Method for testing severity of rheumatoid arthritis

Abstract

The present invention relates to a method for testing the severity of rheumatoid arthritis in a subject or the risk of the subject developing rheumatoid arthritis. A method for testing the severity of rheumatoid arthritis, techniques related to said method, and screening methods for developing therapeutic agents for rheumatoid arthritis can be provided in accordance with the present invention. Therefore, the present invention can be used in medical fields related to rheumatoid arthritis and in the pharmaceutical industry.

Description

Method for testing the severity of rheumatoid arthritis

[0001] The present invention relates to a method for testing the severity of rheumatoid arthritis and the risk of developing rheumatoid arthritis and techniques related thereto. The present invention further relates to a method for screening therapeutic agents for rheumatoid arthritis.

[0002] Genomic traits acquired during human evolution include autoimmune diseases, but the molecular mechanisms underlying the human immune response regulated by these traits remain unclear (Non-Patent Document 1). Rheumatoid arthritis (RA) is an autoimmune disease that causes systemic arthritis accompanied by synovial hyperplasia, resulting in joint deformity and destruction. Active lymphocytic infiltration and the formation of tertiary lymphoid structures (TLS) occur in the synovium of RA patients, leading to an increase in in situ adaptive immune responses, including peripheral helper T cells (Tph cells) providing B cell help (Non-Patent Document 2). Furthermore, the monocyte-macrophage system and fibroblast-like synoviocytes (FLS), which are crucial players in innate immunity, contribute to the vicious inflammatory cycle of RA, leading to synovial hyperplasia, production of matrix-degrading enzymes, and induction of osteoclasts (Non-Patent Documents 3, 4, and 5).

[0003] The involvement of autoantibodies with common epitopes and MHC class II in rheumatoid arthritis suggests that CD4-positive T cells are strongly involved in the development of rheumatoid arthritis (Non-Patent Document 6).

[0004] 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)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).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)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)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)McInnes, I. B. & Schett, G. The Pathogenesis of Rheumatoid Arthritis. New Engl J Med 365, 2205-2219, doi:10.1056 / nejmra1004965 PMID - 22150039 (2011).

[0005] However, how human CD4+ T cells regulate the inflammatory immune response in patients with rheumatoid arthritis continues to be studied.

[0006] The present invention has been made in view of the above circumstances, and aims to provide a method for testing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject. Establishing a method for testing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject will not only enable clinical confirmation of the condition of the subject, but also serve as an effective tool for developing therapeutic drugs for rheumatoid arthritis. Furthermore, the present invention relates to providing a method for screening therapeutic drugs for rheumatoid arthritis.

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

[13] : [1] A method for testing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject, comprising the steps of measuring the amount of IGFL2 protein or the expression level of IGFL2 gene in a sample derived from the subject suffering from rheumatoid arthritis or suspected of suffering from rheumatoid arthritis, and comparing the measured value with a threshold value corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene. [2] The method according to [1], wherein a measured value equal to or greater than the threshold value indicates that the subject has a high severity of rheumatoid arthritis or a high risk of developing rheumatoid arthritis. [3] The method according to [1] or [2], wherein the sample is a body fluid, blood cells, or tissue. [4] A method for providing information for determining and / or monitoring the severity of rheumatoid arthritis in a subject suffering from rheumatoid arthritis or the risk of a subject suspected of having rheumatoid arthritis developing rheumatoid arthritis, comprising the steps of measuring the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from the subject, and providing information related to the measured value. [5] The method according to [4] above, wherein the sample is a body fluid, blood cells, or tissue. [6] Use of IGFL2 protein or the IGFL2 gene in a sample derived from the subject as a biomarker for determining the severity of rheumatoid arthritis in a subject suffering from rheumatoid arthritis or the risk of a subject suspected of having rheumatoid arthritis developing rheumatoid arthritis, comprising the step of comparing the measured value of the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from the subject with a threshold value corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene. [7] The use according to [6], wherein a measured value equal to or greater than the threshold value indicates that the subject has a high level of severity of rheumatoid arthritis or a high risk of developing the disease. [8] The use according to [6] or [7], wherein the sample is a body fluid, blood cells, or tissue.[9] A method for evaluating the effectiveness of a treatment for rheumatoid arthritis, comprising the steps of measuring the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from a subject after treatment with a test therapeutic drug or therapy, and comparing the measured value with a value derived from the subject before the treatment.

