any of the 10th to 90th percentile values, any of the 30th to 70th percentile values, or any of the 40th to 60th percentile values.

[0074] 2. Determination of therapeutic effect of treatment for inflammatory bowel disease The present invention relates to a method for determining the therapeutic effect of a therapeutic agent for inflammatory bowel disease, comprising the step of detecting a biomarker in a biological sample taken from a subject, wherein the biomarker includes proHp and / or LRG.

[0075] Based on the determination result of the therapeutic effect of the therapeutic drug using the above determination method or the inspection method of the present invention described above, a therapeutic drug can be selected and / or the selected therapeutic drug can be administered to the subject.

[0076] 3. Diagnostic reagents for inflammatory bowel disease. In one embodiment, the present invention relates to a diagnostic reagent (sometimes referred to as "the diagnostic reagent of the present invention" in this specification) for use in the testing method or determination method of the present invention, comprising at least one selected from the group consisting of proHp-binding molecules, LRG-binding molecules, and CRP-binding molecules. This will be described below.

[0077] The diagnostic reagent of the present invention may be in the form of a composition containing a target protein-binding molecule. The composition may optionally contain other components. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, and the like.

[0078] The diagnostic reagent of the present invention may be in the form of a kit containing a target protein-binding molecule. The kit may include instruments, reagents, etc., that can be used to carry out the diagnostic method of the present invention.

[0079] The target protein-binding molecule can also be immobilized on any solid phase. Therefore, the diagnostic reagent of the present invention can be provided in the form of a substrate on which the target protein-binding molecule is immobilized (for example, a microarray chip on which a probe is immobilized; another example being an ELISA plate on which an antibody is immobilized).

[0080] The solid phase used for immobilization is not particularly limited as long as it can immobilize antibodies, etc., and examples include glass plates, nylon membranes, microbeads, silicon chips, capillaries, or other substrates. The method of immobilizing the detection agent onto the solid phase is not particularly limited.

[0081] Examples of equipment include test tubes, microtiter plates, agarose particles, latex particles, purification columns, epoxy-coated glass slides, and gold colloid-coated glass slides.

[0082] Examples of reagents include labeled antibodies and standard samples (positive control, negative control).

[0083] As labeled antibodies, various commercially available antibodies can be used depending on the type of protein-binding molecule (e.g., isotype) of the target protein.

[0084] The target protein is used as the standard sample. The target protein can be obtained, for example, by culturing cells into which a target protein expression vector has been introduced, and then purifying the cells or the culture supernatant.

[0085] In one embodiment, the diagnostic reagent of the present invention is preferably at least one companion diagnostic reagent selected from the group consisting of Th17 inhibitors, TNF-α inhibitors, IL-12 / 23p40 inhibitors, CN / JAK inhibitors, and lymphocyte migration inhibitors (e.g., anti-integrin inhibitors). That is, the diagnostic reagent of the present invention can be used to select therapeutic drugs for subjects with inflammatory bowel disease.

[0086] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0087] Test Example 1. Biomarker Analysis 1: The performance of proHp, LRG, and CRP as biomarkers for inflammatory bowel disease was analyzed.

[0088] <Example 1-1. Patient Data and Biomarker Values ​​Used in Analysis> Data from patients with inflammatory bowel disease (UC) were analyzed using ELISA, including age, sex, endoscopic severity score, and serum inflammatory marker (CRP, LRG, prohaptoglobin (proHp)) secretion levels (ulcerative colitis (UC): 165 patients, Crohn's disease (CD): 106 patients). The endoscopic score (MES) for ulcerative colitis was encoded in four stages (0-3) from normal to severe, and the endoscopic score (CDEIS) for Crohn's disease was encoded in four stages: less than 3 as Inactive (normal), 3 to less than 7 as Mild (mild), 7 to less than 16 as Moderate (moderate), and 16 or more as Severe (severe). Furthermore, since proHp secretion could not be adequately measured by ELISA from serum samples of patients with the haptoglobin gene polymorphism (Hp1-1), the final analysis data consisted of samples excluding data from patients with this polymorphism (ulcerative colitis: 149 patients, Crohn's disease: 89 patients). In addition, for inflammatory markers, a power-law transformation using the fourth root was performed to approximate the distribution of secretion levels to a normal distribution. The results are shown in Figure 1.

