Method for testing severity of rheumatoid arthritis

Abstract

The present invention relates to a method for testing a severity of rheumatoid arthritis or a risk of developing rheumatoid arthritis in a subject. Since a method for testing a severity of rheumatoid arthritis and a technique relating to the same and a screening method for development of a therapeutic agent for rheumatoid arthritis can be provided according to the present invention, the present invention can be used in a medical field or a pharmaceutical field associated with rheumatoid arthritis.

Description

DESCRIPTION TITLE OF THE INVENTION: METHOD FOR TESTING SEVERITY OF RHEUMATOID ARTHRITIS TECHNICAL FIELD

[0001] The present invention relates to a method for testing a severity of rheumatoid arthritis or a risk of developing rheumatoid arthritis, and technique relating to the same. Further, the present invention relates to a method for screening a therapeutic agent for rheumatoid arthritis. BACKGROUND ART

[0002] Although genomic traits acquired during evolution of a human include an autoimmune disease, a molecular level mechanism of a human immune response controlled by these traits has not yet been elucidated (Non-Patent Publication 1). Rheumatoid arthritis (RA) is one of autoimmune diseases and causes systemic arthritis accompanying hyperplasia of a synovial tissue. Consequently, deformation or deconstruction of joints is caused. Aggressive lymphocytes infiltration or a formation of tertiary lymphoid structure (TLS) occurs at a synovium of a patient with rheumatoid arthritis, leading enhancement of in situ adaptive immune response such as help of B cells by peripheral helper T cells (Tph cells) (Non-Patent Publication 2). Further, a monocyte-macrophage system and fibroblast-like synovial cells (FLS) which play an extremely important role in an innate immunity constitute a malignant inflammation cycle of rheumatoid arthritis and lead thickness of synovium, production of matrix-degrading enzyme and induction of osteoclast (Non-Patent Publication 3, Non-Patent Publication 4 and Non-Patent Publication 5).

[0003] Involvement of an autoantibody and MHC class II having common epitope in rheumatoid arthritis suggests that CD4 positive T cells are strongly associated with development of rheumatoid arthritis (Non-Patent Publication 6). PRIOR ART REFERENCES NON-PATENT PUBLICATIONS

[0004] Non-Patent Publication 1: Benton, M. L. et al. The influence of evolutionary history on human health and disease. Nat Rev Genet 22, 269- 283, doi:10.1038 / s41576-020-00305-9 (2021) Non-Patent Publication 2: Rao, D. A. et al. Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis. Nature 542, 110-114, doi:10.1038 / nature20810 (2017). Non-Patent Publication 3: Mizoguchi, F. et al. Functionally distinct disease-associated fibroblast subsets in rheumatoid arthritis. Nature communications 9, 789, doi:10.1038 / s41467-018-02892-y (2018) Non-Patent Publication 4: Alivernini, S. et al. Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis. Nature medicine 26, 1295-1306, doi: 10.1038 / s41591-020-0939-8 (2020) Non-Patent Publication 5: Nygaard, G. & Firestein, G. S. Restoring synovial homeostasis in rheumatoid arthritis by targeting fibroblast-like synoviocytes. Nature reviews. Rheumatology 16, 316-333, doi:10.1038 / s41584-020-0413-5 (2020) Non-Patent Publication 6: McInnes, I. B. & Schett, G. The Pathogenesis of Rheumatoid Arthritis. New Engl J Med 365, 2205-2219, doi:10.1056 / nejmra1004965 PMID - 22150039 (2011) SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0005] However, as to how human CD4 positive T cells control an inflammatory immune response in a patient with rheumatoid arthritis, a research has been continued. <semantics>⌈0006⌉<annotation encoding="application / x-tex">\lceil 0006 \rceil< / annotation>< / semantics> The present invention was made by taking the circumstances into account, and an object thereof is to provide a method for testing a severity of rheumatoid arthritis or a risk of developing rheumatoid arthritis in a subject. Establishment of a method for testing a severity of rheumatoid arthritis or a risk of developing rheumatoid arthritis in a subject is an effective tool not only for confirming a clinical pathology of a subject but also developing a therapeutic agent for rheumatoid arthritis. Further, the present invention relates to provision of a method for screening a therapeutic agent for rheumatoid arthritis. MEANS TO SOLVE THE PROBLEMS

[0007] The present invention relates to the following [1] to

[13] : [1] A method for testing a severity of rheumatoid arthritis or a risk of developing rheumatoid arthritis in a subject including: measuring an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample derived from a subject with rheumatoid arthritis or a subject suspected to develop rheumatoid arthritis, and comparing the measured value with a threshold value corresponding to the amount of the IGFL2 protein or the expression amount of the IGFL2 gene. [2] The method according to the above [1], wherein a case where the measured value is equal to or higher than the threshold value indicates a high severity of rheumatoid arthritis or a high risk of developing rheumatoid arthritis in the subject. [3] The method according to the above [1] or [2], wherein the sample is a body fluid, blood cells, or a tissue. [4] A method for providing information for judging and / or monitoring a severity of rheumatoid arthritis in a subject with rheumatoid arthritis or a risk of developing rheumatoid arthritis in a subject suspected to develop rheumatoid arthritis including: measuring an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample derived from the subject, and providing information relating to the measured value. [5] The method according to the above [4], wherein the sample is a body fluid, blood cells, or a tissue. [6] A use of an IGFL2 protein or an IGFL2 gene in a sample derived from a subject with rheumatoid arthritis or suspected to develop rheumatoid arthritis as a biomarker for judging a severity of rheumatoid arthritis or a risk of developing rheumatoid arthritis in the subject, wherein the judgment includes comparing the measured value of an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample derived from the subject with a threshold value corresponding to the amount of the IGFL2 protein or the expression amount of the IGFL2 gene. [7] The use according to the above [6], wherein a case where the measured value is equal to or higher than the threshold value indicates a high severity of rheumatoid arthritis or a high risk of developing rheumatoid arthritis in the subject. [8] The use according to the above [6] or [7], wherein the sample is a body fluid, blood cells, or a tissue. [9] A method for evaluating effectiveness of a treatment of rheumatoid arthritis including: measuring an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample derived from a subject after receiving a treatment with a therapeutic agent or a therapy to be tested, and comparing the measured value with a measured value derived from the subject before receiving the treatment. 10 The method according to the above [9], wherein a case where the measured value is lower than the measured value derived from the subject before receiving the treatment indicates an effective treatment. |11| The method according to the above [9] or