[10] The method of [9], wherein the treatment is effective if the measured value is lower than the value derived from the subject before the treatment.

[11] The method of [9] or

[10] , wherein the sample is a body fluid, blood cells, or tissue.

[12] A method for screening for a therapeutic drug for rheumatoid arthritis, comprising the steps of administering IGFL2 protein and a test therapeutic drug to cells expressing an IGFL2 receptor, and measuring the amount of a physiologically active substance produced by the cells.

[13] The screening method of

[12] , wherein the physiologically active substance is a physiologically active substance produced by stimulation of IGFL2 protein.

[0008] The present invention provides a method for testing and determining the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis using measured values ​​of IGFL2 protein or IGFL2 gene expression in a subject, and a method for evaluating the effectiveness of treatment for rheumatoid arthritis using the measured values.The present invention also provides a method for screening for therapeutic agents for rheumatoid arthritis.

[0009] Figure 1 is a scatter plot showing the correlation between the mean expression level of the IGFL2 gene by synovial CD4+ T cells and disease activity (DAS28-ESR or ESR). Each dot in the figure represents an individual patient. Figure 2 is a graph showing serum IGFL2 protein concentrations between healthy control and rheumatoid arthritis patient groups. In Figure 2, data are presented as dots and mean ± SD, and an unpaired two-tailed t-test was performed. Figure 3 is a receiver operating characteristic curve (ROC) curve and AUC values ​​for the determination of rheumatoid arthritis patients based on serum IGFL2 protein concentrations between healthy control and rheumatoid arthritis patient groups. Figure 4 is a graph showing serum IGFL2 protein concentrations between rheumatoid arthritis patients in remission and non-remission. In Figure 4, data are presented as dots and mean ± SD, and an unpaired two-tailed t-test was performed. Figure 5 shows the ROC curve and AUC values ​​for the determination of rheumatoid arthritis remission based on serum IGFL2 protein concentrations in patients in remission and non-remission. Figure 6 shows a graph depicting the amount of various physiologically active substances in the culture supernatant. Culture supernatants obtained with IGFL2 protein are designated IGFL2+, while those obtained without IGFL2 protein are designated IGFL2-. Data are presented as dots and mean ± SD, and unpaired two-tailed t-tests were performed. Figure 7 shows a graph depicting the amount of various physiologically active substances in the culture supernatant. Culture supernatants obtained with IGFL2 protein are designated IGFL2+, while those obtained without IGFL2 protein are designated IGFL2-. Data are presented as dots and mean ± SD, and unpaired two-tailed t-tests were performed. Figure 8 shows a graph depicting the amount of various physiologically active substances in the culture supernatant. Culture supernatants obtained with IGFL2 protein are designated IGFL2+, while those obtained without IGFL2 protein are designated IGFL2-. Data are shown as dots and mean ± SD, and unpaired two-tailed t-test was performed. Figure 9 shows the effect of anti-IGFLR1 antibody on CXCL10 production by THP1 cells constitutively expressing the IGFL2 receptor. In the figure, data are shown as dots and mean ± SD, and unpaired two-tailed t-test was performed.In the figure, the concentration of CXCL10 when human recombinant IGFL2 protein and an isotype control antibody were added is shown as IGFL2IgG1, and the concentration of CXCL10 when human recombinant IGFL2 protein and FLR, an IGFLR1 neutralizing antibody, were added is shown as IGFL2IGFLR1.

[0010] In the present invention, "rheumatoid arthritis (RA)" refers to, but is not limited to, an autoimmune disease that causes systemic arthritis accompanied by synovial tissue hyperplasia. For example, it generally refers to a disease that meets the 1987 American College of Rheumatology classification criteria or the 2010 ACR / EULAR rheumatoid arthritis classification criteria.

[0011] In the present invention, "suffering from rheumatoid arthritis" refers to, but is not particularly limited to, a subject having the above-mentioned state of rheumatoid arthritis, and can be determined, for example, by the above-mentioned criteria.

[0012] In the present invention, the term "subject" is not particularly limited to, but refers to, for example, a human or a non-human ape, and includes not only those who have rheumatoid arthritis but also those who may be affected. A human with rheumatoid arthritis may be referred to as a patient. Subjects suspected of having rheumatoid arthritis include those who the subject himself / herself subjectively suspects, those for whom tests related to the present invention are included in the test items of a health checkup, those based on some objective evidence, and / or those who a doctor has determined as a result of examination to have a reasonable possibility of being affected. In the present invention, "risk" refers to the risk of a subject suspected of having rheumatoid arthritis developing rheumatoid arthritis.

