Methods for testing the severity of rheumatoid arthritis

Abstract

This invention relates to a method for testing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject. According to the invention, methods for testing the severity of rheumatoid arthritis and related technologies, as well as screening methods for developing therapeutic drugs for rheumatoid arthritis, can be provided. Therefore, this invention can be used in the medical and pharmaceutical industries related to rheumatoid arthritis.

Description

Technical Field

[0001] This invention relates to methods and related technologies for testing the severity of rheumatoid arthritis and the risk of developing rheumatoid arthritis. Furthermore, this invention also relates to methods for screening therapeutic agents for rheumatoid arthritis. Background Technology

[0002] Autoimmune diseases are among the genomic phenotypes acquired during human evolution; however, the molecular mechanisms by which these phenotypes regulate human immune responses remain unclear (Non-Patent Literature 1).

[0003] Rheumatoid arthritis (RA) is an autoimmune disease that causes systemic arthritis accompanied by synovial tissue hyperplasia. This results in joint deformity and destruction. In patients with rheumatoid arthritis, active lymphocyte infiltration and the formation of tertiary lymphoid structures (TLS) occur in the synovium, leading to an increased in situ adaptive immune response, such as peripheral helper T cells (Tph cells) assisting B cells (Non-Patent Literature 2).

[0004] Furthermore, as crucial participants in innate immunity, mononuclear-macrophage cell lines and fibroblast-like synovial cells (FLS) constitute the malignant inflammatory cycle of rheumatoid arthritis, leading to synovial thickening, the production of matrix-degrading enzymes, and the induction of osteoclasts (Non-Patent Literature 3, Non-Patent Literature 4, Non-Patent Literature 5).

[0005] In rheumatoid arthritis, the presence of autoantibodies with common epitopes and the involvement of MHC class II suggests that CD4-positive T cells are closely related to the pathogenesis of rheumatoid arthritis (Non-Patent Literature 6).

[0006] Existing technical documents Non-patent literature Non-patent literature 1: Benton, ML 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 literature 2: Rao, DA et al. Pathologically expanded peripheral Thelper cell subset drives B cells in rheumatoid arthritis. Nature 542, 110-114, doi:10.1038 / nature20810 (2017). Non-patent literature 3: Mizoguchi, F. et al. Functionally distinct disease-associated fibroblast subsets inrheumatoid arthritis. Nature communications 9, 789, doi:10.1038 / s41467-018-02892-y (2018) Non-patent literature 4: Alivernini, S. et al. Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis. Naturemedicine 26, 1295-1306, doi: 10.1038 / s41591-020-0939-8 (2020) Non-patent literature 5: Nygaard, G. & Firestein, GS Restoring synovialhomeostasis in rheumatoid arthritis by targeting fibroblast-likesynoviocytes. Nature reviews. Rheumatology 16, 316-333, doi:10.1038 / s41584-020-0413-5 (2020) Non-patent literature 6: McInnes, IB & 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

[0007] The problem that the invention aims to solve However, research on how human CD4-positive T cells control the inflammatory immune response in patients with rheumatoid arthritis is still ongoing.

[0008] This invention was made in view of the above circumstances, and its object is to provide a method for testing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject. By establishing a method for testing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject, not only can the subject's condition be confirmed clinically, but it will also become an effective tool for the development of therapeutic drugs for rheumatoid arthritis. Furthermore, this invention also relates to providing a method for screening therapeutic drugs for rheumatoid arthritis.

[0009] Methods for solving problems This invention relates to the following [1] to

[13] .

[0010] [1] Methods for testing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in subjects, including: The steps for determining the amount of IGFL2 protein or the expression level of the IGFL2 gene in samples derived from subjects with or suspected of having rheumatoid arthritis, and The step of comparing the aforementioned measured values ​​with a threshold corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene.

[0011] [2] According to the method described in [1] above, when the aforementioned measured value is above the aforementioned threshold, it indicates that the aforementioned subject has a high degree of rheumatoid arthritis or a high risk of developing the disease.

[0012] [3] The method described in [1] or [2] above, wherein the aforementioned sample is body fluid, blood cells or tissue.

[0013] [4] A method for providing information for determining and / or monitoring the severity of rheumatoid arthritis in subjects with rheumatoid arthritis or the risk of rheumatoid arthritis in subjects suspected of having rheumatoid arthritis, the method comprising: The steps for determining the amount of IGFL2 protein or the expression level of the IGFL2 gene in samples derived from the aforementioned subjects, and The steps to provide information related to the aforementioned measured values.

[0014] [5] According to the method described in [4] above, wherein the aforementioned sample is body fluid, blood cells or tissue.

[0015] [6] The use of IGFL2 protein or IGFL2 gene from samples derived from the aforementioned subjects as biomarkers for determining the severity of rheumatoid arthritis in subjects with rheumatoid arthritis or the risk of rheumatoid arthritis in subjects suspected of having rheumatoid arthritis, wherein, The aforementioned determination includes the step of comparing the measured value of the amount of IGFL2 protein or the expression level of the IGFL2 gene in the sample derived from the aforementioned subject with a threshold corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene.

