Method for examining inflammatory bowel disease and method for screening therapeutic agent

Abstract

The present invention provides an examination method for identifying a biomarker capable of detecting the remission phase of an inflammatory bowel disease, and assisting in the diagnosis of the inflammatory bowel disease, the monitoring of disease progression, or the determination of therapeutic effect.　Focusing attention on myeloid immune cells that play an important role in the pathogenesis of inflammatory bowel disease, the present inventors considered that young myeloid immune cells potentially increase even in the remission phase and may induce transition to the active phase by progression of the disease. The present inventors then proceeded with intensive studies thereon. As a result, the present inventors found that CD11b, CD33, and CD84-positive (CD11b+CD33+CD84+) cells, which are precursor cells of myeloid immune cells, can serve as blood markers for inflammatory bowel disease. Further investigation revealed that Membrane-Spanning 4-Domains A3 (Ms4a3)-positive (Ms4a3+) cells, and Ms4a3 and CD84-positive (Ms4a3+CD84+) cells significantly increase not only in the active phase but also in the remission phase, and are significantly greater in number in the active phase than in the remission phase.

Description

Testing methods and screening methods for inflammatory bowel disease

[0001] The present invention relates to a method for testing for inflammatory bowel disease, and more specifically, to a testing method for assisting in the diagnosis of inflammatory bowel disease, monitoring of disease progression, or determination of therapeutic effects, and to a method for screening active ingredients of therapeutic agents for inflammatory bowel disease. This application claims priority to Japanese Patent Application No. 2025-005167, as incorporated herein by reference.

[0002] Inflammatory bowel disease (IBD), exemplified by ulcerative colitis and Crohn's disease, affects more than 6 million people worldwide, and the number of patients is increasing in Japan as it is designated as an intractable disease. Inflammatory bowel disease is characterized by alternating periods of active disease, in which gastrointestinal symptoms such as diarrhea and bloody stools are prominent, and remission, in which symptoms subside. While symptomatic treatments to suppress the worsening of symptoms during the active period have been developed, a fundamental cure has not yet been achieved. Furthermore, conventional maintenance therapies have only been able to partially prevent the transition from remission to the active period (Non-patent Literature 1).

[0003] To date, diagnostic markers capable of distinguishing the active phase, including LRG (leucine-rich α2-glucoprotein), have been developed (Non-Patent Document 2). Patent Document 1 discloses a preventive and / or therapeutic agent for ulcerative colitis containing an LRG expression or function inhibitor, and reports that LRG can be used as an inflammatory marker. Patent Document 2 discloses an immunological method for measuring LRG in biological samples.

[0004] However, no marker has yet been discovered that can sensitively detect potential disease progression during remission.

[0005] Patent No. 6226315, Patent No. 7421711

[0006] Shen et al., Lancet Gastroenterol Hepatol, 2022 Revised Edition Ulcerative Colitis / Crohn's Disease Diagnostic Standards and Treatment Guidelines (Health, Labor and Welfare Scientific Research Grant, Intractable Disease Policy Research Project "Survey Research on Intractable Inflammatory Intestinal Disorders" (Hisamatsu Group) Shared Research Report in 2024), 2024

[0007] The object of the present invention is to identify a biomarker that can detect the remission phase of inflammatory bowel disease and to provide a testing method to assist in the diagnosis of inflammatory bowel disease, monitoring of disease progression, or evaluation of treatment effectiveness.

[0008] In view of the above problems, the inventors focused on myeloid immune cells, which play an important role in the pathogenesis of inflammatory bowel disease, and hypothesized that even during remission, immature myeloid immune cells may be potentially increasing, leading to disease progression and inducing a transition to the active phase. They diligently investigated this possibility. As a result, they found that myeloid immune cell precursors that are positive for CD11b, CD33, and CD84 (CD11b + CD33 + CD84 + ) We found that these cells could serve as a blood marker for inflammatory bowel disease. Further investigation revealed that Membrane-Spanning 4-Domains A3 (Ms4a3) positive (Ms4a3 + ) cells and Ms4a3 and CD84-positive (Ms4a3 + CD84 + We discovered that the number of ) cells increases significantly not only during the active phase but also during the remission phase, and that they are significantly more numerous during the active phase than during the remission phase, thus completing the present invention.

[0009] In other words, the present invention comprises the following: 1. A testing method for assisting in the diagnosis of inflammatory bowel disease, monitoring of disease progression, or determination of therapeutic effect, comprising the step of measuring the expression level of at least one selected from the group consisting of Ms4a3 and CD84 in a biological sample collected from a subject. 2. The testing method according to item 1, further comprising the step of measuring the expression level of Ms4a3 in a biological sample collected from a subject. 3. The testing method according to item 1 or 2, further comprising the step of measuring the expression levels of Ms4a3 and CD84 in a biological sample collected from a subject. 4. The testing method according to any one of items 1 to 3, wherein if the expression level is higher than a reference value or the expression level in a biological sample obtained from a healthy person, it indicates that the person has inflammatory bowel disease, or that a decrease or maintenance of the expression level indicates maintenance of remission or therapeutic effect of inflammatory bowel disease. 5. 1. A method for testing according to any one of paragraphs 1 to 4, wherein if the expression level is higher than a first reference value or the expression level in a biological sample obtained from a healthy person, and lower than a second reference value, it indicates that the patient is in remission from inflammatory bowel disease, and / or if it is higher than the second reference value, it indicates that the patient is in the active phase of inflammatory bowel disease. 6. The method according to any one of paragraphs 1 to 5, wherein the biological sample is a blood-derived sample. 7. A method for testing according to any one of paragraphs 1 to 6, wherein the inflammatory bowel disease is selected from the group consisting of ulcerative colitis and Crohn's disease. 8. A method for screening active ingredients for an inflammatory bowel disease treatment, comprising the steps of: (1) contacting Ms4a3-expressing cells with a test substance; (2) measuring the expression level or activity of Ms4a3 in the cells; and (3) comparing the expression level or activity with that of Ms4a3-expressing cells that have not been contacted with the test substance, and selecting a test substance that reduces the expression level or activity of Ms4a3 as an active ingredient or candidate substance for an inflammatory bowel disease treatment.9. The Ms4a3-expressing cells are cells that further express CD84. In the step (2), the expression level or activity of Ms4a3 and the expression level or activity of CD84 are measured. In the step (3), for each of the expression level or activity of Ms4a3 and the expression level or activity of CD84, they are compared with Ms4a3 and CD84-expressing cells that have not been contacted with the test substance, and a test substance that reduces the expression level or activity of either Ms4a3 or CD84 is selected as an active ingredient of a therapeutic agent for inflammatory bowel disease or a candidate substance thereof. The screening method according to item 8 above, which includes this step.