[0013] In the present invention, the term "healthy subject" refers to a subject in whom the presence of any autoimmune disease has been ruled out, and is determined by a doctor's diagnosis based on symptoms such as autoantibodies, joint swelling, joint tenderness, inflammatory reactions, X-ray findings, and stiffness in the hands.

[0014] In the present invention, the term "derived from" is not particularly limited, but refers to something that can be collected from a subject by, for example, blood sampling, arthroscopy, puncture, biopsy, or surgery.

[0015] In the present invention, the "sample" is not particularly limited, but is preferably, for example, a body fluid, blood cells, or tissue. Examples of body fluids include whole blood, plasma, serum, synovial fluid, urine, and saliva. Examples of blood cells include peripheral blood mononuclear cells, peripheral blood T cells, and peripheral blood CD4-positive T cells. Examples of tissues include synovial membranes and ligaments.

[0016] In the present invention, "IGFL2 protein" refers to insulin-like growth factor (IGF) family member 2. Sequence information for human IGFL2 protein and the IGFL2 gene is publicly known and can be found, for example, from publicly known databases such as NCBI (National Center for Biotechnology Information). Specifically, the sequence information for the human IGFL2 gene is set forth as NM_001002915.3. Currently, the IGFL2 gene has been found in humans and apes, as shown in Table 1 below.

[0017]

[0018] In the present invention, the method for measuring the amount of IGFL2 protein is not particularly limited, and examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and Western blot assay. The IGFL2 protein to be measured may be the full-length protein or a protein fragment. Furthermore, the measured value may be either an absolute amount or a concentration.

[0019] In the present invention, the method for measuring the expression level of the IGFL2 gene is not particularly limited, and examples thereof include RNA sequencing, single-cell RNA sequencing, quantitative PCR, LAMP, etc. Targets for measuring the IGFL2 gene include DNA encoding the entire gene, a part thereof, cDNA, and RNA.

[0020] The measured value of the amount of IGFL2 protein or the expression level of the IGFL2 gene can be, for example, an average of several measurements. Furthermore, the measured value may be a value measured for a single type of subject, or may be a value calculated using multiple subjects. This calculation may further include any coefficients and / or constants, as necessary.

[0021] The present invention was completed based on the relationship between rheumatoid arthritis and the amount of IGFL2 protein or the expression level of the IGFL2 gene, and specific embodiments of the present invention are exemplified below.

[0022] <Method for assessing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject> In one aspect, the present invention provides a method for assessing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject, comprising the steps of measuring the amount of IGFL2 protein or the expression level of IGFL2 gene in a sample derived from the subject having rheumatoid arthritis or a subject suspected of having rheumatoid arthritis, and comparing the measured value with a threshold value corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene.

[0023] In the "step of measuring the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample", these biomarkers are quantified.

[0024] In the "step of comparing the measured value with a threshold value corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene," the measured value is compared with the threshold value. These two steps constitute the testing method of the present invention. In the testing method of the present invention, a measured value equal to or greater than the threshold value indicates that the subject has a high level of severity of rheumatoid arthritis or is at a high risk of developing the disease.

[0025] In the present invention, the "threshold" corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene is not particularly limited and can be set appropriately. For example, a predetermined threshold can be empirically set by accumulating data on the amount of IGFL2 protein or the expression level of the IGFL2 gene in patients with rheumatoid arthritis. More specifically, samples are collected from multiple patients diagnosed with rheumatoid arthritis and multiple healthy subjects, and measurements of the amount of IGFL2 protein or the expression level of the IGFL2 gene are obtained. The value that most accurately distinguishes between a patient group and a healthy group is then determined, and this value is set as the threshold. When setting the threshold, it is preferable to consider sensitivity, specificity, positive predictive value, negative predictive value, etc.

[0026] In the present invention, the "severity of rheumatoid arthritis" can be evaluated, for example, by disease activity. In the present invention, "disease activity" refers to a value expressed as, for example, DAS-28-ESR or ESR, and a DAS-28-ESR value of greater than 5.1 is classified as severe, 3.2-5.1 as moderate, less than 3.2 as mild, and less than 2.6 as remission.