[0089] <Example 1-2. ROC Analysis> To examine the characteristics of inflammatory markers, ROC analysis was performed on the discrimination of endoscopic scores.

[0090] In the case of differentiating ulcerative colitis, an inflammatory bowel disease (MES: 2,3>0,1), the AUC (CI) was 0.827 (0.762-0.893) when CRP alone was used as an indicator, 0.892 (0.840-0.944) when LRG alone was used as an indicator, and 0.922 (0.879-0.964) when proHp alone was used as an indicator. In contrast, when CRP and proHp were combined using a logistic regression model as an indicator, the AUC was 0.936 (0.898-0.974), and when LRG and proHp were combined using a logistic regression model as an indicator, the AUC was 0.944 (0.910-0.978). Thus, it was found that combining CRP and proHp or LRG and proHp allows for more accurate diagnosis of ulcerative colitis than using either indicator alone.

[0091] On the other hand, when CRP and LRG were combined using a logistic regression model as the index, the AUC (CI) was 0.892 (0.840-0.944), which was about the same as when LRG was used alone (0.892 (0.840-0.944)).

[0092] Similar results were obtained in the differentiation of Crohn's disease, another inflammatory bowel disease. For example, in the differentiation of CDEIS: Moderate, Sevire > Inactive, Mild, the AUC (CI) was 0.782 (0.687-0.877) when CRP alone was used as the indicator, and 0.775 (0.675-0.875) when proHp alone was used as the indicator, while it was 0.809 (0.720-0.897) when CRP and proHp were combined using a logistic regression model.

[0093] <Example 1-3. Principal Component Analysis> The correlation between three inflammatory markers was examined using principal component analysis. The results are shown in Figure 2. For both ulcerative colitis and Crohn's disease, the first principal component (PC1) had an explanatory power of approximately 80% of the variance. From the plot of principal component scores and the biplot of loadings in Figure 2, it was confirmed that an increase in the secretion of the three inflammatory markers (positive direction on the PC1 axis) corresponds to an increase in the endoscopic score. Furthermore, the second principal component (PC2), with an explanatory power of approximately 10%, showed a higher correlation with proHp compared to CRP and LRG, and its sign was also reversed. This suggests that factors more specific to proHp and contrary to the secretion of CRP and LRG are involved in the secretion of these inflammatory markers. This result is considered to be the reason why inflammatory bowel disease can be diagnosed with higher accuracy by combining CRP and proHp or LRG and proHp compared to using them individually.

[0094] <Example 1-4. Prediction of the therapeutic effect of molecularly targeted therapies using inflammatory markers> Aiming to predict the improvement of symptoms after administration of various molecularly targeted therapies based on the secretion levels of inflammatory markers, and to select the optimal antibody preparation for each patient based on this, a model was constructed using a logistic regression model to predict the probability of response of antibody preparations. As data for this purpose, samples containing descriptions of the administration of various antibody preparations (inhibitors) and subsequent therapeutic effects were extracted from the analysis data (ulcerative colitis: 94 patients, Crohn's disease: 61 patients).

[0095] Using data from ulcerative colitis, a logistic regression model was constructed to predict the effect of the inhibitor used in treatment (Failure: no response, Success: response) by incorporating the interaction between inflammatory marker secretion and inhibitors as explanatory variables, in addition to sex (Sex: male, female. Female is a dummy variable with Female as the baseline), inflammatory marker secretion levels (LRG, proHp), inhibitors used in prior treatment (Pretreatment: None, TNF (TNF-α inhibitor), Other (other inhibitor). None is a dummy variable with None as the baseline), endoscopic severity score (MES. numerical value from 0 to 3), and inhibitors used in treatment (Inhibitor: TNF (TNF-α inhibitor), IL12 (IL-12 / 23p40 inhibitor), SMD (CN / JAK inhibitor), Other (other inhibitor). TNF is a dummy variable with TNF as the baseline). Furthermore, variable selection was performed using a stepwise method to obtain the following model that minimizes AIC.