[0010] According to the method for examining inflammatory bowel disease of the present invention, it is possible to assist in the diagnosis of inflammatory diseases, the monitoring of disease progression, or the determination of treatment effects. In addition, it is possible to sensitively detect potential disease progression during the remission period of inflammatory bowel disease. Furthermore, according to the screening method of the present invention, it is possible to screen an active ingredient of a therapeutic agent for inflammatory bowel disease or a candidate substance thereof.

[0011] Protein expression (CD11b + CD33 + CD84 + ) in a sample derived from a patient with inflammatory bowel disease is shown. (Example 1) Protein expression (granulocyte CD11b + CD33 + CD84 + , monocyte CD11b + CD33 + CD84 + ) in a sample derived from a patient with inflammatory bowel disease is shown. (Example 2) Protein expression (CD84 + , CD11b + CD84 + , CD33 + CD84 + ) in a sample derived from a patient with inflammatory bowel disease is shown. (Example 3) Protein expression (CD11b + CD33 + Ms4a3 + , CD11b + CD33 + CD84 + Ms4a3 +The results of the analysis are shown. (Example 4) Protein expression in samples from inflammatory bowel disease patients (Ms4a3 + The results of the analysis are shown. (Example 5) Protein expression in samples from inflammatory bowel disease patients (Ms4a3 + CD84 + The results of the analysis are shown. (Example 6) Protein expression (CD11b) in samples from inflammatory bowel disease patients in the estimated active and estimated remission phases. + CD33 + CD84 + CD84 + Ms4a3 + Ms4a3 + CD84 + ) The results of the analysis are shown. (Example 7) The results of the verification of the CDK2 inhibitory effect using an inflammatory bowel disease model animal are shown. (Example 8)

[0012] The present invention relates to a testing method (hereinafter sometimes simply referred to as "the testing method of the present invention") for assisting in the diagnosis, monitoring of disease progression, or evaluation of therapeutic effects for inflammatory bowel disease, and a screening method for active ingredients of therapeutic agents for inflammatory bowel disease (hereinafter sometimes simply referred to as "the screening method of the present invention"). The present invention also relates to a method for measuring the expression levels of Ms4a3 and / or CD84 in a biological sample taken from a subject, which is used to assist in the diagnosis, monitoring of disease progression, or evaluation of therapeutic effects for inflammatory bowel disease. The present invention also relates to a testing composition (hereinafter sometimes simply referred to as "the testing composition of the present invention") for measuring the expression levels of Ms4a3 and / or CD84, which is used to assist in the diagnosis, monitoring of disease progression, or evaluation of therapeutic effects for inflammatory bowel disease. The present invention also relates to a testing kit (hereinafter sometimes simply referred to as "the testing kit of the present invention") for assisting in the diagnosis, monitoring of disease progression, or evaluation of therapeutic effects for inflammatory bowel disease.

[0013] In this specification, "inflammatory bowel disease" refers to a general term for diseases that cause chronic inflammation or ulcers in the mucous membrane of the large or small intestine, such as ulcerative colitis, Crohn's disease, microscopic colitis (collagenosic colitis, lymphocytic colitis), eosinophilic gastroenteritis, Behçet's disease, ischemic colitis, radiation enteritis, drug-induced enteritis, and intestinal graft-versus-host disease (GVHD). During the remission phase of inflammatory bowel disease, therapeutic drugs are used to maintain the remission state, but during the active phase, more potent therapeutic drugs are necessary. If the diagnostic method of the present invention can grasp the potential progression of the disease during the remission phase, it will be possible to select a more appropriate therapeutic drug.

[0014] Ms4a3 is a member of the membrane-spanning 4A gene family, primarily localized to the cell surface, and its expression has been reported in hematopoietic cells and tumor cells. It is thought to function as a cell surface signaling molecule and intracellular adapter protein, performing diverse functions (J Cell Mol Med. 2011 Jan;15(1):86-93). In the examples described below, Ms4a3 was shown to be a marker capable of determining the remission and active phases of inflammatory bowel disease, which cannot be distinguished by LRG, a conventional marker for the active phase of inflammatory bowel disease. Therefore, the expression level of Ms4a3 correlates with the degree of remission, a decrease in Ms4a3 expression level during the active phase indicates a transition to the remission phase, an increase in Ms4a3 expression level during the remission phase indicates a transition to the active phase, and a decrease in Ms4a3 expression level during the remission phase indicates a more robust remission. According to the testing method of the present invention, the degree of remission and treatment strategy for inflammatory bowel disease can be determined by measuring the expression level of Ms4a3 in a biological sample collected from a subject.

[0015] CD84 (Cluster of Differentiation 84) is a member of the immunoglobulin superfamily and has been reported to be expressed in granulocytes, lymphocytes, and monocytes. It is thought to be involved in cell interactions and signal transduction (Blood. 1997 Sep 15;90(6):2398-405). In the examples described below, it was shown that CD84 alone can serve as a marker for inflammatory bowel disease, and that combining Ms4a3 with CD84 can further improve the accuracy of determining the remission and active phases of inflammatory bowel disease. In this specification, "Ms4a3" and "CD84" are used as concepts that include not only the Ms4a3 protein and the CD84 protein, respectively, but also, depending on the context, transcripts derived from the genes encoding these proteins. The protein may include its isoforms, post-translational modifications, or variants having substantially the same biological properties as the protein (including those detectable by antibodies that specifically bind to the protein). The transcript may include mRNA derived from the gene, splicing variants, or nucleic acids containing parts of their sequences.