[10] , wherein the sample is a body fluid, blood cells, or a tissue. 1121 A method for screening a therapeutic agent for rheumatoid arthritis including: dosing an IGFL2 protein and a therapeutic agent to be tested to cells expressing an IGFL2 receptor, and measuring an amount of a bioactive substance produced by the cells. <semantics>⌈13⌉<annotation encoding="application / x-tex">\lceil 13 \rceil< / annotation>< / semantics> The method according to the above

[12] , wherein the bioactive substance is a bioactive substance produced by an IGFL2 protein stimulation. EFFECTS OF THE INVENTION

[8000] According to the present invention, a method for testing and judging a severity of rheumatoid arthritis or a risk of developing rheumatoid arthritis using a measured value of an expression of an IGFL2 protein or an IGFL2 gene in a subject, and a method for evaluating effectiveness of a treatment of rheumatoid arthritis using the above measured value can be provided. Further, the present invention relates to provision of a method for screening a therapeutic agent for rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] [FIG. 1] FIG. 1 is a scatter plot showing a correlation between an average of an expression amount of an IGFL2 gene by CD4 positive T cells in a synovium and a disease activity (DAS28-ESR or ESR). Each dot in the figure represents an individual patient. [FIG. 2] FIG. 2 is a graph showing the concentration of an IGFL2 protein in a serum between a group of healthy controls and a group of patients with rheumatoid arthritis. In the FIG. 2, data was shown by dots and mean <semantics>±<annotation encoding="application / x-tex">\pm< / annotation>< / semantics> SD and unpaired two-tailed t-test was carried out. [FIG. 3] FIG. 3 is an ROC curve and an AUC value relating to judgement of a patient with rheumatoid arthritis based on a concentration of an IGFL2 protein in a serum from a group of healthy controls and a group of patients with rheumatoid arthritis. [FIG. 4] FIG. 4 is a graph showing a concentration of an IGFL2 protein in a serum of a group of patients with remission of rheumatoid arthritis and a group of patients with non-remission of rheumatoid arthritis. In FIG. 4, data was represented by a dot and mean <semantics>±<annotation encoding="application / x-tex">\pm< / annotation>< / semantics> SD, and an unpaired two-tailed t-test was carried out. [FIG. 5] FIG. 5 is an ROC curve and an AUC value relating to judgement of remission of rheumatoid arthritis based on a concentration of an IGFL2 protein in a serum of a group of patients with remission of rheumatoid arthritis and a group of patients with non-remission of rheumatoid arthritis. [FIG. 6] FIG. 6 is a graph showing an amount of various bioactive substances in a culture supernatant. A culture supernatant obtained by adding an IGFL2 protein was shown as IGFL2 +, and a culture supernatant without addition was shown as IGFL2 -. Data was represented by a dot and mean <semantics>±<annotation encoding="application / x-tex">\pm< / annotation>< / semantics> SD, and an unpaired two-tailed t-test was carried out. [FIG. 7] FIG. 7 is a graph showing an amount of various bioactive substances in a culture supernatant. A culture supernatant obtained by adding an IGFL2 protein was shown as IGFL2 +, and a culture supernatant without addition was shown as IGFL2 -. Data was represented by a dot and mean <semantics>±<annotation encoding="application / x-tex">\pm< / annotation>< / semantics> SD, and an unpaired two-tailed t-test was carried out. [FIG. 8] FIG. 8 is a graph showing an amount of various bioactive substances in a culture supernatant. A culture supernatant obtained by adding an IGFL2 protein was shown as IGFL2 +, and a culture supernatant without addition was shown as IGFL2 -. Data was represented by a dot and mean <semantics>±<annotation encoding="application / x-tex">\pm< / annotation>< / semantics> SD, and an unpaired two-tailed t-test was carried out. [FIG. 9] FIG. 9 is a figure showing an action which an anti-IGFLR1 antibody gives to production of CXCL10 by IGFL2 receptor-constitutively expressing THP1 cells. In the figure, data was represented by a dot and mean <semantics>±<annotation encoding="application / x-tex">\pm< / annotation>< / semantics> SD, and an unpaired two-tailed t-test was carried out. In the figure, a concentration of CXCL10 when adding a human recombinant IGFL2 protein and an isotype control antibody is shown as IGFL2IgG1, and a concentration of CXCL10 when adding a human recombinant IGFL2 protein and FLR which is an IGFLR1 neutralizing antibody is shown as IGFL2IGFLR1. MODES FOR CARRYING OUT THE INVENTION

[0010] In the present invention, the "rheumatoid arthritis (RA)" is, but not particularly limited to, one of autoimmune diseases and causes systemic arthritis accompanying hyperplasia of a synovial tissue. For example, the disease generally refers to a disease satisfying a classification criteria of American College of Rheumatology, 1987 or a classification criteria of ACR / EULAR Rheumatoid Arthritis, 2010. <semantics>⌈0011⌉<annotation encoding="application / x-tex">\lceil 0011 \rceil< / annotation>< / semantics> In the present invention, the "developing rheumatoid arthritis" refers to, but not particularly limited to, a case where a subject has a condition of rheumatoid arthritis mentioned above, and for example, can be judged according to the above-mentioned judgement criteria.