[0027] <Method for providing information for determining and / or monitoring the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject> In one aspect, the present invention provides a method for providing information for determining and / or monitoring the severity of rheumatoid arthritis in a subject suffering from rheumatoid arthritis, or the risk of developing rheumatoid arthritis in a subject suspected of having rheumatoid arthritis, the method comprising the steps of measuring the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from the subject, and providing information related to the measured value.

[0028] In the present invention, "assessment" and / or "monitoring" of the "severity of rheumatoid arthritis" or the "risk of developing rheumatoid arthritis" refers to, for example, determining which of the above-described severity levels of rheumatoid arthritis a subject falls into ("assessment"), or determining changes over time ("monitoring"). The assessment includes determining the presence or absence of rheumatoid arthritis, determining the risk of developing rheumatoid arthritis, determining the severity of rheumatoid arthritis in a subject, determining the preventive effect of rheumatoid arthritis in a subject, determining the therapeutic effect in patients with rheumatoid arthritis, determining the presence or absence of recurrence of rheumatoid arthritis in a subject, and determining the risk of recurrence of rheumatoid arthritis in a subject.

[0029] The "step of measuring the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample" is the same as above.

[0030] In the "step of providing information related to measurement values" of the present invention, "providing information" means, for example, providing information related to the measurement values ​​obtained in the measurement step (e.g., measurement values, threshold values) to a physician or the like. The information can be provided to a party other than a physician, for example, a laboratory technician. The physician can use the provided information to assess and / or monitor the severity of rheumatoid arthritis, for example, in a situation independent of the steps of the present invention. The method of providing information of the present invention is carried out by these two steps.

[0031] <Use of IGFL2 protein or IGFL2 gene in a sample as a biomarker> In one aspect, the present invention relates to the use of IGFL2 protein or IGFL2 gene in a sample derived from a subject as a biomarker for determining the severity of rheumatoid arthritis in the subject suffering from the disease, or the risk of a subject suspected of suffering from the disease to develop rheumatoid arthritis, wherein the determination comprises a step of comparing a measured value of the amount of IGFL2 protein or the expression level of the IGFL2 gene in the sample derived from the subject with a threshold value corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene.

[0032] In the present invention, using the IGFL2 protein or the IGFL2 gene as a biomarker means, for example, using the measured value of the IGFL2 protein or the measured value of the expression level of the IGFL2 gene as an indicator value for determining the severity of rheumatoid arthritis or for determining the risk of developing rheumatoid arthritis.

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

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

[0035] <Method for evaluating the effectiveness of treatment for rheumatoid arthritis> In one aspect, the present invention provides a method for evaluating the effectiveness of treatment for rheumatoid arthritis, comprising the steps of measuring the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from a subject after treatment with a test therapeutic agent or therapy, and comparing the measured value with the measured value derived from the subject before treatment.

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

[0037] In the present invention, "effectiveness of treatment" refers to, for example, the occurrence of reduction in the severity, improvement, or remission of rheumatoid arthritis as described above. Evaluation of whether or not these have occurred is, for example, referred to as "evaluation of the effectiveness of treatment."

[0038] In the present invention, test therapeutic agents or treatments include, but are not limited to, steroids, NSAIDs (analgesics), conventional small molecule antirheumatic drugs (csDMARDS), molecularly targeted antirheumatic drugs (tsDMARDS), LCAP, synovectomy, etc. A subject who has undergone such treatment is referred to as a "subject after treatment."

[0039] The "step of measuring the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from a subject after treatment with the test therapeutic drug or therapy" is the same as the "step of measuring the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from a subject" described above.

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

[0041] The method for evaluating the effectiveness of a treatment for rheumatoid arthritis is carried out through these two steps. In the evaluation method of the present invention, if the measured value is lower than the measured value derived from the subject before the treatment, it is indicated that the treatment is effective. Specifically, if an unpaired two-tailed t-test shows that the value is significantly lower than that of the control group, it is indicated that the treatment is effective.

[0042] <Method for screening for therapeutic agents for rheumatoid arthritis> A further aspect of the present invention is a method for screening for therapeutic agents for rheumatoid arthritis, which method comprises the steps of administering an IGFL2 protein and a test therapeutic agent to cells expressing an IGFL2 receptor, and measuring the amount of a physiologically active substance produced by the cells.

[0043] In the "step of administering an IGFL2 protein and a test therapeutic agent to cells expressing an IGFL2 receptor," the cells expressing the IGFL2 receptor are preferably those that constitutively express the IGFL2 receptor. Such cells can be constitutively expressed by gene transfer using a known method, such as lentivirus, into human cells that express the IGFL2 receptor, such as THP1 cells, Jurkat cells, or peripheral blood mononuclear cells from healthy individuals. More specifically, the cells can be prepared by the method described in the Examples.