[0016] In this specification, “subject” means a human or non-human mammal (e.g., monkey, sheep, pig, horse, cattle, mouse, rat, cat, dog, etc.), preferably a human. The subject is not limited to patients with inflammatory bowel disease, but also includes healthy individuals, individuals suspected of having inflammatory bowel disease, and individuals undergoing or who have completed treatment.

[0017] In this specification, "biological sample" is not particularly limited to any sample taken from a subject and includes, but is not limited to, samples processed or analyzed outside the body. Examples of biological samples include bodily fluids and tissues taken from a subject, and cultures obtained by culturing cells or tissues taken from a living organism. Examples of bodily fluids include blood-derived samples such as whole blood, plasma, serum, cell fractions or liquid fractions separated from blood, lymph fluid, ascites, urine, tears, saliva, bone marrow fluid, and papillary secretions. Examples of tissues include tissue excisions obtained by surgery or biopsy, and tissue biopsy materials obtained by preoperative endoscopic examination. Among these, blood-derived samples are preferred from the viewpoint of low invasiveness, and samples containing leukocytes are more preferred. The method for obtaining a sample containing leukocytes is not particularly limited and can be obtained, for example, by hemolyzing the red blood cells in a whole blood sample. Biological samples may be subjected to hemolysis, centrifugation, washing, or other treatments as needed. Furthermore, the method for collecting and pre-treating biological samples is not particularly limited, and any method, including known methods and methods to be developed in the future, can be used.

[0018] In this specification, "expression level" means the level of expression, abundance, or detection intensity of mRNA of a gene or the protein encoded by that gene, and may be either an absolute or relative value.

[0019] In this specification, "decreased expression level" means an expression level lower than the reference value or the previously measured expression level of the subject. The criteria for determining a "decreased expression level" are not particularly limited, but for example, an expression level may be determined to be "decreased expression level" if it is 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more lower than the reference value or the previously measured expression level of the subject, and it may be determined to be "maintained expression level" if it is below the criteria. The criteria for determining an "increase in expression level" are not particularly limited, but for example, an increase in expression level may be determined if the expression level is 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, or 150% or more higher than the reference value or the subject's previous expression level, while a maintenance of the expression level may be determined if it is below the criteria.

[0020] The present invention's testing method includes the step of measuring the expression level of at least one selected from the group consisting of Ms4a3 and CD84 in a biological sample collected from a subject.

[0021] In one embodiment, the testing method of the present invention includes the step of measuring the expression level of Ms4a3 in a biological sample collected from a subject.

[0022] In one embodiment, the testing method of the present invention includes the step of measuring the expression level of CD84 in a biological sample collected from a subject.

[0023] In one embodiment, the testing method of the present invention includes the step of measuring the expression levels of Ms4a3 and CD84 in a biological sample collected from a subject.

[0024] Expression levels can be measured by known methods or methods to be developed in the future, by measuring the expression level, abundance, or detection intensity of gene transcripts or proteins. The amount of gene transcripts can be measured by nucleic acid detection methods, such as quantitative PCR, digital PCR, Northern hybridization, in situ hybridization, and whole transcriptome sequencing. Protein amounts can be measured by immunological methods, such as flow cytometry, imaging cytometry, immunohistochemistry (immunostaining), Western blotting, ELISA, radioimmunoassay, immunoprecipitation, and antibody array analysis.

[0025] According to the testing method of the present invention, the expression levels of Ms4a3 and / or CD84 may be measured in the same subject at any one or more time points (for example, at the time of the initial diagnosis of inflammatory bowel disease, before and after surgery, during follow-up, during induction therapy, and during maintenance therapy).

[0026] In one embodiment, according to the testing method of the present invention, if the expression level measured in the step of measuring the expression level of Ms4a3 and / or CD84 is found to be higher than the reference value or the expression level in a biological sample obtained from a healthy person, it can be used to assist in the diagnosis or determination of inflammatory bowel disease (including the remission and active phases). When measuring the expression levels of Ms4a3 and CD84, "the expression level measured in the step of measuring the expression level of Ms4a3 and CD84 is higher than the reference value or the expression level in a biological sample obtained from a healthy person" means that the expression levels of Ms4a3 and CD84 measured in the measurement step are both higher than the reference value or the expression level in a biological sample obtained from a healthy person. In this specification, "expression level in a biological sample obtained from a healthy person" is not particularly limited, but preferably a representative value based on the measurement results of biological samples derived from multiple healthy persons, and may be, for example, the mean or median in a predetermined group of healthy persons.

[0027] In one embodiment, according to the testing method of the present invention, if a decrease or maintenance of the expression level measured in the step of measuring the expression level of Ms4a3 and / or CD84 is confirmed, it can be used to assist in determining whether there is maintenance of remission or therapeutic effect in relation to monitoring the progression of inflammatory bowel disease or therapeutic effect. When measuring the expression levels of Ms4a3 and CD84, "a decrease or maintenance of the expression level measured in the step of measuring the expression level of Ms4a3 and CD84" means a decrease or maintenance of the expression levels of both Ms4a3 and CD84 measured in the measurement step. For example, if the expression level measured in the step of measuring the expression level of Ms4a3 and / or CD84 is lower than the reference value or the subject's previous expression level, it can be used to assist in determining whether there is maintenance of remission or therapeutic effect in inflammatory bowel disease. Furthermore, if the expression levels measured in the step for measuring the expression levels of Ms4a3 and / or CD84 maintain the subject's previous expression levels, it can assist in determining whether the remission phase of inflammatory bowel disease is being maintained or whether there is a therapeutic effect. Here, the subject is not particularly limited, but may be a person who was diagnosed with remission of inflammatory bowel disease at the time of the previous measurement of expression levels and / or is undergoing remission maintenance therapy.