[0012] In the present invention, the "subject" refers to, but not particularly limited to, for example, a human or non-human ape, and the subject includes not only ones developing rheumatoid arthritis but also ones which can develop rheumatoid arthritis. A human with rheumatoid arthritis may be described as a patient. The subject suspected to develop rheumatoid arthritis includes a person who subjectively suspects to develop rheumatoid arthritis by himself, a person in which an examination such as a physical examination relating to the present invention is encompassed in examination items, a person based on some objective supports and / or a person who is judged to have reasonable development possibility by a physician as a result of an observation. In the present invention, the "risk" refers to a risk of developing rheumatoid arthritis by a subject suspected to develop rheumatoid arthritis. In the present invention, the "healthy person" refers to a subject in which the entirety of the autoimmune diseases are denied, and is determined by a physician's diagnosis based on conditions such as an autoantibody, a joint swelling, a joint tenderness, an inflammation reaction, an X-ray findings and a stiffness in hands. <semantics>⌈0014⌉<annotation encoding="application / x-tex">\lceil 0014 \rceil< / annotation>< / semantics> In the present invention, the "derivation" refers to, but not particularly limited to, for example, ones which can harvested from a subject by a blood collection, an arthroscopy, a puncture, a biopsy or an operation. In the present invention, the "sample" is, but not particularly limited to, for example, preferably a body fluid, blood cells or a tissue. The example of the body fluid includes whole blood, plasma, serum, synovial fluid, urine and saliva. The examples of the blood cells include peripheral blood mononuclear cells, peripheral blood T cells and peripheral blood CD4 positive T cells. The example of the tissue includes synovium and ligament.

[0016] In the present invention, the "IGFL2 protein" refers to an insulin- like growth factor (IGF) family member 2. The sequence information of a human IGFL2 protein and a human IGFL2 gene is known and can be found from a known database, for example, NCBI (National Center for Biotechnology Information) and the like. Specifically, the sequence information of a human IGFL2 gene is shown in NM_001002915.3. At present, an IGFL2 gene is found in a human and an ape as shown in the following Table 1.

[0017] [Table 1] . Table 1 [Image disponible dans le document PDF, Image available in the PDF document]

[0018] The method of measuring an amount of an IGFL2 protein in the present invention includes, but not particularly limited to, for example, a measurement method by an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), and a western blotting. The measurement subject of an IGFL2 protein may be a full-length protein or a fragment of the protein. In addition, the measured value may be an absolute amount or a concentration.

[0019] The measurement method of an expression amount of an IGFL2 gene in the present invention includes, but not particularly limited to, for example, an RNA sequencing, a single cell RNA sequencing, a quantitative PCR, a LAMP method and the like. The measurement subject of the IGFL2 gene includes a DNA coding an entirety of a gene or a part thereof, a cDNA, or an RNA. As the measured value of the amount of an IGFL2 protein or the expression amount of an IGFL2 gene, for example, an average of several times of measurements can be used. In addition, the measured value may be a measured value of one measurement subject or a value calculated by using plural measurement subjects. In this calculation, any coefficients and / or constants may be further used as needed. <semantics>⌈0021⌉<annotation encoding="application / x-tex">\lceil 0021 \rceil< / annotation>< / semantics> The present invention is completed based on the relationship between rheumatoid arthritis and an amount of an IGFL2 protein or an expression amount of an IGFL2 gene, and the specific embodiment of the present invention is exemplified hereinbelow. <semantics>⌈0022⌉<annotation encoding="application / x-tex">\lceil 0022 \rceil< / annotation>< / semantics> < Method for Testing Severity of Rheumatoid Arthritis or Risk of Developing Rheumatoid Arthritis in Subject > In one embodiment, the present invention is a method for testing a severity of rheumatoid arthritis or a risk of developing rheumatoid arthritis in a subject including: measuring an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample derived from a subject with rheumatoid arthritis or a subject suspected to develop rheumatoid arthritis, and comparing the measured value with a threshold value corresponding to the amount of the IGFL2 protein or the expression amount of the IGFL2 gene.

[0023] In the step of "measuring an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample," these biomarkers are quantified. In the step of "comparing the measured value with a threshold value corresponding to the amount of the IGFL2 protein or the expression amount of the IGFL2 gene," the measured value is compared with the threshold value. The test method of the present invention is carried out by the two steps. Moreover, in the test method of the present invention, a case where the measured value is equal to or higher than the threshold value indicates a high severity of rheumatoid arthritis or a high risk of developing the disease in a subject. In the present invention, "threshold value" corresponding to the amount of the IGFL2 protein or the expression amount of the IGFL2 gene is not particularly limited, and can be appropriately set. For example, the given threshold value can be empirically set by accumulating data of the amount of the IGFL2 protein or the expression amount of the IGFL2 gene in a patient with rheumatoid arthritis. More specifically, samples are collected from plural patients diagnosed to have rheumatoid arthritis and plural healthy persons, to provide a measured value of the amount of the IGFL2 protein or the expression amount of the IGFL2 gene. Moreover, a value which can most precisely distinguish a group of patients from a group of healthy persons is obtained, and the value is set as a threshold value. When setting the threshold value, it is preferred to consider a sensitivity, a specificity, a positive predictive value, a negative predictive value and the like.