[0044] This step can be carried out, for example, by culturing cells expressing the IGFL2 receptor and adding the IGFL2 protein and the test therapeutic agent to the medium. Other detailed culture conditions, culture methods, etc., can be determined using known conditions, methods, etc., without particular limitations. Furthermore, the concentration, timing, and method of administration of the IGFL2 protein and the test therapeutic agent can be appropriately determined taking into consideration the physical properties of the test therapeutic agent used, the toxicity of the test therapeutic agent to living organisms, etc.

[0045] The IGFL2 receptor is a structure capable of binding to the IGFL2 protein, and examples thereof include IGFLR1. The sequence information of the IGFLR1 gene is publicly known and can be found, for example, from publicly known databases such as NCBI (National Center for Biotechnology Information). Specifically, the sequence information of the human IGFLR1 gene is shown in NM_024660.4.

[0046] Cells that constitutively express the IGFL2 receptor may be modified so that they express a fluorescent protein such as GFP or a luciferase protein such as Renilla luciferase upon stimulation by the IGFL2 protein, and measuring the expression levels of these proteins using such modified cells can serve as an alternative to quantifying physiologically active substances.

[0047] The physiologically active substance to be measured in the "step of measuring the amount of a physiologically active substance produced by the cells" is preferably a physiologically active substance such as a soluble protein, such as a chemokine or cytokine, or a small molecule signal transmitter, which is produced in response to stimulation by IGFL2 protein. Specific examples of such physiologically active substances include CXCL10, CXCL1, CXCL11, TNF, CXCL5, CCL20, GM-CSF, CCL24, CXCL8, CCL2, and CCL15. IGFL2 protein stimulation can be carried out, for example, by adding IGFL2 protein to the culture medium of cells expressing the IGFL2 receptor.

[0048] The method for measuring a physiologically active substance is not particularly limited, and examples thereof include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and Western blot assay. The value of a physiologically active substance to be actually measured includes, for example, the amount and concentration. The measured value of a physiologically active substance can be, for example, an average of several measurements. Furthermore, the measured value of a physiologically active substance may be the measured value of a single physiologically active substance, or may be a value calculated using multiple physiologically active substances. Any coefficients and / or constants may also be used in this calculation, as necessary.

[0049] As a criterion for determining whether or not a test therapeutic drug is a therapeutic drug for rheumatoid arthritis, for example, if the "measurement value of a physiologically active substance obtained by administering IGFL2 protein and the test therapeutic drug" is less than the "mean value -3SD of the measurement value of a physiologically active substance obtained by administering IGFL2 protein but not the test therapeutic drug," the test therapeutic drug can be determined to be a therapeutic drug for rheumatoid arthritis.

[0050] In relation to the above-described embodiments, the present invention further discloses the following diagnostic methods: [1] A method for diagnosing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject, comprising the steps of measuring the amount of IGFL2 protein or the expression level of IGFL2 gene in a sample derived from the subject suffering from rheumatoid arthritis or suspected of having rheumatoid arthritis, and comparing the measured value with a threshold value corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene. [2] The method according to [1] above, wherein a measured value equal to or greater than the threshold value indicates that the subject has a high severity of rheumatoid arthritis or a high risk of developing rheumatoid arthritis. [3] The method according to [1] or [2] above, wherein the sample is a body fluid, blood cells, or tissue.

[0051] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples, etc. Furthermore, ethical approval for the experiments according to the present invention was obtained from the Ethics Committee of the Graduate School of Medicine and Faculty of Medicine, Kyoto University, and written informed consent was obtained from all participants.

[0052] Statistical Analysis Statistical analysis was performed using Prism 10 software (GraphPad, San Diego, CA, USA) or R (v40.5). A p-value <0.05 was considered statistically significant.

[0053] [Disease activity] Disease activity in rheumatoid arthritis patients was assessed using DAS28-ESR and ESR.

[0054] [Patients and healthy subjects] Joint samples were obtained from surplus materials generated during joint surgery or at outpatient medical facilities for rheumatoid arthritis patients. Furthermore, among rheumatoid arthritis patients, those with a DAS28-ESR score of less than 2.6 were defined as in remission, and those with a DAS28-ESR score of 2.6 or higher were defined as not in remission. The healthy subject group was selected from the group whose autoimmune disease was ruled out by a doctor's diagnosis after testing at a medical facility.