[0028] In this specification, “reference value” means a predetermined standard used as a reference for evaluating values ​​or data obtained from biological samples taken from subjects. Reference values ​​may be absolute values, relative values, values ​​with upper or lower limits, values ​​within a range, mean values, or median values. Reference values ​​may also be values ​​based on confidence intervals calculated by statistical methods, for example, they may be set as the upper or lower limit of a confidence interval (e.g., a 95% confidence interval) for expression levels. Reference values ​​may be based on a large number of samples obtained from a group of inflammatory bowel disease patients and / or a group of healthy individuals. Preferably, reference values ​​are set separately for the expression levels of Ms4a3 and CD84. As an example, reference values ​​may be set from a range that is higher than the expression level in biological samples taken from healthy individuals and lower than the expression level in biological samples taken from inflammatory bowel disease patients, based on the results of measuring expression levels in biological samples taken from inflammatory bowel disease patients and healthy individuals who have been previously confirmed not to have inflammatory bowel disease. As another example, the reference value may be set based on the results of measuring the expression levels of biological samples taken from multiple inflammatory bowel disease patients who received treatment, within a range that is higher than the expression levels of biological samples taken from those who responded to treatment and lower than the expression levels of biological samples taken from those who did not respond to treatment. As yet another example, the reference value may be set based on the results of measuring the expression levels of biological samples taken from multiple healthy individuals and classifying them into those who did not develop inflammatory bowel disease within a predetermined follow-up period and those who did, within a range that is higher than the expression levels of biological samples taken from those who did not develop inflammatory bowel disease within a predetermined period and lower than the expression levels of biological samples taken from those who developed inflammatory bowel disease within a predetermined period.

[0029] In one embodiment, according to the testing method of the present invention, if the expression level measured in the measurement step of the expression level of Ms4a3 and / or CD84, preferably the expression level of Ms4a3, more preferably the expression level of both Ms4a3 and CD84, is higher than a first reference value or the expression level in a biological sample obtained from a healthy person, and lower than a second reference value, it can be used to assist in the diagnosis or determination of being in remission of inflammatory bowel disease, with regard to the diagnosis of inflammatory bowel disease or monitoring of disease progression. When measuring the expression levels of Ms4a3 and CD84, "the expression level measured in the measurement step of the expression level of Ms4a3 and CD84 is higher than a first reference value or the expression level in a biological sample obtained from a healthy person, and lower than a second reference value" means that the expression levels of Ms4a3 and CD84 measured in the measurement step are both higher than a first reference value or the expression level in a biological sample obtained from a healthy person, and lower than a second reference value.

[0030] In one embodiment, according to the testing method of the present invention, if the expression level measured in the step of measuring the expression level of Ms4a3 and / or CD84, preferably the expression level of Ms4a3, more preferably the expression level of both Ms4a3 and CD84, is higher than a second reference value, it can be used to assist in the diagnosis or determination of an active phase of inflammatory bowel disease, in relation to the diagnosis of inflammatory bowel disease or monitoring of disease progression. When measuring the expression levels of Ms4a3 and CD84, "the expression level measured in the step of measuring the expression levels of Ms4a3 and CD84 is higher than a second reference value" means that the expression levels of both Ms4a3 and CD84 measured in the measurement step are higher than a second reference value.

[0031] In one embodiment, according to the testing method of the present invention, if the expression level measured in the step of measuring the expression level of Ms4a3 and / or CD84, preferably the expression level of Ms4a3, more preferably the expression level of both Ms4a3 and CD84, is higher than a first reference value or the expression level in a biological sample obtained from a healthy person, and lower than a second reference value, it can assist in the diagnosis or determination that the patient is in the remission phase of inflammatory bowel disease, in relation to the diagnosis of inflammatory bowel disease or monitoring the progression of the disease. Furthermore, if the expression level measured in the step of measuring the expression level of Ms4a3 and / or CD84 is higher than the second reference value, it can assist in the diagnosis or determination that the patient is in the active phase of inflammatory bowel disease, in relation to the diagnosis of inflammatory bowel disease or monitoring the progression of the disease. The meanings of "the expression level measured in the Ms4a3 and CD84 expression level measurement step is higher than the first reference value or the expression level in a biological sample obtained from a healthy person, and lower than the second reference value" and "the expression level measured in the Ms4a3 and CD84 expression level measurement step is higher than the second reference value" when measuring the expression levels of Ms4a3 and CD84 are as described above.

[0032] In this specification, the method for setting the "first reference value" and the "second reference value" is not particularly limited, but it is preferable that they be set separately for the respective expression levels of Ms4a3 and CD84. In one embodiment, the "first reference value" may be a value set from a range that is higher than the expression level in biological samples taken from healthy individuals and lower than the expression level in biological samples taken from patients in remission of inflammatory bowel disease, based on the results of measuring the expression levels in biological samples taken from patients in remission of inflammatory bowel disease and healthy individuals who have been previously confirmed not to have inflammatory bowel disease. In another embodiment, the "second reference value" may be a value set from a range that is higher than the expression level in biological samples taken from patients in remission and lower than the expression level in biological samples taken from patients in the active phase, based on the results of measuring the expression levels in biological samples taken from patients in remission of inflammatory bowel disease and patients in the active phase of inflammatory bowel disease.

[0033] In one embodiment, the screening method of the present invention includes the following steps: (1) contacting Ms4a3-expressing cells with a test substance; (2) measuring the expression level or activity of Ms4a3 in the cells; and (3) comparing the expression level or activity with that of Ms4a3-expressing cells that have not been contacted with the test substance, and selecting a test substance that reduces the expression level or activity of Ms4a3 as an active ingredient or a candidate substance thereof.