[0016] [7] According to the use described in [6] above, when the aforementioned measured value is above the aforementioned threshold, it indicates that the aforementioned subject has a high degree of rheumatoid arthritis or a high risk of developing the aforementioned disease.

[0017] [8] According to the use described in [6] or [7] above, wherein the aforementioned sample is a body fluid, blood cells or tissue.

[0018] [9] Methods for evaluating the effectiveness of treatment for rheumatoid arthritis, including: The steps for determining the amount of IGFL2 protein or the expression level of the IGFL2 gene in samples from subjects who have received an investigational treatment drug or treatment, and The step of comparing the aforementioned measured values ​​with the measured values ​​derived from the aforementioned subjects before receiving the aforementioned treatment.

[0019]

[10] According to the method described in [9] above, the aforementioned measured value is lower than the measured value of the aforementioned subject before receiving the aforementioned treatment, indicating that the aforementioned treatment is effective.

[0020]

[11] The method described in [9] or

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

[0021]

[12] A screening method for drugs to treat rheumatoid arthritis, which includes: The steps for administering IGFL2 protein and experimental therapeutic drugs to cells expressing the IGFL2 receptor, and The steps for determining the amount of physiologically active substances produced by the aforementioned cells.

[0022]

[13] According to the screening method described in

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

[0023] Invention Effects According to the present invention, methods can be provided for: testing and determining the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis using measurements of IGFL2 protein or IGFL2 gene expression in subjects; and methods for evaluating the effectiveness of treatment for rheumatoid arthritis using the aforementioned measurements. Furthermore, the present invention relates to a method for screening therapeutic drugs for rheumatoid arthritis. Attached Figure Description

[0024] Figure 1 : Figure 1 This is a scatter plot showing the correlation between mean IGFL2 gene expression levels in CD4-positive T cells of the synovium and disease activity (DAS28-ESR or ESR). Each point in the plot represents an individual patient.

[0025] Figure 2 : Figure 2 This is a graph showing the serum IGFL2 protein concentration between a healthy control group and a rheumatoid arthritis patient group. Figure 2 In this study, data are expressed as points and mean ± SD, and unpaired two-tailed t-tests were performed.

[0026] Figure 3 : Figure 3 The ROC curves and AUC values ​​are based on the concentration of IGFL2 protein in the serum of healthy controls and rheumatoid arthritis patients, and are relevant to the determination of rheumatoid arthritis patients.

[0027] Figure 4 : Figure 4 This is a graph showing the serum IGFL2 protein concentration between patients in remission and those in non-remission groups of rheumatoid arthritis. Figure 4 In this study, data are expressed as points and mean ± SD, and unpaired two-tailed t-tests were performed.

[0028] Figure 5 : Figure 5 The ROC curves and AUC values ​​are based on the concentration of IGFL2 protein in the serum of rheumatoid arthritis patients in remission and non-remission groups, which are related to the determination of rheumatoid arthritis remission.

[0029] Figure 6 : Figure 6 This is a graph showing the amounts of various physiologically active substances in the culture supernatant. Culture supernatant with the addition of IGFL2 protein is shown as IGFL2+, and culture supernatant without the addition is shown as IGFL2-. Data are expressed as points and mean ± SD, and unpaired two-sided t-tests were performed.

[0030] Figure 7 : Figure 7This is a graph showing the amounts of various physiologically active substances in the culture supernatant. Culture supernatant with the addition of IGFL2 protein is shown as IGFL2+, and culture supernatant without the addition is shown as IGFL2-. Data are expressed as points and mean ± SD, and unpaired two-sided t-tests were performed.

[0031] Figure 8 : Figure 8 This is a graph showing the amounts of various physiologically active substances in the culture supernatant. Culture supernatant with the addition of IGFL2 protein is shown as IGFL2+, and culture supernatant without the addition is shown as IGFL2-. Data are expressed as points and mean ± SD, and unpaired two-sided t-tests were performed.

[0032] Figure 9 : Figure 9 This figure illustrates the effect of anti-IGFLR1 antibody on CXCL10 production in THP1 cells stably expressing the IGFL2 receptor. Data in the figure are presented as points and mean ± SD, and an unpaired two-sided t-test was performed. In the figure, the concentration of CXCL10 when recombinant human IGFL2 protein and isotype control antibody are added is represented as IGFL2IgG1, and the concentration of CXCL10 when recombinant human IGFL2 protein and IGFLR1 neutralizing antibody FLR are added is represented as IGFL2IGFLR1. Detailed Implementation

[0033] In this invention, "rheumatoid arthritis (RA)" is not specifically defined, but it is a type of autoimmune disease that causes systemic arthritis accompanied by synovial tissue hyperplasia. For example, it usually refers to diseases that meet the 1987 American College of Rheumatology classification criteria or the 2010 ACR / EULAR classification criteria for rheumatoid arthritis.