[0096]

[0097] Significant main effects were detected for MES, LRG, proHp, pretreatment (TNF), and inhibitor (IL12), and significant interactions were detected for LRG × inhibitor and proHp × inhibitor (Table 1). This means that the efficacy of each inhibitor differs depending on the amount of LRG and proHp secreted (Figure 3).

[0098]

[0099] Example 2. Biomarker Analysis 2: The mechanism of proHp production in enteritis was analyzed.

[0100] <Test Example 2-1. Method> Western blot [Sample preparation] Culture supernatant…Amicon Ultra-4 30K (Merck, Darmstadt, Germany) was centrifuged at 4,000G for 20 minutes at 4°C. After centrifugation, it was collected in a 1.5 mL tube and used for Western blot.

[0101] Cell lysate…The culture supernatant was removed from the dish and plate, washed with 1 mL of PBS(-), and then 1 mL of PBS(-) was added to detach the cells using a cell scraper (BM equipment, Tokyo, Japan). After centrifugation (4°C, 200 G, 5 min), the supernatant was discarded, and 30 μL of TNE buffer (10 mM Tris-HCl pH 7.8, 1% Nonidet p-40, 0.5 mM NaCl, 1 mM EDTA, Protease Inhibitor Cocktail (nacalai tesque), Phosphatase Inhibitor Cocktail (nacalai tescue)) was added. The supernatant was sonicated using Bransonic® (Yamato Scientific, Tokyo, Japan) (room temperature, 5 min), and then centrifuged (4°C, 14,000 G, 10 min) to collect the supernatant as lysate. Protein levels were measured using the Protein Assay BCA Kit (nacalai tesque) according to the manufacturer's recommended protocol.

[0102] The concentrated culture supernatant was prepared at 10 μL / well, and the cell lysate was prepared at 20 μg / well of protein. The electrophoresis samples were prepared by adjusting the 1× Loading dye to 5% 2-mercaptoethanol (nacalai tesque) and reducing it (95°C, 5 min).

[0103] [SDS-PAGE] Samples were applied to the wells and electrophoresis was performed on a 10% polyacrylamide gel (230 V, 10 mA, 120 min).

[0104] [Blotting] The sample was transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, USA) (20 V, 200 mA, 60 min).

[0105] [Blocking, Primary Antibody Reaction, Secondary Antibody Reaction] After blocking with 3% bovine serum albumin (BSA; nacalai tesque) in TBST (Tris-buffered saline containing Tween 20; 20 mM Tris, 0.5 M NaCl, pH 7.5 containing 0.05% Tween 20) or 5% Skim Milk (nacalai tesque) in TBST (room temperature, 1 hour), the samples were reacted with the primary antibody (4°C, overnight). After washing with TBST (room temperature, 1 hour), the samples were reacted with 5,000-fold diluted Anti-mouse IgG-HRP or Anti-rabbit IgG-HRP (Promega) (room temperature, 1 hour). After washing with TBST (room temperature, 1 hour), Chemi-Lumi One Super (nacalai tesque) was reacted according to the manufacturer's recommended protocol, and the target band was detected using the FUSION Chemiluminescence imaging system (Vilber-Lourmat, Collegien, France).

[0106] RNA was recovered using the Real-time RT-PCR [RNA extraction] RNeasy® Mini Kit (QIAGEN, Venlo, Netherlands). RNA concentration was measured using Nano Drop (Thermo Fisher Scientific, Tokyo, Japan).

[0107] [cDNA Synthesis] Extracted RNA was mixed with Nuclease-Free Water until the RNA concentration reached 500 ng / 10 μL, resulting in a final volume of 10 μL. RNA denaturation was performed using a Thermal Cycler (TaKaRa, Shiga, Japan) at 70 °C for 5 min, followed by 4 °C for 5 min. A GoScript™ReverseTranscriptionMix (4 μL Nuclease-Free Water, 4 μL GoScript™ReactionBufferOligo (dT) (Promega, Madison, USA) per sample, and 2 μL GoScript™EnzymeMix (Promega, Madison, USA)) was prepared, and 10 μL of the prepared mix was added and mixed. cDNA was synthesized using a thermal cycler (25°C, 5 min, 1 cycle → 42°C, 60 min, 1 cycle → 70°C, 15 min, 1 cycle → 4°C, ∞). 20 μL of the synthesized cDNA was mixed with 80 μL of nucleotide-free water to make a concentration of 0.5 μg / 100 μL.