[0034] In step (1), the test substance is brought into contact with Ms4a3-expressing cells. The method of bringing the test substance into contact with Ms4a3-expressing cells is not particularly limited; for example, the Ms4a3-expressing cells may be cultured in the presence of the test substance, or the contact may be carried out in a buffer after removing the culture medium. The concentration of the test substance brought into contact with the cells is not particularly limited; for example, it may be 0.1 pM to 1000 μM, 0.1 nM to 100 μM, 0.1 μM to 10 μM, etc. The time and temperature of contact between the cells and the test substance are not particularly limited and may be selected as appropriate. A control group is provided in which Ms4a3-expressing cells are not brought into contact with the test substance. Examples of Ms4a3-expressing cells include naturally occurring cells that express Ms4a3, recombinant cells genetically engineered to express Ms4a3, or cultures thereof. Naturally occurring cells that express Ms4a3 can be isolated, for example, from patients with inflammatory bowel disease using a cell sorter. Recombinant cells genetically engineered to express Ms4a3 can be produced, for example, by transforming cells using a vector incorporating the gene encoding human Ms4a3. In one embodiment, Ms4a3-expressing cells may also express CD84. Examples of Ms4a3 and CD84-expressing cells include native cells expressing Ms4a3 and CD84, genetically engineered recombinant cells expressing Ms4a3 and CD84, or cultures thereof. Native cells expressing Ms4a3 and CD84 can be isolated, for example, from patients with inflammatory bowel disease using a cell sorter. Recombinant cells genetically engineered to express Ms4a3 and CD84 can be produced, for example, by transforming cells using a vector incorporating the genes encoding human Ms4a3 and human CD84.

[0035] The test substance is not particularly limited and may be a known compound or a novel compound. Examples include nucleic acids, carbohydrates, lipids, proteins, peptides, organic small molecule compounds, compound libraries obtained by combinatorial chemistry or conventional synthesis techniques, random peptide libraries prepared by solid-phase synthesis or phage display, and natural components derived from microorganisms, plants and animals, marine organisms, etc. The test substance may be used dissolved or suspended in a buffer such as water, phosphoric acid, or Tris, or in a solvent such as ethanol, acetone, dimethyl sulfoxide, or a mixture thereof.

[0036] The method for measuring the expression level or activity of Ms4a3 in step (2) is not particularly limited, and known methods can be appropriately selected and used. The expression level can be measured in the same way as the expression level measurement in the testing method of the present invention described above. Specifically, for example, the protein expression level may be measured using a labeled antibody, or the gene expression level may be measured using quantitative PCR. The activity of Ms4a3 may be measured, for example, by measuring the inhibitory activity against a substance that has agonist activity against Ms4a3 (e.g., CD20), or the amount of downstream signal of Ms4a3 (e.g., phosphorylated CDK2 amount) may be measured as the activity of Ms4a3. When Ms4a3 and CD84 expressing cells are used, the expression level or activity of Ms4a3 and the expression level or activity of CD84 are measured in step (2). The method for measuring the expression level or activity of CD84 is not particularly limited, and can be measured in the same way as the method for measuring the expression level or activity of Ms4a3 described above. The activity of CD84 may be measured, for example, by measuring the binding inhibitory activity with a substance that has agonist activity against CD84.

[0037] In step (3), based on the measurement results from step (2), the expression level or activity of Ms4a3 is compared with Ms4a3-expressing cells that have not been in contact with the test substance, and the test substance that reduces the expression level or activity of Ms4a3 is selected as an active ingredient or candidate substance for an inflammatory bowel disease treatment. The degree to which the expression level or activity of Ms4a3 is reduced is not particularly limited, but for example, a test substance that reduces it by 90% or less, more preferably 70% or less, more preferably 50% or less, more preferably 30% or less, and more preferably 10% or less compared with Ms4a3-expressing cells that have not been in contact with the test substance can be selected as an active ingredient or candidate substance for an inflammatory bowel disease treatment. When using Ms4a3 and CD84 expressing cells, in step (3), the expression level or activity of Ms4a3 and CD84 is compared with that of Ms4a3 and CD84 expressing cells that have not been in contact with the test substance, and the test substance that reduces the expression level or activity of both Ms4a3 and CD84 is selected as the active ingredient or candidate substance for the inflammatory bowel disease treatment. The degree to which the expression level or activity of Ms4a3 and CD84 is reduced is not particularly limited, but for example, a test substance that reduces the expression level or activity of both Ms4a3 and CD84 by 90% or less, more preferably 70% or less, more preferably 50% or less, more preferably 30% or less, and more preferably 10% or less compared with that of Ms4a3 and CD84 expressing cells that have not been in contact with the test substance can be selected as the active ingredient or candidate substance for the inflammatory bowel disease treatment.

[0038] In one embodiment, the screening method of the present invention includes the following steps: (A) contacting a test substance with CDK2; (B) measuring the activity of CDK2; and (C) comparing the activity with that of a test substance that has not been in contact with the test substance, and selecting a test substance that reduces the activity as an active ingredient or a candidate substance thereof.

[0039] In step (A), the test substance is brought into contact with CDK2. CDK2 may be a CDK-cyclin complex such as CDK2 / cyclin A1, CDK2 / cyclin A2, or CDK2 / cyclin E1. The concentration of the test substance is not particularly limited and may be, for example, 0.1 pM to 1000 μM, 0.1 nM to 100 μM, or 0.1 μM to 10 μM. The time and temperature of contact with the test substance are not particularly limited and may be selected as appropriate. The test substance is as described above. A control group is provided in which CDK2 is not brought into contact with the test substance.

[0040] The method for measuring CDK2 activity in step (B) is not particularly limited, and known methods can be appropriately selected and used. For example, CDK2 activity may be measured by measuring the kinase activity of CDK-cyclin complexes such as CDK2 / cyclin A1, CDK2 / cyclin A2, and CDK2 / cyclin E1, or a commercially available kit such as the CDK2 Assay Kit (manufactured by BPS Bioscience) may be used.

[0041] In step (C), based on the measurement results from step (B), a test substance that reduces CDK2 activity compared to when CDK2 is not in contact with the test substance is selected as an active ingredient or candidate substance for an inflammatory bowel disease treatment. The degree to which CDK2 activity is reduced is not particularly limited, but for example, a test substance that reduces CDK2 activity by 90% or less, more preferably 70% or less, more preferably 50% or less, more preferably 30% or less, and more preferably 10% or less compared to when the test substance is not present can be selected as an active ingredient or candidate substance for an inflammatory bowel disease treatment. Candidate substances for active ingredients of inflammatory bowel disease treatments can also be used as lead compounds for synthesizing novel compounds with improved physical properties, pharmacokinetics, and / or safety.