[0026] In the present invention, the "severity of rheumatoid arthritis" can be evaluated by, for example, a disease activity. In the present invention, the "disease activity" is, for example, a value represented as DAS-28-ESR or ESR, and the disease activity is classified to high when the value of DAS-28-ESR is higher than 5.1, the disease activity is classified to moderate when the value is from 3.2 to 5.1, the disease activity is classified to mild when the value is smaller than 3.2, and the disease activity is classified to remission when the value is smaller than 2.6. < Method for Providing Information for Judging and / or Monitoring Severity of Rheumatoid Arthritis or Risk of Developing Rheumatoid Arthritis in Subject > In one embodiment, the present invention is a method for providing information for judging and / or monitoring a severity of rheumatoid arthritis in a subject with rheumatoid arthritis or a risk of developing rheumatoid arthritis in a subject suspected to develop rheumatoid arthritis including: measuring an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample derived from the subject, and providing information relating to the measured value. In the present invention, "judgement" and / or "monitoring" of "severity of rheumatoid arthritis" or "risk of developing rheumatoid arthritis" refers to, for example, determining which severity of rheumatoid arthritis represented as mentioned above the subject falls under ("judgement"), and determining the temporal change ("monitoring"). The judgement includes judgement of presence or absence of rheumatoid arthritis, judgement of a risk of developing rheumatoid arthritis, judgement of a severity of rheumatoid arthritis in a subject, judgement of a preventive effect of rheumatoid arthritis in a subject, judgement of a therapeutic effect in a patient with rheumatoid arthritis, judgement of presence or absence of recurrence of rheumatoid arthritis in a subject, and judgement of a risk of recurrence of rheumatoid arthritis in a subject.

[0029] The step of "measuring an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample" is same as the above. In the step of "providing information relating to the measured value" of the present invention, "providing information" is, for example, provision of information relating to the measured value obtained in the above step of measuring (e.g. the measured value and the threshold value) to a physician and the like. Information can be provided to, for example, a medical technologist in addition to a physician. The physician, for example, can use the information provided in judgement and / or monitoring of a severity of rheumatoid arthritis in a situation independent from the step of the present invention. The method for providing information of the present invention is carried out by the above two steps. < Use of IGFL2 Protein or IGFL2 Gene in Sample as Biomarker > In one embodiment, the present invention is a use of an IGFL2 protein or an IGFL2 gene in a sample derived from a subject with rheumatoid arthritis or suspected to develop rheumatoid arthritis as a biomarker for judging a severity of rheumatoid arthritis or a risk of developing rheumatoid arthritis in the subject, wherein the judgement includes: comparing the measured value of an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in the sample derived from the subject with a threshold value corresponding to the amount of the IGFL2 protein or the expression amount of the IGFL2 gene. <semantics>⌈0032⌉<annotation encoding="application / x-tex">\lceil 0032 \rceil< / annotation>< / semantics> In the present invention, the use of the IGFL2 protein or the IGFL2 gene as a biomarker refers to, for example, a use of the measured value of the IGFL2 protein or the measured value of the expression amount of IGFL2 gene as an index for judging a severity of rheumatoid arthritis or judging a risk of developing rheumatoid arthritis.

[0033] The "measured value of an amount of an IGFL2 protein or an expression amount of an IGFL2 gene" can be obtained by, for example, the step of "measuring an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample" mentioned above.

[0034] The step of "comparing the measured value of an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample derived from the subject with a threshold value corresponding to the amount of the IGFL2 protein or the expression amount of the IGFL2 gene" is same as the step of "comparing the measured value with a threshold value corresponding to the amount of the IGFL2 protein or the expression amount of the IGFL2 gene" mentioned above.

[0035] < Method for Evaluating Effectiveness of Treatment of Rheumatoid Arthritis > In one embodiment, the present invention is a method for evaluating effectiveness of a treatment of rheumatoid arthritis including: measuring an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample derived from a subject after receiving a treatment with a therapeutic agent or a therapy to be tested, and comparing the measured value with a measured value derived from the subject before receiving the treatment.

[0036] In the present invention, "treatment of rheumatoid arthritis" refers to, for example, reducing, improving or allowing to be remission of the severity of rheumatoid arthritis mentioned above.

[0037] In the present invention, the "effectiveness of treatment" refers to, for example, occurrence of reduction, improvement or remission of the severity of rheumatoid arthritis mentioned above. Evaluation of these occurrences is referred to as, for example, "evaluation of effectiveness of treatment."

[0038] In the present invention, the therapeutic agent or the therapy to be tested includes, but not particularly limited to, a steroid, a NSAID (Non- Steroidal Anti-Inflammatory Drug), a conventional synthetic molecular disease-modifying anti-rheumatic drug (csDMARDS), a targeted synthetic disease-modifying anti-rheumatic drug (tsDMARDS), an LCAP, synovectomy, and the like. In addition, a subject receiving them is referred to as a subject after receiving a treatment. <semantics>⌈0039⌉<annotation encoding="application / x-tex">\lceil 0039 \rceil< / annotation>< / semantics> The step of "measuring an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample derived from a subject after receiving a treatment with a therapeutic agent or a therapy to be tested" is similar to the step of "measuring an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample derived from a subject" mentioned above.

[0040] In the step of "comparing the measured value with a measured value derived from the subject before receiving the treatment," the measured value obtained in the above step is compared with a measured value derived from the subject before receiving the treatment. In the present invention, a measured value derived from the subject before receiving a treatment refers to, for example, a measured value of an expression of an IGFL2 protein or a gene before receiving the treatment.

[0041] The method for evaluating effectiveness of a treatment of rheumatoid arthritis is carried out by the above two steps. Moreover, in the evaluation method of the present invention, a case where the measured value is lower than the measured value derived from the subject before receiving the treatment indicates an effective treatment. Specifically, a case where the measured value is shown to be significantly lower than that of a control group by unpaired two-tailed t- test indicates an effective treatment.

[0042] < Method for Screening Therapeutic Agent for Rheumatoid Arthritis > An additional embodiment of the present invention is a method for screening a therapeutic agent for rheumatoid arthritis including: dosing an IGFL2 protein and a therapeutic agent to be tested to cells expressing an IGFL2 receptor, and measuring the amount of a bioactive substance produced by the cells.