[0108] [Real-time RT-PCR] To 1 μL of synthesized cDNA, a forward primer (final concentration 0.25 μM), a reverse primer (final concentration 0.25 μM), and 2×THUNDERBIRD® Next SYBR qPCR Mix (final concentration 1×) were mixed and adjusted in MQ to a total volume of 20 μL. Analysis was performed using MxPro ET QPCR Software (Agilent, Tokyo, Japan). The Ct value was calculated as the intersection of the threshold and the amplification curve, and the expression level of the target gene was divided by the expression level of the housekeeping gene RPL4 to perform relative quantification. The primers used are shown in Table 2.

[0109]

[0110] 10-7G ELISA: 50 μL of culture supernatant was added to an ELISA plate immobilized with a 10-7G monoclonal antibody (Nishino K et al, Oncotarget 9(16), 12732-44, 2018), an antibody against haptoglobin and its precursor proHp. After incubation at room temperature for 1 hour, the plate was washed three times with 250 μL of TBST. 50 μL of a 3-25-1C monoclonal antibody (HRP conjugate) that recognizes the haptoglobin β-chain was added and incubated at room temperature for 1 hour. The plate was washed three times with 250 μL of TBST, and the moisture was thoroughly absorbed and removed with Kimwipes. 50 μL of TMB (Wako) was added and incubated at room temperature for 15 minutes, and the absorbance was measured.

[0111] <Test Example 2-2. Results> Figure 4 shows the HP gene expression graph quantified by Real-time RT-PCR after stimulating Caco-2 and THP-1 with each cytokine (20 ng / mL) for 24 hours. Figure 5 shows the Western blot results of the culture supernatant after stimulating Caco-2 and THP-1 with each cytokine (20 ng / mL) for 24 hours. Figure 6 shows the 10⁻⁷G values ​​of the culture supernatant after stimulating Caco-2 and THP-1 with each cytokine (20 ng / mL) for 24 hours. Figure 7 shows the results of the phosphorylated STAT3 antibody Western blot of cell lysates after stimulating Caco-2 with each cytokine (20 ng / mL) for 24 hours. Figure 8 shows the results of the phosphorylated STAT3 antibody Western blot of cell lysates after stimulating Caco-2 with IL-22 (20 ng / mL) in the presence of a STAT3 inhibitor (10 ng / mL) for 24 hours. These results indicate that IL-22 stimulation enhances proHp production in intestinal cells. This suggests that proHp, which is increased in inflammatory bowel disease, is secreted from intestinal epithelial cells via IL-22-mediated STAT3 signaling. Therefore, it is considered possible to evaluate the effectiveness of Th17 inhibitors (inhibitors of differentiation into Th17 cells, and inhibitors of Th17-producing cytokines) using proHp as an indicator.