[0055] [Isolation of CD4+ T cells] Synovial tissue from rheumatoid arthritis patients (including those in remission) and healthy controls was finely minced and treated with 100 μg / mL Liberase TL (Roche) and 100 μg / mL DNase I (Roche) at 37°C for 30 minutes. Peripheral blood monocytes or synovial monocytes were then collected using Lymphocyte Separation Solution 1.077 (Nacalai Tesque). Furthermore, CD4+ T cells from synovial tissue and serum were isolated from rheumatoid arthritis patients and healthy controls using a CD4+ T Cell Isolation Kit (Miltenyi).

[0056] [Production of recombinant IGFL2 protein] Optimized double-stranded DNA encoding human IGFL2 protein flanked by a C-terminal His-tag was synthesized by IDT (San Diego, CA) and inserted into CSII-EF-IERS-Venus (obtained from the RIKEN BioResource Center), a lentiviral vector developed by Atsushi Miyawaki that expresses the YFP gene and mutant forms of the Venus gene.

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

[0058] Recombinant IGFL2 protein bearing a C-terminal His-tag was purified from the culture supernatant of the IGFL2-producing HEK293T cells using Dynabeads His-Tag Isolation & Pulldown (Thermo Scientific). The concentration of the recombinant IGFL2 protein was measured using a His Tag ELISA Detection Kit (GenScript). The endotoxin content of the recombinant protein was confirmed to be less than 0.1 EU / μg using a ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript).

[0059] The amino acid sequence of human IGFL2 protein is as follows: PAGSEPWLCQPAPRCGDKIYNPLEQCCYNDAIVSLSETRQCGPPCTFWPCFELCCLDSFGLTNDFVVKLKVQGVNSQCHSSPISSKCESRRRFP (SEQ ID NO: 1).

[0060] Establishment of a THP1 cell line constitutively expressing the IGFL2 receptor: Optimized double-stranded DNA encoding human IGFLR1 (NM_024660.4) was synthesized by IDT (San Diego, CA) and inserted into CSII-EF-IERS-puro (obtained from the RIKEN BioResource Center). THP1 cells were transduced with lentiviral particles (containing the CSII-EF-IERS-puro inserted double-stranded DNA) and cultured for 4 days in the presence of 2 μg / mL puromycin. Transfected THP1R cells were selected to establish a THP1 cell line constitutively expressing the IGFL2 receptor.

[0061] [ELISA] The protein concentrations of cytokines and chemokines in monocyte supernatants were assessed using the Bio-Plex Pro Human Chemokine 40-Plex Assay Kit (Bio-Rad) and the Bio-Plex 200 system (Bio-Rad).

[0062] For detection of IGFL2 protein, anti-IGFL2 rabbit polyclonal antibody was produced at Eurofins Genomics (Tokyo, Japan). Maxisorp 96-well plates (Thermo Scientific) were coated with the anti-IGFL2 capture antibody diluted in Coating Buffer (BioLegend). After washing four times with washing buffer (1x PBS, 0.05% Tween-20), the plates were blocked with Assay Diluent (Biolegend) for 1 hour. Serum samples diluted 4-fold in Assay Diluent were left on the plates for 2 hours. Anti-IGFL2 biotinylated detection antibody, Avidin-HRP (Thermo Scientific), and Ultra TMB (Thermo Scientific) were added sequentially to the plates, followed by terminating the reaction with 2N sulfuric acid (Nacalai Tesque). The absorbance at 450 nm was measured using Spectra Max iD3 (Molecular Devices) to detect the captured IGFL2 protein.

[0063] Quantification of IGFL2 by quantitative PCR: mRNA was extracted using MyOne Silane (Thermo). cDNA was synthesized using the SuperPrep® RT Kit for qPCR (Toyobo). qRT-PCR was performed for the following two genes using THUNDERBIRD™ SYBR® qPCR Mix (Toyobo) on a C1000 Touch (Bio-Rad).

[0064] The following primers 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'-CAGCGGGAGCGATGACT-3' (SEQ ID NO: 5) mRNA expression was normalized by the expression of h18srRNA.