[0034] In this invention, "having rheumatoid arthritis" is not specifically limited, but refers to the subject having the aforementioned rheumatoid arthritis condition, which can be determined by the aforementioned judgment criteria.

[0035] In this invention, the term "subject" is not specifically limited and refers to, for example, humans or non-human apes, including not only those suffering from rheumatoid arthritis but also those who may have it. Sometimes, people suffering from rheumatoid arthritis are referred to as patients.

[0036] Subjects suspected of having rheumatoid arthritis include: those who subjectively suspect it, those whose health diagnoses include examinations related to this invention, those based on certain objective evidence, and / or those whose examination results indicate a reasonable probability of having the disease.

[0037] In this invention, "risk" refers to the risk that a subject suspected of having rheumatoid arthritis will develop rheumatoid arthritis.

[0038] In this invention, "healthy person" refers to a subject who has excluded all autoimmune diseases and is determined by a doctor based on symptoms such as autoantibodies, joint swelling, joint tenderness, inflammatory response, X-ray findings, and hand stiffness.

[0039] In this invention, "derived from" is not specifically limited, but can refer to those that can be collected by the subject through blood collection, arthroscopy, puncture, biopsy or surgery.

[0040] In this invention, the term "sample" is not particularly limited, but is preferably, for example, body fluids, blood cells, or tissues. 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 synovium and ligaments.

[0041] In this invention, "IGFL2 protein" refers to member 2 of the insulin-like growth factor (IGF) family. The sequence information of the human IGFL2 protein and IGFL2 gene is publicly known, for example, and can be found in well-known databases such as NCBI (National Center for Biotechnology Information). Specifically, the sequence information of the human IGFL2 gene is shown in NM_001002915.3.

[0042] Currently, as shown in Table 1 below, the IGFL2 gene has been found in humans and apes.

[0043] [Table 1] The method for determining the amount of IGFL2 protein in this invention is not particularly limited, and examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and Western blot-based assays. The IGFL2 protein being measured can be a full-length protein or a protein fragment. Furthermore, the measured value can be an absolute amount or a concentration.

[0044] The method for determining the expression level of the IGFL2 gene in this invention is not particularly limited, and examples include RNA sequencing, single-cell RNA sequencing, quantitative PCR, and LAMP. The target for IGFL2 gene measurement can be the DNA encoding the entire gene or a portion thereof, cDNA, or RNA.

[0045] The amount of IGFL2 protein or the expression level of the IGFL2 gene can also be measured using, for example, the average of several measurements. Furthermore, the measurement can be from a single test subject or calculated using multiple test subjects. This calculation can be further performed using any coefficients and / or constants as needed.

[0046] This invention is based on the relationship between rheumatoid arthritis and the amount of IGFL2 protein or the expression level of the IGFL2 gene. Specific embodiments of this invention are illustrated below.

[0047] <Methods for testing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in subjects> In one embodiment, the present invention is a method for testing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject, comprising: The steps for determining the amount of IGFL2 protein or the expression level of the IGFL2 gene in samples derived from subjects with or suspected of having rheumatoid arthritis, and The step of comparing the aforementioned measured values ​​with a threshold corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene.

[0048] In the step of "determining the amount of IGFL2 protein or the expression level of the IGFL2 gene in the sample", these biomarkers are quantified.

[0049] In the step of “comparing the aforementioned measured value with a threshold corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene”, the measured value is compared with the threshold.

[0050] The testing method of the present invention is implemented through the above two steps. Subsequently, in the testing method of the present invention, when the aforementioned measured value is above the aforementioned threshold, it indicates that the aforementioned subject has a high degree of rheumatoid arthritis severity or a high risk of developing the disease.

[0051] In this 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 appropriately set. For example, by accumulating data on the amount of IGFL2 protein or the expression level of the IGFL2 gene in patients with rheumatoid arthritis, a predetermined threshold can be empirically set.

[0052] More specifically, samples were collected from multiple patients diagnosed with rheumatoid arthritis and multiple healthy individuals to determine the amount of IGFL2 protein or the expression level of the IGFL2 gene. Then, the value that most accurately distinguishes the patient group from the healthy group was determined and set as a threshold. In setting the threshold, sensitivity, specificity, positive predictive value, and negative predictive value are preferably considered.

[0053] In this invention, the severity of rheumatoid arthritis can be evaluated, for example, by disease activity.

[0054] In this invention, "disease activity" refers to a value expressed, for example, in the form of DAS-28-ESR or ESR. A DAS-28-ESR value greater than 5.1 is classified as high, 3.2-5.1 as moderate, less than 3.2 as mild, and less than 2.6 as remission.

[0055] <The 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 embodiment, the present invention provides a method for determining and / or monitoring the severity of rheumatoid arthritis in a subject with rheumatoid arthritis or the risk of rheumatoid arthritis in a subject suspected of having rheumatoid arthritis, the method comprising: The steps for determining the amount of IGFL2 protein or the expression level of the IGFL2 gene in samples derived from the aforementioned subjects, and The steps to provide information related to the aforementioned measured values.