[0112] Study Example 3. Biomarker Analysis 3: The predictive performance of proHp and LRG in predicting the therapeutic effect of inflammatory bowel disease (IBD) treatments was analyzed. <Methods> A model was constructed to predict the effectiveness of drug therapy in IBD patients using the patients' serum LRG and proHp concentrations. To construct this model, data from 192 UC patients and 111 CD patients who received advanced treatment were used. In the logistic regression model, LRG and proHp concentrations, the choice of advanced treatment and its interaction were set as independent variables, and the treatment outcome was set as the dependent variable. Age, sex, and prior treatment were included as covariates. Continuous variables (LRG, proHp, age) were standardized with z-scores. Other classification variables were dummy-coded. Sex was set as female=0, male=1. Prior treatment was set as untreated=0, treated=1. For the choice of advanced treatment, antiintegrin drugs were defined as the reference category (value 0) for both UC and CD. Therefore, in UC, anti-TNF, anti-IL-12 / 23, and JAKi were coded as dummy indicator variables, while in CD, anti-TNF and anti-IL-12 / 23 underwent similar processing. The model fitted to the current data can calculate the probability of success outcomes for each advanced treatment selection based on the covariates and specific values ​​of biomarker concentrations in individual patients. This ensures that the drug with the highest probability is selected as the most effective treatment for the patient. To improve predictive performance, this logistic regression model was further refined in the following way: First, to correct for class imbalance in outcomes (the ratio of success to failure outcomes is 1.46:1 in UC and 1.92:1 in CD), a weighted logistic regression model was constructed with weights introduced according to the reciprocal of the ratio of outcomes. Next, variable selection was performed on the covariates of the two logistic regression models (unweighted and weighted) using Leave-One-Out Cross-Validation (LOOCV). LOOCV provides a nearly unbiased estimate of the predictive accuracy of treatment outcomes. Based on the most effective treatment predicted by LOOCV, patients were classified into those who received the most effective treatment (matched patients) and those who did not (unmatched patients). The odds ratios of the success outcome between matched and unmatched patients were used as criteria for model and variable selection.The models ultimately selected were an unweighted logistic regression model without covariates for UC and a weighted logistic regression model without covariates for CD. The predicted proportion of successful patients was compared to the level of chance using a one-sided binomial test. The difference in the predicted proportion of successful patients between the concordant and misconductant groups was evaluated using a chi-square test. A p-value of less than 0.05 was considered statistically significant. <Results> The results are shown in Figures 9-12. To evaluate the clinical utility of LRG and proHp as guidelines for treatment selection, a logistic regression model was developed that incorporated the interaction between biomarker levels and treatment classes, adjusted for baseline covariates (age, sex, prior treatment). Leave-one-out cross-validation identified the optimal model, which included LRG, proHp, and interaction terms between each biomarker and drug class. This model accurately predicted the treatment response at week 8, particularly in patients where the level of any biomarker was above the cohort mean. Forest plot analysis revealed that LRG showed a significant negative main effect on the overall response probability, while proHp showed a significant contribution through interactions with specific drug classes. In CD, high LRG levels were associated with a better response to anti-TNF therapy, and elevated proHp was associated with improved efficacy of anti-IL-12 / 23 agents. In UC, elevated proHp predicted better outcomes with JAK inhibitors, and LRG showed a positive interaction with anti-IL-12 / 23 agents. Response probability maps plotted in the LRG-proHp space revealed clear therapeutic areas: in CD, anti-TNF agents were recommended in LRG-dominant profiles, and IL-23 inhibitors were recommended in proHp-dominant cases; in UC, a single high pattern coincided with integrins or IL-12 / 23 inhibitors, and a dual high profile corresponded to the maximum effect with JAK inhibitors. Patients who received the optimal drug predicted by the model (Matched group) showed a significantly higher response rate compared to patients who received alternative therapy (Unmatched group), with an overall response rate of 80% vs. 50%, and a particularly significant effect of 85% vs. 45% in highly active patients. On the other hand, patients with low disease activity and low biomarker levels consistently showed high response rates (70% vs. 70%) across all treatment options.This suggests the possibility of a "dual-low subgroup" exhibiting a biotolerable state where multiple mechanisms of action are equally effective, even without stratification information. These results indicate that elevated LRG or proHp levels provide mechanistically meaningful guidance in treatment selection. Furthermore, a combined interpretation of both improves predictive accuracy and enables a dual-axis framework to support precision medicine in IBD.

[0113] Study Example 4. Biomarker Analysis 4 Principal component analysis of the correlation between serum cytokines and serum biomarkers revealed that, independently of IL-6, serum IL-8 (a substance that promotes lymphocyte migration) and proHp correlated. This suggests that patients with high proHp levels also have high levels of IL-8, which promotes lymphocyte migration, and that lymphocyte migration inhibitors may be effective. Similarly, it was found that, independently of IL-6, serum IL-17 and proHp also correlated.