[0065] Example 1 Relationship between the human IGFL2 gene and disease activity in rheumatoid arthritis patients The present inventors investigated the relationship between the IGFL2 gene expressed in CD4+ T cells of rheumatoid arthritis patients and disease activity in rheumatoid arthritis patients. CD4+ T cells were isolated from synovial tissue (ST) of 11 rheumatoid arthritis patients, and the expression level of the IGFL2 gene expressed by CD4+ T cells was measured by single-cell RNA sequencing.

[0066] Meanwhile, disease activity in the same rheumatoid arthritis patients was assessed using DAS28-ESR and ESR. Figure 1 shows a scatter plot showing the relationship between the mean expression level of IGFL2 gene and disease activity. Figure 1 shows that IGFL2 gene expression was significantly correlated with disease activity (DAS28-ESR: r=0.621, p=0.041; ESR: R=0.827, p=0.002).

[0067] Example 2: Expression of IGFL2 protein in serum from rheumatoid arthritis patients. The present inventors investigated the expression of IGFL2 protein in peripheral blood. (1) The expression level (concentration) of IGFL2 protein in serum from peripheral blood obtained from age- and sex-matched healthy subjects and rheumatoid arthritis patients was quantified by ELISA. The results showed that the serum IGFL2 protein concentration was significantly higher in the rheumatoid arthritis patient group (RA) than in the healthy subject group (Control) (Figure 2).

[0068] (2) Based on the IGFL2 protein concentrations in the serum of healthy subjects and rheumatoid arthritis patients obtained in (1) above, the optimal threshold for distinguishing between rheumatoid arthritis patients and healthy subjects was determined. The sensitivity, specificity, and area under the curve (AUC) of the determination using the set threshold (119.3 pg / mL) were calculated. The resulting ROC curve is shown in Figure 3. This determination can distinguish between rheumatoid arthritis patients and healthy subjects with a sensitivity of 96% and a specificity of 76%. Table 2 summarizes the threshold (pg / mL), determination sensitivity (%), specificity (%), and AUC.

[0069]

[0070] The results show that the AUC value for the determination based on serum IGFL2 protein concentration was sufficiently high at 0.92, indicating that serum IGFL2 protein concentration can be used as a biomarker to distinguish between rheumatoid arthritis patients and healthy individuals.

[0071] Example 3 [IGFL2 Protein Expression in Serum of Patients with Rheumatoid Arthritis in Remission] The present inventors investigated IGFL2 protein expression in peripheral blood in the same manner as in Example 2. (1) A group of rheumatoid arthritis patients was divided into a group of patients in remission and a group of patients not in remission. The expression level (concentration) of IGFL2 protein in serum from peripheral blood obtained from age- and sex-matched patients in the remission and non-remission groups was quantified by ELISA. The results showed that serum IGFL2 protein concentration was significantly higher in the active group than in the remission group (Figure 4).

[0072] (2) Using the IGFL2 protein concentrations in the serum of patients with and without rheumatoid arthritis remission obtained in (1) above, the optimal threshold for distinguishing between patients with and without rheumatoid arthritis remission was determined. The sensitivity, specificity, and area under the curve (AUC) of the determination using the determined threshold (989 pg / mL) were calculated. The resulting ROC curve is shown in Figure 5. This determination can distinguish between patients with and without rheumatoid arthritis remission with a sensitivity of 76% and a specificity of 77%. Table 3 summarizes the threshold (pg / mL), the determination sensitivity (%), specificity (%), and AUC.

[0073]

[0074] The results show that the AUC value for the determination based on serum IGFL2 protein concentration was sufficiently high at 0.8, indicating that serum IGFL2 protein concentration can be used as a biomarker to distinguish between patients in remission and those in rheumatoid arthritis remission.

[0075] The results of Examples 2 and 3 demonstrated a strong correlation between serum IGFL2 protein concentration and the severity of rheumatoid arthritis. Therefore, measuring serum IGFL2 protein concentration can be used to (1) test the severity of rheumatoid arthritis in patients with rheumatoid arthritis, and to test the risk of rheumatoid arthritis in patients suspected of having rheumatoid arthritis. (2) provide information for determining the severity of rheumatoid arthritis in patients with rheumatoid arthritis and / or for monitoring the disease, and to determine the risk of rheumatoid arthritis in patients suspected of having rheumatoid arthritis and / or for monitoring the disease. (3) identify subjects suspected of having rheumatoid arthritis, diagnose patients with rheumatoid arthritis, and evaluate the effectiveness of treatments with drugs or the like for patients with rheumatoid arthritis.