[0043] In the step of "dosing an IGFL2 protein and a therapeutic agent to be tested to cells expressing an IGFL2 receptor," the cells expressing an IGFL2 receptor are preferably cells constitutively expressing an IGFL2 receptor. The cells can constitutively express an IGFL2 receptor by transducing cells expressing an IGFL2 receptor among human cells, for example, THP1 cells, Jurkat cells or peripheral blood mononuclear cells of a healthy person using a known method, for example, a lentivirus or the like. More specifically, the cells can be produced by the method described in Examples. The step can be carried out by, for example, culturing cells expressing an IGFL2 receptor, and adding an IGFL2 protein and a therapeutic agent to be tested to a medium. Additionally, as to the detailed culture condition, culture method and the like, a known condition, a method and the like can be used without being particularly limited. In addition, a dosing concentration, a timing of dosing, a dosing method or the like of an IGFL2 protein or a therapeutic agent to be tested may be appropriately set by considering physical properties of the used therapeutic agent to be tested, toxicity of the therapeutic agent to be tested to a living body, or the like. The IGFL2 receptor is a structure having a function which can be bound to an IGFL2 protein, and includes IGFLR1 and the like. The sequence information of the IGFLR1 gene is known, and can be seen from a known database, for example, NCBI (National Center for Biotechnology Information) and the like. Specifically, the sequence information of a human IGFLR1 gene is shown in NM 024660.4.

[0046] The cells constitutively expressing an IGFL2 receptor may be modified to express a fluorescent protein such as GFP or a luciferase protein such as Renilla Luciferase by a stimulation with an IGFL2 protein, and measurement of an expression amount of these proteins using the modified cells can be used to quantify a bioactive substance.

[0047] The bioactive substance to be measured in the step of "measuring an amount of a bioactive substance produced by the cells" is preferably a soluble protein such as a chemokine or a cytokine or a bioactive substance such as a small molecular signal transmitter produced by a stimulation with an IGFL2 protein. The specific example of the bioactive substance includes, for example, CXCL10, CXCL11, CXCL11, TNF, CXCL5, CCL20, GM-CSF, CCL24, CXCL8, CCL2 and CCL15. The stimulation with an IGFL2 protein can be carried out by, for example, adding an IGFL2 protein to a medium of the cells expressing an IGFL2 receptor. <semantics>⌈0048⌉<annotation encoding="application / x-tex">\lceil 0048 \rceil< / annotation>< / semantics> The measurement method of the bioactive substance include, but not particularly limited to, for example, a measurement method by an enzyme- linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), a western blotting or the like. A measured value of the bioactive substance in practice includes, for example, an amount and a concentration. As a measured value of the bioactive substance, for example, an average of several measured values can be used. In addition, the measured value of the bioactive substance may be a measured value of one bioactive substance or a value calculated by using plural bioactive substances. The calculation may use any coefficients and / or constants, as needed.

[0049] As a criteria for judging whether the therapeutic agent to be tested is a therapeutic agent of rheumatoid arthritis or not, for example, when "the measured value of the bioactive substance obtained by dosing an IGFL2 protein and a therapeutic agent to be tested" is less than "an average - 3SD of the measured value of the bioactive substance obtained by dosing an IGFL2 protein without dosing a therapeutic agent to be tested," the therapeutic agent to be tested can be judged as being a therapeutic agent for rheumatoid arthritis. <semantics>⌈0050⌉<annotation encoding="application / x-tex">\lceil 0050 \rceil< / annotation>< / semantics> With respect to the embodiments mentioned above, the present invention further discloses the following diagnosing methods: <semantics>⌈1⌉<annotation encoding="application / x-tex">\lceil 1 \rceil< / annotation>< / semantics> A method for diagnosing a severity of rheumatoid arthritis or a risk of developing rheumatoid arthritis in a subject with rheumatoid arthritis or suspected to develop rheumatoid arthritis, including: measuring an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample derived from the subject; and comparing the measured value with a threshold value corresponding to the amount of the IGFL2 protein or the expression amount of the IGFL2 gene. [2] The method according to the above [1], wherein a case where the measured value is equal to or higher than the threshold value indicates a high severity of rheumatoid arthritis or a high risk of developing the disease in the subject. [3] The method according to the above [1] or [2], wherein the sample is a body fluid, blood cells, or a tissue. EXAMPLES <semantics>⌈0051⌉<annotation encoding="application / x-tex">\lceil 0051 \rceil< / annotation>< / semantics> Hereinafter, the present invention will be particularly described by showing Examples and the like, without intending to limit the scope of the present invention to the following Examples and the like. In addition, an ethical approval of experimentations according to the present invention was obtained by Ethics Committee, Kyoto University Graduate School and Faculty of Medicine, and a written informed consent was obtained from all participants. <semantics>⌈0052⌉<annotation encoding="application / x-tex">\lceil 0052 \rceil< / annotation>< / semantics> [Statistical Analysis] Statistical analysis was carried out using Prism 10 software (GraphPad, San Diego, CA, US) or R (v40.5). The p value < 0.05 was considered as statistically significant.

[0053] [Disease Activity] The disease activity of the patient with rheumatoid arthritis was judged by DAS28-ESR and ESR.

[0054] [Patients and Healthy Persons] An excess produced during a surgery of a joint of a patient with rheumatoid arthritis or at a medical institute for outpatient was used as a sample of a joint. In addition, among the patients with rheumatoid arthritis, patients having DAS28-ESR less than 2.6 were defined as a remission person, and persons having DAS28-ESR equal to or higher than 2.6 were defined as a non-remission person. After inspection at a medical institute, a group of healthy persons was obtained from a group which were denied being affected by an autoimmune disease according to a diagnosis by a physician.