[0056] In this invention, the “determination” and / or “monitoring” of “severity of rheumatoid arthritis” and “risk of developing rheumatoid arthritis” means, for example, determining (“determining”) which of the severity levels of rheumatoid arthritis a subject belongs to as described above, or determining (“monitoring”) changes over time.

[0057] The determinations include: the presence or absence of rheumatoid arthritis, the risk of developing rheumatoid arthritis, the severity of rheumatoid arthritis in the subjects, the effectiveness of rheumatoid arthritis prevention in the subjects, the effectiveness of treatment in patients with rheumatoid arthritis, the presence or absence of rheumatoid arthritis recurrence in the subjects, and the risk of rheumatoid arthritis recurrence in the subjects.

[0058] The procedure for "determining the amount of IGFL2 protein or the expression level of the IGFL2 gene in the sample" is the same as described above.

[0059] In the "step of providing information related to the measured value" of the present invention, "providing information" means, for example, providing information related to the measured value (e.g., measured value, threshold) obtained in the above-mentioned measurement step to a doctor or the like.

[0060] The information can be provided not only to doctors, but also, for example, to examination technicians. Doctors may, for example, use the provided information to determine and / or monitor the severity of rheumatoid arthritis, independently of the steps of this invention.

[0061] The method for providing information according to the present invention is implemented through the above two steps.

[0062] <Use of IGFL2 protein or IGFL2 gene in samples as biomarkers> In one embodiment, the present invention utilizes the IGFL2 protein or IGFL2 gene derived from the aforementioned subject's sample as a biomarker for determining the severity of rheumatoid arthritis in subjects with rheumatoid arthritis or the risk of rheumatoid arthritis in subjects suspected of having rheumatoid arthritis. The aforementioned determination includes the step of comparing the measured value of the amount of IGFL2 protein or the expression level of the IGFL2 gene in the sample derived from the aforementioned subject with a threshold corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene.

[0063] In this invention, the IGFL2 protein or IGFL2 gene is used as a biomarker. For example, the measured value of the IGFL2 protein or the measured value of the expression level of the IGFL2 gene is used as an indicator value for determining the severity of rheumatoid arthritis or for determining the risk of developing rheumatoid arthritis.

[0064] The amount of IGFL2 protein or the expression level of the IGFL2 gene can be obtained, for example, by the steps described above for "measuring the amount of IGFL2 protein or the expression level of the IGFL2 gene in the sample".

[0065] The step of “comparing the measured value of the amount of IGFL2 protein or the expression level of the IGFL2 gene in the sample derived from the aforementioned subject with a threshold corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene” is the same as the step of “comparing the aforementioned measured value with a threshold corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene” described above.

[0066] <Methods for evaluating the effectiveness of rheumatoid arthritis treatment> In one embodiment, the present invention is a method for evaluating the therapeutic efficacy of rheumatoid arthritis, comprising: The steps for determining the amount of IGFL2 protein or the expression level of the IGFL2 gene in samples from subjects who have received an investigational treatment drug or treatment, and The step of comparing the aforementioned measured values ​​with the measured values ​​derived from the aforementioned subjects before receiving the aforementioned treatment.

[0067] In this invention, "treatment of rheumatoid arthritis" refers to, for example, reducing, improving, or alleviating the severity of the aforementioned rheumatoid arthritis.

[0068] In this invention, "treatment effectiveness" refers to, for example, a reduction, improvement, or relief of the severity of the aforementioned rheumatoid arthritis. The evaluation of whether these conditions occur is referred to, for example, "evaluation of treatment effectiveness."

[0069] In this invention, the experimental therapeutic drugs or methods are not particularly limited, and examples include steroids, NSAIDs (analgesics), conventional low molecular weight antirheumatic drugs (csDMARDs), molecularly targeted antirheumatic drugs (tsDMARDs), LCAP, synovectomy, etc. Furthermore, subjects receiving these treatments are referred to as post-treatment subjects.

[0070] The procedure for "determining the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from a subject who has received the investigational therapeutic drug or treatment" is the same as the procedure for "determining the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from a subject" described above.

[0071] In the step of “comparing the measured value with the measured value derived from the aforementioned subject before receiving the aforementioned treatment”, the measured value obtained in the above steps is compared with the measured value derived from the aforementioned subject before receiving the aforementioned treatment.

[0072] In this invention, the measured values ​​derived from the aforementioned subjects before receiving the treatment refer to, for example, the measured values ​​of IGFL2 protein or gene expression before receiving the aforementioned treatment.

[0073] The above two steps constitute a method for evaluating the effectiveness of rheumatoid arthritis treatment. Furthermore, in the evaluation method of this invention, if the aforementioned measured value is lower than the measured value derived from the aforementioned subject before receiving the aforementioned treatment, it indicates that the aforementioned treatment is effective.