[0042] The test composition of the present invention comprises at least one detection component for measuring the expression level of Ms4a3 and / or CD84 in a biological sample taken from a subject. The detection component is not particularly limited as long as it can measure the expression level of Ms4a3 and / or CD84, but can be any that is commonly used by those skilled in the art, such as an antibody that specifically binds to Ms4a3 and / or CD84, an antigen-binding fragment, a probe, or a mixture containing these. The detection component may be labeled with a labeling substance such as a fluorescent dye or an enzyme. The detection component can be a commercially available product, and can be used by adjusting the combination and concentration as needed. In addition to the detection component, the test composition of the present invention may optionally contain other components such as buffers, stabilizers, preservatives, diluents, and carriers. Depending on the type of detection component, the test composition of the present invention can be manufactured according to general methods, including known antibody production techniques and purification methods.

[0043] The test kit of the present invention includes at least one detection means for measuring the expression level of Ms4a3 and / or CD84, and may optionally include a buffer, a standard substance, a reaction vessel, instructions for use, etc. The detection means is not particularly limited as long as it is capable of measuring the expression level of Ms4a3 and / or CD84, but for example, the test composition may be used, and the measurement may be performed using an optical detection device (e.g., a flow cytometer, a microplate reader, etc.) or analytical software, etc., as needed. These devices and software do not necessarily have to be included in the test kit and may be provided separately. The test composition and the test kit of the present invention can be used to obtain information related to the state of inflammatory bowel disease, and can be used, for example, in the test method of the present invention.

[0044] To aid in understanding the present invention, the present invention will be specifically described below with reference to examples, but it goes without saying that the present invention is not limited to these examples. The following examples were approved by the Ethics Committee of Fujita Health University, and written informed consent was obtained from each patient. All procedures were carried out in accordance with approved guidelines and regulations.

[0045] (Example 1) Protein expression in samples derived from patients with inflammatory bowel disease (CD11b + CD33 + CD84 + 600 μL of blood was collected in EDTA2Na blood collection tubes from 18 healthy individuals and 21 patients with inflammatory bowel disease (29 with ulcerative colitis (UC) and 21 with Crohn's disease (CD)). 19 mL of ACK buffer (0.15 M NH4Cl, 0.1 M KHCO3, 0.0001 M EDTA2Na) was added to each sample, and the cells were incubated at room temperature for 5 minutes to lyse red blood cells. The cells were centrifuged at 500 g for 5 minutes at room temperature, the supernatant was removed, and 5 mL of PBS was added. The cells were centrifuged at 500 g for 5 minutes at room temperature, the supernatant was removed, and 50 μL of Fc Blocking Reagent human (Miltenyi Biotec) was added to the cell pellet. After incubation at room temperature for 5 minutes, 1 mL of PBS was added. The cells were centrifuged at 500g for 5 minutes at room temperature, the supernatant was removed, and antibody solutions (FITC-labeled anti-CD3 antibody (Biolegend), FITC-labeled anti-CD19 antibody (Biolegend), FITC-labeled anti-CD235a antibody (Biolegend), PE-Cy7-labeled anti-CD11b antibody (Biolegend), APC-labeled anti-CD33 antibody (Biolegend), and PE-labeled anti-CD84 antibody (Biolegend)) were added to the cell pellet. The cells were stained by incubation at 4°C for 20 minutes. After incubation, 1 mL of PBS was added. The cells were centrifuged at 500g for 5 minutes at room temperature, the supernatant was removed, and a 1% BSA / PBS solution containing 7-Amino-Actinomycin D (7-AAD; Biolegend) was added to the cell pellet to suspend it, and the sample was prepared for flow cytometry (Beckman CytoFLEX). Live cells were negative for CD3, CD19, and CD235a (CD3 - CD19 - CD235a - ) The cell fraction is separated, and from this fraction, CD11b and CD33-positive (CD11b + CD33 + The cell fractions were isolated, and CD84 expression was analyzed in each fraction.

[0046] As a result, both ulcerative colitis patients and Crohn's disease patients were positive for CD11b, CD33, and CD84 (CD11b + CD33 + CD84 + The rate was significantly higher in CD11b cells than in healthy individuals (Figure 1, ****P < 0.0001). "%Total" in Figure 1 represents the total number of CD11b cells detected by flow cytometry. + CD33 + CD84 + This refers to the percentage of cell counts. Therefore, the combination of CD11b, CD33, and CD84 has been shown to be a potential blood marker for inflammatory bowel disease.

[0047] (Example 2) Protein expression in samples from patients with inflammatory bowel disease (granulocyte CD11b + CD33 + CD84 + monocyte CD11b + CD33 + CD84 + The cell pellet isolated in Example 1 was stained in the same manner as in Example 1, and the CD3, CD19, and CD235a negative cell fractions were separated from the viable cells. Further, the CD11b and CD33 positive cell fractions were separated from these fractions, and CD84 expression was analyzed in these fractions. The CD84 positive cell population was then analyzed using a method with high side scatter (SSC) high ) A population of granulocytes (polymorphonuclear leukocytes: PMNs) and low (SSCs) low They were classified into a population of monocyte-type cells (M).

[0048] As a result, both ulcerative colitis patients and Crohn's disease patients had CD11b, CD33, and CD84-positive granulocytes and CD11b, CD33, and CD84-positive monocytes (CD11b + CD33 + CD84 + SSC high and CD11b + CD33 + CD84 + SSC lowThe ratio of + CD33 + CD84 + SSC high the number of cells (left graph) and + CD33 + CD84 + SSC low was higher in patients with inflammatory bowel disease than in healthy subjects, and the ratio of CD11b, CD33, and CD84-positive granulocytes was particularly high (Figure 2, ****P < 0.0001, ***P < 0.001). "% Total" in Figure 2 means the ratio (%) of the number of CD11b cells (right graph) among all the cells detected by flow cytometry. Therefore, it was shown that CD11b, CD33, and CD84-positive granulocytes can be particularly excellent blood markers for inflammatory bowel disease.