[0055] [Isolation of CD4 Positive T Cells] A synovial tissue derived from patients with rheumatoid arthritis (including remission) and healthy persons was finely cut, and the tissue was subjected to an enzymatic treatment with 100 µg / mL of LiberaseTL (Roche) and 100 μg / mL of DNaseI (Roche) for 30 minutes at 37°C. Then, peripheral blood monocytes or monocytes in synovium were collected by using Lymphocyte Separation Solution 1.077 (Nacalai Tesque). Further, for each of patients with rheumatoid arthritis and healthy persons, CD4+ T cells derived from a synovial tissue and CD4+ T cells derived from a serum were isolated by using CD4 + T Cell Isolation Kit (Miltenyi Biotec). <semantics>⌈0056⌉<annotation encoding="application / x-tex">\lceil 0056 \rceil< / annotation>< / semantics> [Production of Recombinant IGFL2 Protein] Optimized double-stranded DNA encoding a human IGFL2 protein which was flanked with a C-terminus His-tag was synthesized at IDT (San Diego, CA), and it was inserted into CSII-EF-IERS-Venus (obtained from RIKEN BioResource Center). CSII-EF-IERS-Venus is a lentivirus vector which has been developed by Atsushi Miyawaki and expresses a mutant YFP gene and a mutant Venus gene.

[0057] HEK293T cells were transduced with lentivirus particles (containing CSII-EF-IERS-Venus inserted with the double-stranded DNA), and were sorted with FACSMelody (BD Biosciences), to establish Venus+ IGFL2- producing HEK293T cells.

[0058] The recombinant IGFL2 protein with a C-terminus His-tag was purified from a culture supernatant of the IGFL2-producing HEK293T cells mentioned above using Dynabeads His-Tag Isolation & Pulldown (Thermo Scientific). The concentration of the recombinant IGFL2 protein was measured with His Tag ELISA Detection Kit (GenScript), to confirm that an endotoxin in the recombinant protein was less than 0.1 EU / µg using ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript). <semantics>⌈0059⌉<annotation encoding="application / x-tex">\lceil 0059 \rceil< / annotation>< / semantics> Incidentally, the amino acid sequence of human IGFL2 protein is as follows: PAGSEPWLCQPAPRCGDKIYNPLEQCCYNDAIVSLSETRQCGPPCT FWPCFELCCLDSFGLTNDFVVKLKVQGVNSQCHSSPISSKCESRRR FP (SEQ ID NO: 1)

[0060] [Establishment of IGFL2 Receptor-Constitutively Expressing THP1 Cell Line Optimized double-stranded DNA encoding a human IGFLR1 (NM 024660.4) protein was synthesized at IDT (San Diego, CA), and it was inserted into CSII-EF-IERS-puro (obtained from RIKEN BioResource Center). THP1 cells were transduced with lentivirus particles (containing CSII-EF-IERS-puro inserted with the double-stranded DNA), then the cells were cultured for 4 days in the presence of 2 µg / mL puromycin, and THP1R cells with gene introduction were selected, to provide an IGFL2 receptor-constitutively expressing THP1 cell line.

[0061] [ELISA] The protein concentrations of a cytokine and a chemokine in a monocyte supernatant were evaluated using Bio-Plex Pro Human Chemokine 40-Plex Assay Kit (Bio-Rad) and Bio-Plex 200 system (Bio- Rad).

[0062] For the purpose of detection of IGFL2 protein, an anti-IGFL2 rabbit polyclonal antibody was produced at Eurofins Genomics (Tokyo, Japan). Maxisorp 96-Well Plates (Thermo Scientific) was coated with anti-IGFL2 capturing antibody diluted with Coating Buffer (BioLegend). The plate was washed four times with wash buffer (1xPBS, 0.05% Tween-20), and thereafter, the plate was blocked with Assay Diluent (Biolegend) over 1 hour. The serum sample diluted with Assay Diluent by 4 folds was allowed to stand on the plate for 2 hours. An anti-IGFL2 biotinylated- detection antibody, Avidin-HRP (Thermo Scientific) and Ultra TMB (Thermo Scientific) were sequentially added in this order to the plate to react, and then the reaction was terminated with 2N sulfuric acid (Nacalai Tesque). Absorbance at 450 nm was measured with Spectra Max iD3 (Molecular Devices), to detect the captured IGFL2 protein.

[0063] [Quantification of IGFL2 by Quantitative PCR] The mRNA was extracted using MyOne Silane (Thermo). The cDNA was synthesized with SuperPrepTM RT Kit for qPCR (TOYOBO). The following two genes were subjected to qRT-PCR on C1000 Touch (Bio-Rad) by using THUNDERBIRD® SYBRTM qPCR Mix (TOYOBO).

[0064] As primers, the followings were used: for human 18srRNA gene (h18srRNA) 5'-AACTTTCGATGGTAGTCGCCG-3' (SEQ ID NO: 2) 5'-CCTTGGATGTGGTAGCCGTTT-3' (SEQ ID NO: 3) for human IGFL2 gene (hIGFL2) 5'-GACTACCCCAGGAGTGTGCT-3' (SEQ ID NO: 4) 5'-CAGCGGAGCGATGACT-3' (SEQ ID NO: 5) The expression of mRNA was standardized by the expression of h18srRNA.

[0065] < Example 1 > [Relationship of Human IGFL2 Gene with Disease Activity of Patient with Rheumatoid Arthritis] The present inventors inspected the relationship of IGFL2 gene expressed by CD4 positive T cells of patients with rheumatoid arthritis with disease activity of patients with rheumatoid arthritis. CD4 positive T cells were isolated from synovial tissue (ST) of 11 patients with rheumatoid arthritis, and an expression amount of an IGFL2 gene expressed by CD4 positive T cells was measured by single- cell RNA sequencing.