[0074] Specifically, if the value is significantly lower than that of the control group by an unpaired two-sided t-test, it indicates that the aforementioned treatment is effective.

[0075] <Methods for screening drugs for the treatment of rheumatoid arthritis> A further aspect of the present invention is a method for screening therapeutic drugs for rheumatoid arthritis, the method comprising: The steps for administering IGFL2 protein and experimental therapeutic drugs to cells expressing the IGFL2 receptor, and The steps for determining the amount of physiologically active substances produced by the aforementioned cells.

[0076] In the step of "administering IGFL2 protein and experimental therapeutic agent to cells expressing the IGFL2 receptor", the cells expressing the IGFL2 receptor are preferably those that stably express the IGFL2 receptor.

[0077] This cell can be generated by introducing and stably expressing the IGFL2 receptor into human cells such as THP1 cells, Jurkat cells, or peripheral blood mononuclear cells of healthy individuals using known methods, such as lentiviruses. More specifically, it can be prepared using the methods described in the examples.

[0078] This step can be performed, for example, by culturing cells expressing the IGFL2 receptor, adding the IGFL2 protein and the experimental therapeutic drug to the culture medium. Furthermore, known conditions and methods can be used without particular restriction regarding detailed culture conditions and methods.

[0079] In addition, the concentration, timing, and method of administration of IGFL2 protein and the test therapeutic drug can be appropriately set by taking into account the physical properties of the test therapeutic drug and its toxicity to organisms.

[0080] The IGFLR1 receptor is a structure that can bind to the IGFLR2 protein; examples include IGFLR1. The sequence information of the IGFLR1 gene is publicly available, for example, from databases such as NCBI (National Center for Biotechnology Information). Specifically, the sequence information of the human IGFLR1 gene is shown in NM_024660.4.

[0081] Cells that stably express the IGFL2 receptor can be modified to express fluorescent proteins such as GFP or luciferase proteins such as Renilla Luciferase upon stimulation by the IGFL2 protein. The expression levels of these proteins can be measured using these modified cells to replace the quantification of physiologically active substances.

[0082] The physiologically active substances to be measured in the "step of measuring the amount of physiologically active substances produced by the aforementioned cells" are preferably soluble proteins such as chemokines or cytokines produced by stimulation of the IGFL2 protein, or low-molecular-weight signal transduction substances. Specific examples of such physiologically active substances include, for example, CXCL10, CXCL1, CXCL11, TNF, CXCL5, CCL20, GM-CSF, CCL24, CXCL8, CCL2, and CCL15.

[0083] Stimulation of the IGFL2 protein can be achieved, for example, by adding the IGFL2 protein to the culture medium of cells expressing the IGFL2 receptor.

[0084] There are no particular limitations on the method for determining physiologically active substances, and examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and Western blotting-based methods. The value of the physiologically active substance actually measured can be, for example, the amount or concentration. The measured value of a physiologically active substance can also be, for example, the average of several measurements. Furthermore, the measured value of a physiologically active substance can be the value of a single physiologically active substance or a value calculated using multiple physiologically active substances. This calculation can be further modified using any coefficients and / or constants as needed.

[0085] As a standard for determining whether an experimental therapeutic drug is a treatment for rheumatoid arthritis, for example, if the "measured value of physiologically active substances obtained after administration of IGFL2 protein and experimental therapeutic drug" is less than the "average value of physiologically active substances obtained after administration of IGFL2 protein but without administration of experimental therapeutic drug - 3SD", then the experimental therapeutic drug can be determined to be a treatment for rheumatoid arthritis.

[0086] Regarding the above embodiments, the present invention further discloses the following diagnostic methods.

[0087] [1] Methods for diagnosing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject, including: The steps for determining the amount of IGFL2 protein or the expression level of the IGFL2 gene in samples derived from subjects with or suspected of having rheumatoid arthritis, and The step of comparing the aforementioned measured values ​​with a threshold corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene.

[0088] [2] According to the method described in [1] above, when the measured value is above the aforementioned threshold, it indicates that the subject has a high degree of rheumatoid arthritis or a high risk of developing the disease.

[0089] [3] The method described in [1] or [2] above, wherein the sample is a body fluid, blood cells or tissue. Example

[0090] The present invention will be specifically described below with examples, but the present invention is not limited to the examples described below. In addition, the experiments of the present invention have been approved by the Ethics Committee of the Graduate School of Medicine, Kyoto University, and written informed consent has been obtained from all participants.

[0091] [Statistical Analysis] Statistical analyses were performed using Prism 10 software (GraphPad, San Diego, California, USA) or R (v40.5). A p-value < 0.05 was considered statistically significant.

[0092] [Disease activity] Disease activity in patients with rheumatoid arthritis is determined using DAS28-ESR and ESR.