Claims

1. (1) A method for testing for inflammatory bowel disease, comprising the step of detecting a biomarker in a biological sample taken from a subject, wherein the biomarker includes proHp and LRG, or proHp and CRP.

2. The method according to claim 1, wherein the biomarkers include proHp and LRG.

3. The method according to claim 1, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

4. The method according to claim 1, wherein the biological sample is at least one selected from the group consisting of blood, serum, and plasma.

5. The method according to claim 1, wherein the subject is a human.

6. The method according to claim 1, wherein the examination for inflammatory bowel disease includes determining at least one of the following: the presence of inflammatory bowel disease, the severity of inflammatory bowel disease, and the therapeutic effect of a drug for treating inflammatory bowel disease.

7. The method according to any one of claims 1 to 6, further comprising (2) determining, based on the amount or concentration of the biomarker detected in step (1) or an index value calculated based on the amount or concentration, at least one selected from the group consisting of the presence of inflammatory bowel disease, the severity of inflammatory bowel disease, and the therapeutic effect of a drug for treating inflammatory bowel disease in the subject.

8. Step (2) includes: (2a) determining that the subject has inflammatory bowel disease and / or has a high severity of inflammatory bowel disease if the amount or concentration of the biomarker detected in step (1) or the index value calculated based on the amount or concentration is equal to or greater than a cutoff value; (2b) determining that the subject does not have inflammatory bowel disease and / or has a low severity of inflammatory bowel disease if the amount or concentration of the biomarker detected in step (1) or the index value calculated based on the amount or concentration is less than or equal to a cutoff value; (2c) determining that the subject has a high therapeutic effect of Th17 inhibitors, a low therapeutic effect of TNF-α inhibitors, a low therapeutic effect of IL-12 / 23p40 inhibitors, a high therapeutic effect of CN / JAK inhibitors, or a high therapeutic effect of lymphocyte migration inhibitors. (2d) A step in which, if the amount or concentration of proHp detected in step (1) or an index value calculated based on the amount or concentration is less than or equal to a cutoff value, the subject is determined to have a low therapeutic effect on Th17 inhibitors, a high therapeutic effect on TNF-α inhibitors, a high therapeutic effect on IL-12 / 23p40 inhibitors, a low therapeutic effect on CN / JAK inhibitors, or a low therapeutic effect on lymphocyte migration inhibitors; (2e) A step in which, if the amount or concentration of LRG and / or CRP detected in step (1) or an index value calculated based on the amount or concentration is greater than or equal to a cutoff value, the subject is determined to have a high therapeutic effect on TNF-α inhibitors or IL-12 / 23p40 inhibitors; The method according to claim 7, comprising at least one selected from the group consisting of (2f) determining that the subject has a low therapeutic effect on a TNF-α inhibitor or an IL-12 / 23p40 inhibitor when the amount or concentration of LRG and / or CRP detected in step (1) or an index value calculated based on the amount or concentration is less than or equal to a cutoff value.

9. A diagnostic reagent for use in the method of any one of claims 1 to 6, comprising at least one selected from the group consisting of a proHp-binding molecule, an LRG-binding molecule, and a CRP-binding molecule.

10. The diagnostic agent according to claim 9, which is at least one companion diagnostic agent selected from the group consisting of Th17 inhibitors, TNF-α inhibitors, IL-12 / 23p40 inhibitors, CN / JAK inhibitors, and lymphocyte migration inhibitors.

11. (X) A method for determining the therapeutic effect of a therapeutic agent for inflammatory bowel disease, comprising the step of detecting a biomarker in a biological sample taken from a subject, wherein the biomarker includes proHp and / or LRG.

12. A diagnostic reagent for use in the method of claim 11, comprising at least one selected from the group consisting of proHp-binding molecules and LRG-binding molecules.

13. The diagnostic agent according to claim 12, which is at least one companion diagnostic agent selected from the group consisting of Th17 inhibitors, TNF-α inhibitors, IL-12 / 23p40 inhibitors, CN / JAK inhibitors, and lymphocyte migration inhibitors.

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