[0066] On the other hand, disease activity of the same patients with rheumatoid arthritis was judged by DAS28-ESR and ESR. FIG. 1 shows a scatter plot showing a relationship of an average expression amount of an IGFL2 gene with disease activity. It was found from FIG. 1 that the expression of the IGFL2 gene had a significant correlation to disease activity of rheumatoid arthritis (DAS28-ESR: <semantics>r=0.621<annotation encoding="application / x-tex">r = 0.621< / annotation>< / semantics>, <semantics>p=0.041<annotation encoding="application / x-tex">p = 0.041< / annotation>< / semantics>; ESR: <semantics>R=0.827<annotation encoding="application / x-tex">R = 0.827< / annotation>< / semantics>, <semantics>p=0.002<annotation encoding="application / x-tex">p = 0.002< / annotation>< / semantics>).

[0067] < Example 2 > [Expression of IGFL2 Protein in Serum of Patient with Rheumatoid Arthritis] The present inventors inspected an expression of an IGFL2 protein in a peripheral blood. (1) An expression amount of an IGFL2 protein (concentration) in a serum of a peripheral blood obtained from a group of healthy persons and a group of patients with rheumatoid arthritis in which age and sex were fitted was quantified by an ELISA method. Consequently, it was found that the concentration of the IGFL2 protein in a serum was significantly higher in the group of patients with rheumatoid arthritis (RA) than the group of healthy persons (Control) (FIG. 2). (2) Based on the concentration of the IGFL2 protein in the sera of the group of healthy persons and the group of patients with rheumatoid arthritis obtained in the above (1), an optimal threshold value for distinguishing the group of patients with rheumatoid arthritis from the group of healthy persons was obtained. Sensitivity of judgement, specificity and an area under the curve (AUC) using the set threshold value (119.3 pg / mL) were calculated. The resulting ROC curve is shown in FIG. 3. In this judgement, patients with rheumatoid arthritis and healthy persons can be distinguished with 96% of sensitivity and 76% of specificity. The threshold value (pg / mL), sensitivity of judgement (%), specificity (%) and AUC were summarized in Table 2.

[0069] [Table 2] Table 2 [Image disponible dans le document PDF, Image available in the PDF document]

[0070] It is found from the obtained results that in the judgement based on the concentration of an IGFL2 protein in a serum, the value of AUC is sufficiently high as 0.92, and the concentration of an IGFL2 protein in a serum can be used as a biomarker to distinguish patients with rheumatoid arthritis from healthy persons. < Example3 > [Expression of IGFL2 Protein in Serum of Person with Remission of Rheumatoid Arthritis] In the same manner as Example 2, the present inventors inspected an expression of an IGFL2 protein in a peripheral blood. (1) A group of patients with rheumatoid arthritis was divided to a group of persons with remission of rheumatoid arthritis and a group of persons with non-remission of rheumatoid arthritis, and an expression amount of an IGFL2 protein (concentration) in sera of peripheral bloods obtained from a group of persons with remission of rheumatoid arthritis and a group of persons with non-remission of rheumatoid arthritis in which age and sex were fitted was quantified by an ELISA method. Consequently, it was found that the concentration of the IGFL2 protein in sera was significantly higher in the group of persons with non-remission of rheumatoid arthritis (active) than the group of persons with remission of rheumatoid arthritis (remission) (FIG. 4). (2) For the concentrations of the IGFL2 proteins in sera of the group of persons with remission of rheumatoid arthritis and the group of persons with non-remission of rheumatoid arthritis obtained in the above (1), an optimal threshold value to distinguishing the group of persons with remission of rheumatoid arthritis from the group of persons with non- remission of rheumatoid arthritis was obtained. Sensitivity of judgement, specificity and an area under the curve (AUC) using the set threshold value (989 pg / mL) were calculated. The obtained ROC curve is shown in FIG. 5. In this judgement, persons with non-remission of rheumatoid arthritis and persons with remission of rheumatoid arthritis can be distinguished with 76% of sensitivity and 77% of specificity. The threshold value (pg / mL), the sensitivity of judgement (%), the specificity (%) and AUC were summarized in Table 3. <semantics>⌈0073⌉<annotation encoding="application / x-tex">\lceil 0073 \rceil< / annotation>< / semantics> [Table 3] Table 3 [Image disponible dans le document PDF, Image available in the PDF document]

[0074] It is found from the obtained results that in the judgement based on the concentration of the IGFL2 protein in the serum, the value of AUC is sufficiently high as 0.8, and the concentration of the IGFL2 protein in a serum can be used as a biomarker to distinguish persons with non- remission of rheumatoid arthritis from persons with remission of rheumatoid arthritis. <semantics>⌈0075⌉<annotation encoding="application / x-tex">\lceil 0075 \rceil< / annotation>< / semantics> It was found from the results of Example 2 and Example 3 that there is a strong correlation between the concentration of the IGFL2 protein in the serum and a severity of rheumatoid arthritis. Accordingly, the followings: (1) a method for testing a severity of rheumatoid arthritis of a patient with rheumatoid arthritis, and a method for testing a risk of developing rheumatoid arthritis of a patient suspected to develop rheumatoid arthritis, (2) a method for providing information for judging and / or monitoring a severity of rheumatoid arthritis of a patient with rheumatoid arthritis, and a method for providing information for judging and / or monitoring a risk of developing rheumatoid arthritis of a patient suspected to develop rheumatoid arthritis, and (3) a judgement of a subject suspected to be a patient with rheumatoid arthritis, a diagnosis of a patient with rheumatoid arthritis, and a judgement of effectiveness of a treatment of a patient with rheumatoid arthritis with therapeutic agent and the like can be carried out by measuring the concentration of the IGFL2 protein in a serum.