[0093] [Patients and healthy individuals] Joint samples were taken from surgical remnants or artifacts generated during or at outpatient clinics in patients with rheumatoid arthritis. Additionally, among rheumatoid arthritis patients, those with a DAS28-ESR less than 2.6 were defined as those in remission, while those with a DAS28-ESR greater than 2.6 were defined as those not in remission.

[0094] After examination at a medical institution, a healthy group was obtained from the group that was diagnosed by doctors to rule out autoimmune diseases.

[0095] [Isolation of CD4-positive T cells] Synovial tissue from patients (including those in remission) and healthy individuals was minced and enzymatically treated with 100 μg / mL Liberase TL (Roche) and 100 μg / mL DNase I (Roche) at 37°C for 30 minutes. Peripheral blood mononuclear cells or synovial mononuclear cells were then collected using Lymphocyte Separation Solution 1.077 (Nacalai Tesque). Further, CD4+ T cells derived from synovial tissue and serum were isolated from patients with rheumatoid arthritis and healthy individuals, respectively, using the CD4+ T Cell Isolation Kit (Mitenia).

[0096] [Production of recombinant IGFL2 protein] An optimized double-stranded DNA encoding the human IGFL2 protein, with a His-tagged C-terminus, was synthesized by IDT (San Diego, California) and inserted into CSII-EF-IERS-Venus (obtained from RIKEN BioResource Center). CSII-EF-IERS-Venus is a mutant lentiviral vector expressing the YFP and Venus genes, developed by Atsushi Miyawaki.

[0097] HEK293T cells were transformed with lentiviral particles (including CSII-EF-IERS-Venus containing the aforementioned double-stranded DNA) and sorted using FACSMelody (BD Biosciences) to establish Venus+ IGFL2- to generate HEK293T cells.

[0098] Recombinant IGFL2 protein with a C-terminal His-tag was purified from the culture supernatant of HEK293T cells that had previously produced IGFL2 using Dynabeads His-Tag Isolation & Pulldown (Thermo Scientific). The concentration of recombinant IGFL2 protein was determined using the His Tag ELISA Detection Kit (GenScript), and the endotoxin content in the recombinant protein was confirmed to be less than 0.1 EU / μg using the ToxinSensorChromogenic LAL Endotoxin Assay Kit (GenScript).

[0099] It should be noted that the amino acid sequence of the human IGFL2 protein is as follows.

[0100] PAGSEPWLCQPAPRCGDKIYNPLEQCCYNDAIVSLSETRQCGPPCTFWPCFELCLDSFGLTNDFVVKLKVQGVNSQCHSSPISSKCESRRRFP (SEQ ID NO: 1) [Establishment of a cell line stably expressing the IGFL2 receptor THP1] An optimized double-stranded DNA encoding the human IGFLR1 (NM_024660.4) protein was synthesized by IDT (San Diego, California) and inserted into CSII-EF-IERS-puro (obtained from RIKEN BioResource Center). Lentiviral particles (containing CSII-EF-IERS-puro containing the aforementioned double-stranded DNA) were used to transform THP1 cells, which were then cultured for 4 days in the presence of 2 μg / mL puromycin. The gene-transfected cells, THP1R, were selected as a stable THP1 cell line expressing the IGFL2 receptor.

[0101] ELISA The protein concentrations of cytokines and chemokines in monocyte supernatants were evaluated using the Bio-Plex Pro Human Chemokine 40-Plex Assay Kit (Bio-Rad) and the Bio-Plex200 system (Bio-Rad).

[0102] To detect IGFL2 protein, a rabbit polyclonal antibody against IGFL2 was produced by Eurofins Genomics (Tokyo, Japan). Maxisorp 96-well plates (Thermo Scientific) were coated with the anti-IGFL2 capture antibody diluted in Coating Buffer (BioLegend). After four washes with washing buffer (1xPBS, 0.05% Tween-20), the plates were blocked with Assay Diluent (Biolegend) for 1 hour. Serum samples diluted 4-fold with Assay Diluent were incubated on the plates for 2 hours. The anti-IGFL2 biotinylation detection antibody, Avidin-HRP (Thermo Scientific), and Ultra TMB (Thermo Scientific) were added sequentially to the plates for reaction, followed by termination with 2N sulfuric acid (NacalaiTesque). The captured IGFL2 protein was detected by measuring absorbance at 450 nm using a Spectra Max iD3 (Molecular Devices).

[0103] [Quantification of IGFL2 based on quantitative PCR] mRNA was extracted using MyOne Silane (Thermo Scientific). cDNA was synthesized using the SuperPrep RT Kit for qPCR (TOYOBO). qRT-PCR was performed on the following two genes using THUNDERBIRD SYBR qPCR Mix (TOYOBO) on a C1000 Touch (Bio-Rad) system.

[0104] Use the following as primers: Human 18S rRNA gene (h18S rRNA) is used 5'-AACTTTCGATGGTAGTCGCCG-3' (SEQ ID NO: 2) 5'-CCTTGGATGTGGTAGCCGTTT-3' (SEQ ID NO: 3) Human IGFL2 gene (hIGFL2) is used 5'-GACTACCCCAGGAGTGTGCT-3' (SEQ ID NO: 4) 5'-CAGCGGGAGCGATGACT-3' (SEQ ID NO: 5) The expression of mRNA was normalized by the expression of h18s rRNA.