[0049] (Example 3) Protein expression in samples from patients with inflammatory bowel disease (CD84 + , CD11b + CD84 + , CD33 + CD84 + ) After staining the cell pellet isolated in Example 1 in the same manner as in Example 1, the expression of CD84, CD11 and CD84, and CD33 and CD84 in live cells was analyzed.

[0050] As a result, both in patients with ulcerative colitis and Crohn's disease, the rates of CD84-positive (CD + ), CD11b and CD84-positive (CD11b + CD84 + ), and CD33 and CD84-positive (CD33 + CD84 + CD84 + ) were all significantly higher than those in healthy subjects (Figure 3, ****P < 0.0001). "%Total" in Figure 3 means the ratio (%) of the number of CD84 cells (left graph), CD11b + CD84 + cells (middle graph), and CD33 + CD84 + cells (right graph) among all the cells detected by flow cytometry. Therefore, it was shown that CD84 can be a marker for inflammatory bowel disease alone. ​​​​​​​​

[0051] (Example 4) Protein expression in samples from patients with inflammatory bowel disease (CD11b + CD33 + Ms4a3 + , CD11b + CD33 + CD84 + Ms4a3 + ) Analysis The cell pellet isolated in Example 1 was stained with various labeled antibodies similar to those in Example 1 (APC-Cy7 labeled anti-CD3 antibody (Biolegend), APC-Cy7 labeled anti-CD19 antibody (Biolegend), APC-Cy7 labeled anti-CD235a antibody (Biolegend), PE-Cy7 labeled anti-CD11b antibody (Biolegend), APC labeled anti-CD33 antibody (Biolegend) and PE labeled anti-CD84 antibody (Biolegend)) and Alexa Fluor 488 labeled anti-Ms4a3 + antibody (R&D Systems, Inc.), and dead cells were stained with 7-AAD. CD3, CD19 and CD235a negative (CD3 - CD19 - CD235a - ) cell fractions were isolated, and further CD11b and CD33 positive (CD11b + CD33 + ) cell fractions were isolated, and the expression of Ms4a3 and CD84 in this fraction was analyzed.

[0052] As a result, the rates of CD11b, CD33 and Ms4a3 positive (CD11b + CD33 + Ms4a3 + ) and the rates of CD11b, CD3, CD84 and Ms4a3 positive (CD11b + CD33 + CD84[[ID=4I]]<00C0109>Ms4a3 + ) were significantly higher in patients with ulcerative colitis and Crohn's disease than in healthy subjects (*P < 0.05 in Figure 4). "% Total" in Figure 4 represents the number of CD11b + CD33 + Ma4a3 + cells (left graph) and CD11b +CD33 + CD84 + Ma4a3 + This represents the percentage of cell count (right graph). Therefore, the combinations of CD11b, CD33, and Ms4a3, as well as the combinations of CD11b, CD33, CD84, and Ms4a3, have been shown to be potential blood markers for inflammatory bowel disease.

[0053] (Example 5) Protein expression in samples derived from patients with inflammatory bowel disease (Ms4a3 + The cell pellet isolated in Analysis Example 1 was stained in the same manner as in Example 4, and then the expression of Ms4a3 in live cells was analyzed.

[0054] As a result, both ulcerative colitis patients and Crohn's disease patients tested positive for Ms4a3 (Ms4a3 + The rate was significantly higher in Ms4a3 compared to healthy individuals (Figure 5, ***P<0.001, **P<0.01), and Ms4a3 + We confirmed that the cells can serve as a marker for inflammatory bowel disease. "% Total" in Figure 5 represents the number of Ma4a3 cells out of the total number of cells detected by flow cytometry. + This refers to the percentage of cell numbers. Therefore, it was shown that Ms4a3 alone can serve as a marker for inflammatory bowel disease.

[0055] (Example 6) Protein expression in samples derived from patients with inflammatory bowel disease (Ms4a3 + CD84 + The cell pellet isolated in Analysis Example 1 was stained in the same manner as in Example 4, and then the expression of Ms4a3 and CD84 in live cells was analyzed.

[0056] As a result, both ulcerative colitis patients and Crohn's disease patients were positive for Ms4a3 and CD84 (Ms4a3 + CD84 + The rate was significantly higher in CD84 cells than in healthy individuals (Figure 6, **P < 0.01). In Figure 6, "%Total" represents the total number of cells detected by flow cytometry, out of a total CD84 cell count. + Ma4a3 + This refers to the percentage of cell numbers. From the comparison of Figures 1, 3, 5, and 6, Ms4a3 + CD84 +The cells showed the least overlap with those of healthy individuals (HC). Therefore, the combination of Ms4a3 and CD84 was shown to be a particularly sensitive marker for inflammatory bowel disease.

[0057] (Example 7) Protein expression in samples from inflammatory bowel disease patients in the estimated active and estimated remission phases (CD11b + CD33 + CD84 + CD84 + Ms4a3 + Ms4a3 + CD84 + In Analysis Example 1, serum was separated from blood samples collected from 29 UC patients and 21 CD patients. LRG was measured by latex immunoturbidimetry, and CRP was measured by latex agglutination turbidimetry. Since the reference LRG values ​​for healthy individuals obtained from whole blood samples from healthy individuals ranged from 6.48 to 13.92 μg / mL, patients with LRG > 15 μg / mL and CRP > 0.3 mg / dL were determined to be in the estimated active phase (Active), and patients with LRG < 14 μg / mL and CRP < 0.3 mg / dL were determined to be in the estimated remission phase (Rem). The cell pellet obtained in Example 1 was stained in the same manner as in Example 4, and after staining, the expression of CD11b, CD3, and CD84, CD84 expression, Ms4a3 expression, and Ms4a3 and CD84 expression in live cells were analyzed.