[0076] < Example 4 > [Study of Method for Screening Therapeutic Agent for Rheumatoid Arthritis] (1) IGFL2 receptor-constitutively expressing THP1 cells were cultured with 100 ng / mL of human recombinant IGFL2 protein in 10% bovine serum RPMI-1640. After 48 hours, the culture solution was centrifuged at <semantics>400×g<annotation encoding="application / x-tex">400 \times g< / annotation>< / semantics> for 5 minutes, to obtain a culture supernatant. Next, the amounts of various bioactive substances (CXCL10, CXCL1, CXCL11, TNF-α, CXCL5, CCL20, GM-CSF, CCL24, CXCL8, CCL2 and CCL15) (concentration) in the culture supernatant were quantified by a multiplex ELISA method. The results are shown in graphs of FIG. 6 to FIG. 8 as IGFL2 +.

[0077] (2) On the other hand, also for the cultured solution obtained by using IGFL2 receptor-constitutively expressing THP1 cells without adding an IGFL2 protein, a culture supernatant was obtained in the same manner as the above (1), to quantify the amounts of various chemokines. The results are shown in graphs of FIG. 6 to FIG. 8 as IGFL2 -. <semantics>⌈0078⌉<annotation encoding="application / x-tex">\lceil 0078 \rceil< / annotation>< / semantics> As shown in FIG. 6 to FIG. 8, it was found that the expression amounts of various bioactive substances were significantly increased by adding an IGFL2 protein. (3) Further, IGFL2 receptor-constitutively expressing THP1 cells were cultured in 10% bovine serum RPMI-1640. During the culture, any one of the following components (a) to (b): (a) 100 ng / mL of human recombinant IGFL2 protein and 20 μg / mL of isotype control antibody (R&D systems), and (b) 100 ng / mL of human recombinant IGFL2 protein and 20 μg / mL of anti-human IGFLR1 antibody (R&D systems) was added. After 24 hours, the culture solution was centrifuged at <semantics>400×g<annotation encoding="application / x-tex">400 \times g< / annotation>< / semantics> for 5 minutes, to obtain a culture supernatant. Then, an amount of CXCL10 (concentration) in the culture supernatant was quantified by an ELISA method. The result is shown in FIG. 9. In FIG. 9, IGFL2IgG1 corresponds to (a), and IGFL2IGFLR1 corresponds to (b). As shown in FIG. 9, it was found that the concentration of CXCL10 produced by adding anti-human IGFLR1 antibody which was an antibody with a neutralizing activity and an IGFL2 protein was statistically and significantly lower than the concentration of CXCL10 produced by adding an isotype control antibody which was an antibody without a neutralizing activity and an IGFL2 protein. Since the anti-human IGFLR1 antibody is a neutralizing antibody of IGFLR1, the antibody has an inhibitory activity of IGFL2 / IGFL2 receptor pathway. Accordingly, it is found that a substance reducing the production of a bioactive substance such as CXCL10 like the anti-human IGFLR1 antibody is useful as a therapeutic agent for rheumatoid arthritis. Accordingly, it was found that the method of the present invention could provide a method for screening a therapeutic agent for rheumatoid arthritis. INDUSTRIAL APPLICABILITY

[0800] The method for testing a severity of rheumatoid arthritis and technique relating to the same and the method for screening a therapeutic agent for rheumatoid arthritis of the present invention can be used in a medical field or a pharmaceutical field associated with rheumatoid arthritis.

Claims

1. A method for testing a severity of rheumatoid arthritis or a risk of developing rheumatoid arthritis in a subject comprising: measuring an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample derived from a subject with rheumatoid arthritis or a subject suspected to develop rheumatoid arthritis, and comparing the measured value with a threshold value corresponding to the amount of the IGFL2 protein or the expression amount of the IGFL2 gene.

2. The method according to claim 1, wherein a case where the measured value is equal to or higher than the threshold value indicates a high severity of rheumatoid arthritis or a high risk of developing rheumatoid arthritis in the subject.

3. The method according to claim 1 or 2, wherein the sample is a body fluid, blood cells, or a tissue.

4. A method for providing information for judging and / or monitoring a severity of rheumatoid arthritis in a subject with rheumatoid arthritis or a risk of developing rheumatoid arthritis in a subject suspected to develop rheumatoid arthritis comprising: measuring an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample derived from the subject, and providing information relating to the measured value.

5. The method according to claim 4, wherein the sample is a body fluid, blood cells, or a tissue.

6. A use of an IGFL2 protein or an IGFL2 gene in a sample derived from a subject with rheumatoid arthritis or suspected to develop rheumatoid arthritis as a biomarker for judging a severity of rheumatoid arthritis or a risk of developing rheumatoid arthritis in the subject, wherein the judgment comprises comparing the measured value of an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample derived from the subject with a threshold value corresponding to the amount of the IGFL2 protein or the expression amount of the IGFL2 gene.

7. The use according to claim 6, wherein a case where the measured value is equal to or higher than the threshold value indicates a high severity of rheumatoid arthritis or a high risk of developing rheumatoid arthritis in the subject.

8. The use according to claim 6 or 7, wherein the sample is a body fluid, blood cells, or a tissue.

9. A method for evaluating effectiveness of a treatment of rheumatoid arthritis comprising: measuring an amount of an IGFL2 protein or an expression amount of an IGFL2 gene in a sample derived from a subject after receiving a treatment with a therapeutic agent or a therapy to be tested, and comparing the measured value with a measured value derived from the subject before receiving the treatment.

10. The method according to claim 9, wherein a case where the measured value is lower than the measured value derived from the subject before receiving the treatment indicates an effective treatment.

11. The method according to claim 9 or 10, wherein the sample is a body fluid, blood cells, or a tissue.

12. A method for screening a therapeutic agent for rheumatoid arthritis comprising: dosing an IGFL2 protein and a therapeutic agent to be tested to cells 5 expressing an IGFL2 receptor, and measuring an amount of a bioactive substance produced by the cells.

13. The method according to claim 12, wherein the bioactive substance is a bioactive substance produced by an IGFL2 protein stimulation.