[0105] <Example 1> [The relationship between the human IGFL2 gene and disease activity in patients with rheumatoid arthritis] The inventors investigated the relationship between the expression of the IGFL2 gene on CD4-positive T cells in patients with rheumatoid arthritis and the disease activity of these patients.

[0106] CD4-positive T cells were isolated from synovial tissue (ST) of 11 patients with rheumatoid arthritis, and the expression level of the IGFL2 gene expressed in CD4-positive T cells was determined by single-cell RNA sequencing.

[0107] On the other hand, the disease activity of the same rheumatoid arthritis patients can be determined by DAS28-ESR and ESR.

[0108] Figure 1 The image shows a scatter plot illustrating the relationship between the average expression level of the IGFL2 gene and disease activity. Figure 1 It can be seen that the expression of the IGFL2 gene is significantly correlated with the disease activity of rheumatoid arthritis (DAS28-ESR: r=0.621, p=0.041; ESR: R=0.827, p=0.002).

[0109] <Example 2> [Expression of IGFL2 protein in the serum of patients with rheumatoid arthritis] The inventors investigated the expression of IGFL2 protein in peripheral blood.

[0110] (1) The expression level (concentration) of IGFL2 protein in serum peripheral blood obtained from age- and sex-matched healthy individuals and rheumatoid arthritis patients was quantified using ELISA. The results showed that the concentration of IGFL2 protein in serum was significantly higher in the rheumatoid arthritis patient group (RA) than in the healthy group (control). Figure 2 ).

[0111] (2) Based on the serum IGFL2 protein concentrations obtained in (1) above for the healthy group and the rheumatoid arthritis patient group, the optimal threshold for distinguishing between the rheumatoid arthritis patient group and the healthy group was determined. Using the set threshold (119.3 pg / mL), the sensitivity, specificity, and area under the curve (AUC) of the determination were calculated. The resulting ROC curve is shown in... Figure 3 .

[0112] This assay can distinguish between patients with rheumatoid arthritis and healthy individuals with a sensitivity of 96% and a specificity of 76%. Table 2 summarizes the threshold (pg / mL), sensitivity (%), specificity (%), and AUC of the assay.

[0113] [Table 2] The results show that the AUC value of 0.92 is high enough in the determination based on serum IGFL2 protein concentration, and serum IGFL2 protein concentration can be used as a biomarker to distinguish between patients with rheumatoid arthritis and healthy individuals.

[0114] <Example 3> [Expression of IGFL2 protein in the serum of patients in remission of rheumatoid arthritis] Similar to Example 2, the inventors investigated the expression of IGFL2 protein in peripheral blood.

[0115] (1) Rheumatoid arthritis patients were divided into a rheumatoid arthritis remission group and a rheumatoid arthritis non-remission group. The expression level (concentration) of IGFL2 protein in serum peripheral blood obtained from the age- and sex-matched rheumatoid arthritis remission group and the rheumatoid arthritis non-remission group was quantified by ELISA. The results showed that the concentration of IGFL2 protein in serum was significantly higher in the rheumatoid arthritis non-remission group (active) than in the rheumatoid arthritis remission group (remission). Figure 4 ).

[0116] (2) Based on the serum IGFL2 protein concentrations obtained in (1) above for the rheumatoid arthritis remission group and the rheumatoid arthritis non-remission group, the optimal threshold for distinguishing between the two groups was determined. Using the set threshold (989 pg / mL), the sensitivity, specificity, and area under the curve (AUC) were calculated. The resulting ROC curve is shown in... Figure 5 .

[0117] This determination can differentiate between non-remission rheumatoid arthritis patients and those in remission with a sensitivity of 76% and a specificity of 77%. Table 3 summarizes the threshold (pg / mL), sensitivity (%), specificity (%), and AUC of the determination.

[0118] [Table 3] The results show that the AUC value of 0.8 is high enough in the determination based on serum IGFL2 protein concentration, and serum IGFL2 protein concentration can be used as a biomarker to distinguish between non-remission rheumatoid arthritis patients and rheumatoid arthritis patients in remission.

[0119] The results of Examples 2 and 3 show that there is a strong correlation between the concentration of IGFL2 protein in serum and the severity of rheumatoid arthritis.

[0120] Therefore, by measuring the concentration of IGFL2 protein in serum, it is possible to: (1) Methods for testing the severity of rheumatoid arthritis in patients with rheumatoid arthritis and methods for testing the risk of developing rheumatoid arthritis in patients suspected of having rheumatoid arthritis.

[0121] (2) Methods for providing information for determining and / or monitoring the severity of rheumatoid arthritis in patients with rheumatoid arthritis, and methods for providing information for determining and / or monitoring the risk of rheumatoid arthritis in patients suspected of having rheumatoid arthritis.