[0076] Example 4: Screening Method for Rheumatoid Arthritis Therapeutics (1) THP1 cells constitutively expressing IGFL2 receptor were cultured with 100 ng / mL of human recombinant IGFL2 protein in 10% bovine serum RPMI-1640 medium. After 48 hours, the culture medium was centrifuged at 400 × g for 5 minutes to obtain the culture supernatant. The amounts (concentrations) of various physiologically active substances (CXCL10, CXCL1, CXCL11, TNF-α, CXCL5, CCL20, GM-CSF, CCL24, CXCL8, CCL2, and CCL15) in the culture supernatant were then quantified by multiplex ELISA. The results are shown in Figures 6 to 8 as IGFL2 +.

[0077] (2) On the other hand, THP1 cells constitutively expressing IGFL2 receptor were cultured without the addition of IGFL2 protein in the same manner as in (1) above, and the amounts of various chemokines in the culture supernatant were quantified. The results are shown as IGFL2 - in the graphs in Figures 6 to 8.

[0078] As shown in Figures 6 to 8, it was found that the expression levels of various physiologically active substances were significantly increased by adding IGFL2 protein.

[0079] (3) THP1 cells constitutively expressing IGFL2 receptor were cultured in RPMI-1640 medium supplemented with 10% bovine serum. During culture, one of the following components (a) or (b) was added: (a) 100 ng / mL human recombinant IGFL2 protein and 20 μg / mL isotype control antibody (R&D Systems) or (b) 100 ng / mL human recombinant IGFL2 protein and 20 μg / mL anti-human IGFLR1 antibody (R&D Systems). After 24 hours, the culture medium was centrifuged at 400 × g for 5 minutes to obtain the culture supernatant. The amount (concentration) of CXCL10 in the culture supernatant was then quantified by ELISA. The results are shown in Figure 9. In Figure 9, IGFL2IgG1 corresponds to (a), and IGFL2IGFLR1 corresponds to (b). As shown in Figure 9, the concentration of CXCL10 produced by the addition of an anti-human IGFLR1 antibody, which is a neutralizing antibody, and an IGFLR2 protein was statistically significantly lower than the concentration of CXCL10 produced by the addition of an isotype control antibody, which is an antibody without neutralizing activity, and an IGFLR2 protein. Because the anti-human IGFLR1 antibody is a neutralizing antibody of IGFLR1, it has inhibitory activity against the IGFL2 / IGFL2 receptor pathway. Therefore, substances that, like the anti-human IGFLR1 antibody, reduce the production of physiologically active substances such as CXCL10, are useful as therapeutic agents for rheumatoid arthritis. Therefore, it was found that the method of the present invention can provide a method for screening therapeutic agents for rheumatoid arthritis.

[0080] The method of the present invention for testing the severity of rheumatoid arthritis and related techniques, as well as the method for screening therapeutic drugs for rheumatoid arthritis, can be used in the medical field and pharmaceutical industry related to rheumatoid arthritis.

Claims

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

2. The method according to claim 1, wherein a measurement value equal to or greater than the threshold value indicates that the subject has a high level of severity of rheumatoid arthritis or is at a high risk of developing the disease.

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

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

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

6. Use of IGFL2 protein or IGFL2 gene in a sample derived from a subject as a biomarker for determining the severity of rheumatoid arthritis in the subject suffering from the disease or the risk of a subject suspected of suffering from the disease developing rheumatoid arthritis, wherein the determination comprises a step of comparing a measured value of the amount of IGFL2 protein or the expression level of the IGFL2 gene in the sample derived from the subject with a threshold value corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene.

7. The use according to claim 6, wherein a measurement value equal to or greater than the threshold value indicates that the subject has a high level of severity of rheumatoid arthritis or is at a high risk of developing the disease.

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

9. A method for evaluating the effectiveness of a treatment for rheumatoid arthritis, comprising the steps of measuring the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from a subject after treatment with a test therapeutic drug or therapy, and comparing the measured value with the measured value derived from the subject before treatment.

10. The method of claim 9, wherein the treatment is effective if the measured value is lower than the measured value from the subject before receiving the treatment.

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

12. A method for screening for a therapeutic agent for rheumatoid arthritis, comprising the steps of administering an IGFL2 protein and a test therapeutic agent to cells expressing an IGFL2 receptor, and measuring the amount of a physiologically active substance produced by the cells.

13. The screening method according to claim 12, wherein the physiologically active substance is a physiologically active substance produced by stimulation of IGFL2 protein.

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