[0058] As a result, CD11b, CD33, and CD84 were positive during the active and remission phases (CD11b + CD33 + CD84 + ) rate, CD84 positive (CD84 + ) rate, Ms4a3 positive (Ms4a3 + ) rate, and Ms4a3 and CD84 positive (Ms4a3 + CD84 + The rates were significantly higher in both cases than in healthy individuals (Figure 7, ****P < 0.0001, **P < 0.01). "% Total" in Figure 7 represents the total number of CD11b cells detected by flow cytometry. + CD33 + CD84 + Cell count (first graph from the left), CD84 +Cell count (second graph from the left), Ma4a3 + Cell count (third graph from the left) and Ma4a3 + CD84 + The cell count (fourth graph from the left) represents the percentage of the cell count. Therefore, CD84 + Cells and Ms4a3 + The cells were shown to increase not only during the active phase of inflammatory bowel disease but also during the remission phase. Furthermore, Ms4a3-positive (Ms4a3 + ) rate, and Ms4a3 and CD84 positive (Ms4a3 + CD84 + The rate was significantly higher during the active phase than during the remission phase (Figure 7, *P < 0.05, ***P < 0.001), Ms4a3 + CD84 + The cell is Ms4a3 + The overlap area between Ms4a3 and healthy individuals was smaller than that between cells and patients with inflammatory bowel disease (IBD). Therefore, it was shown that Ms4a3 can not only distinguish between healthy individuals and patients with IBD, but also determine the remission and active phases of IBD, which cannot be distinguished by the conventional inflammatory marker LRG. Furthermore, combining Ms4a3 with CD84 can further improve the accuracy of determining the remission and active phases of IBD.

[0059] (Example 8) Verification of CDK2 inhibitory effect using an inflammatory bowel disease model animal The effect of inhibitors targeting CDK2 (Cyclin Dependent Kinase 2), a downstream signaling molecule of Ms4a3 involved in cell proliferation, on improving inflammatory bowel disease was verified using a dextran sulfate sodium (DSS) induced inflammatory bowel disease model animal. C57BL / 6J mice were given an aqueous 2% DSS (dextran sulfate sodium salt; number average molecular weight 36,000-50,000) solution as free drinking water for 7 days. The day on which the DSS intake began was considered day 1. The control group was given water as free drinking water for 7 days. The CDK2 inhibitor CVT-313 was administered intraperitoneally once daily from day 1 to day 7 at a dose of 10 mg / kg, and the effect on improving inflammatory bowel disease was verified using the DAI score. On day 8, the mice were euthanized and the characteristics of their intestines were observed. The rate of weight loss, stool characteristics, and presence or absence of blood in the stool of mice were scored according to the criteria shown in Table 1, and this was used as the DAI score. Weight measurements were taken in the mornings of days 0 to 7. Stool consistency and the presence or absence of blood in the stool were visually checked after collecting fresh stool samples from mice in individual cages.

[0060] As a result, when CDK2 inhibitors were administered, the DAI score from day 5 after inflammation onset was significantly lower and symptoms were significantly reduced compared to a DSS-induced inflammatory bowel disease model that did not receive CDK2 inhibitors (Figure 8, **P < 0.01). This suggests that targeting Ms4a3 or its downstream signaling molecule CDK2, which are potentially increased even during remission, can be used to screen for inflammatory bowel disease treatments that can maintain a more potent and sustained remission.

[0061]

[0062] The present invention's method for testing inflammatory bowel disease can assist in the diagnosis of inflammatory bowel disease, monitoring of disease progression, or evaluation of treatment effectiveness. By assisting in the diagnosis of inflammatory bowel disease, monitoring of disease progression, or evaluation of treatment effectiveness, it can contribute to finding more optimal treatment methods. Furthermore, the screening method for active ingredients of inflammatory bowel disease therapeutic agents according to the present invention is expected to lead to the development of potent and sustained remission maintenance therapies for inflammatory bowel disease.

Claims

1. A diagnostic method for assisting in the diagnosis of inflammatory bowel disease, monitoring of disease progression, or evaluation of treatment effectiveness, comprising the step of measuring the expression level of at least one selected from the group consisting of Ms4a3 and CD84 in a biological sample taken from a subject.

2. The testing method according to claim 1, further comprising the step of measuring the expression level of Ms4a3 in a biological sample collected from a subject.

3. The testing method according to claim 1, further comprising the step of measuring the expression levels of Ms4a3 and CD84 in a biological sample collected from a subject.

4. The testing method according to any one of claims 1 to 3, wherein if the expression level is higher than a reference value or the expression level in a biological sample obtained from a healthy person, it indicates that the person has inflammatory bowel disease, or that a decrease or maintenance of the expression level indicates that there is a maintenance or therapeutic effect on the remission phase of inflammatory bowel disease.

5. The testing method according to any one of claims 1 to 3, wherein if the expression level is higher than a first reference value or the expression level in a biological sample obtained from a healthy person, and lower than a second reference value, it indicates that the patient is in the remission phase of inflammatory bowel disease, and / or if it is higher than the second reference value, it indicates that the patient is in the active phase of inflammatory bowel disease.

6. The method according to any one of claims 1 to 3, wherein the biological sample is a blood-derived sample.

7. The examination method according to any one of claims 1 to 3, wherein the inflammatory bowel disease is selected from the group consisting of ulcerative colitis and Crohn's disease.

8. A method for screening active ingredients for an inflammatory bowel disease treatment, comprising: (1) contacting Ms4a3-expressing cells with a test substance; (2) measuring the expression level or activity of Ms4a3 in the cells; and (3) comparing the expression level or activity with that of Ms4a3-expressing cells that have not been contacted with the test substance, and selecting a test substance that reduces the expression level or activity of Ms4a3 as an active ingredient or candidate substance for an inflammatory bowel disease treatment.

9. The screening method according to claim 8, wherein the Ms4a3-expressing cells are also cells that express CD84, the expression level or activity of Ms4a3 and the expression level or activity of CD84 are measured in step (2), and the expression level or activity of Ms4a3 and the expression level or activity of CD84 are compared with Ms4a3 and CD84-expressing cells that have not been in contact with the test substance in step (3), and the test substance that reduces the expression level or activity of both Ms4a3 and CD84 is selected as an active ingredient or candidate substance for an inflammatory bowel disease treatment drug.

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