[0122] (3) Judgment of whether the subject is suspected of having rheumatoid arthritis, diagnosis of rheumatoid arthritis, and judgment of the therapeutic effect of drugs on rheumatoid arthritis patients.

[0123] <Example 4> [Research on screening methods for drugs to treat rheumatoid arthritis] (1) THP1 cells with stable expression of IGFL2 receptor were cultured together with 100 ng / mL of recombinant human IGFL2 protein in 10% bovine serum RPMI-1640. After 48 hours, the culture medium was centrifuged at 400×g for 5 minutes to obtain the culture supernatant.

[0124] Next, 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 quantified using a multiplex ELISA method. The results were... Figures 6-8 The figure is represented by IGFL2+.

[0125] (2) On the other hand, for those THP1 cells stably expressing the IGFL2 receptor but without the addition of IGFL2 protein, the culture was carried out in the same manner as in (1) above to obtain the culture supernatant, and the amounts of various chemokines were quantified. The results showed that... Figures 6-8 The diagram is represented by IGFL2 -.

[0126] like Figures 6-8 As shown, the expression levels of various physiologically active substances are significantly increased by adding IGFL2 protein.

[0127] (3) Further, THP1 cells with stable expression of IGFL2 receptor were cultured in 10% bovine serum RPMI-1640. During culture, any one of the components (a) to (b) below was added.

[0128] (a) 100 ng / mL of recombinant human IGFL2 protein and 20 μg / mL of isotype control antibody (R&D Systems) (b) 100 ng / mL of recombinant human IGFL2 protein and 20 μg / mL of anti-human IGFLR1 antibody (R&D Systems) Twenty-four hours later, the culture medium was centrifuged at 400×g for 5 minutes to obtain the culture supernatant. Then, the amount (concentration) of CXCL10 in the culture supernatant was quantified by ELISA. The results are shown below. Figure 9 . Figure 9 In the above, IGFL2IgG1 corresponds to (a), and IGFL2IGFLR1 corresponds to (b).

[0129] like Figure 9 As shown, the concentration of CXCL10 produced by adding neutralizing antibodies, namely anti-human IGFLR1 antibody and IGFL2 protein, is statistically significantly lower than the concentration produced by adding non-neutralizing antibodies, namely isotype control antibody and IGFL2 protein. Anti-human IGFLR1 antibody, being a neutralizing antibody against IGFLR1, possesses inhibitory activity against the IGFL2 / IGFL2 receptor pathway. Therefore, substances that reduce the production of physiologically active substances such as CXCL10, similar to anti-human IGFLR1 antibody, can be used as therapeutic agents for rheumatoid arthritis.

[0130] Therefore, it can be seen that the method of the present invention can provide a method for screening drugs for the treatment of rheumatoid arthritis.

[0131] Industrial practicality The method for testing the severity of rheumatoid arthritis according to the present invention, as well as the related technologies and methods for screening drugs for the treatment of rheumatoid arthritis, can be used in the medical field and pharmaceutical industry related to rheumatoid arthritis.

Claims

1. Methods for testing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in subjects, including: The steps for determining the amount of IGFL2 protein or the expression level of the IGFL2 gene in samples derived from subjects with or suspected of having rheumatoid arthritis, and The step of comparing the measured value with a threshold corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene.

2. The method according to claim 1, wherein, When the measured value is above the threshold, it indicates that the subject has a high degree of rheumatoid arthritis severity or a high risk of developing the disease.

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

4. A method for providing information for determining and / or monitoring the severity of rheumatoid arthritis in subjects with rheumatoid arthritis or the risk of rheumatoid arthritis in subjects suspected of having rheumatoid arthritis, the method comprising: The steps for determining the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from the subject, and The step of providing information related to the measured value.

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

6. The use of IGFL2 protein or IGFL2 gene from samples derived from subjects as biomarkers for determining the severity of rheumatoid arthritis in subjects with rheumatoid arthritis or the risk of rheumatoid arthritis in subjects suspected of having rheumatoid arthritis, wherein, The determination includes 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 corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene.

7. The use according to claim 6, wherein, When the measured value is above the threshold, it indicates that the subject has a high degree of rheumatoid arthritis severity or a high risk of developing the disease.

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

9. Methods for evaluating the effectiveness of treatment for rheumatoid arthritis, including: The steps for determining the amount of IGFL2 protein or the expression level of the IGFL2 gene in samples from subjects who have received an investigational treatment drug or treatment, and The step of comparing the measured value with a value derived from the subject prior to receiving the treatment.

10. The method according to claim 9, wherein, The treatment is considered effective when the measured value is lower than the measured value of the subject before receiving the treatment.

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

12. Screening methods for drugs used to treat rheumatoid arthritis, including: The steps for administering IGFL2 protein and experimental therapeutic drugs to cells expressing the IGFL2 receptor, and The step of determining the amount of physiologically active substances produced by the cells.

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

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