Methods for testing the 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

Technical Field

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

Background Art

[0002] Genomic traits acquired during human evolution include autoimmune diseases, but the molecular-level mechanisms of human immune responses regulated by these traits have not yet been elucidated (Non-Patent Document 1). Rheumatoid arthritis (RA) is an autoimmune disease that causes systemic arthritis accompanied by hyperplasia of synovial tissue, which results in deformation and destruction of joints. Active lymphocyte infiltration and formation of tertiary lymphoid structures (TLS) occur in the synovium of rheumatoid arthritis patients, leading to enhanced in situ adaptive immune responses such as B cell help provided by peripheral helper T cells (Tph cells) (Non-Patent Document 2). Furthermore, the monocyte-macrophage system and fibroblast-like synoviocytes (FLS), which are extremely important players in innate immunity, constitute a malignant inflammatory cycle in rheumatoid arthritis, leading to synovial hyperplasia, production of matrix degrading enzymes, and induction of osteoclasts (Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5).

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

Prior Art Literature

Non-Patent Literature

[0004]

Non-Patent Document 1

[0005] However, research is ongoing into how human CD4-positive T cells regulate the inflammatory immune response in patients with rheumatoid arthritis.

[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. By establishing a method for testing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject, it will not only be possible to confirm the clinical condition of the subject, but it will also be an effective tool for the development of therapeutic drugs for rheumatoid arthritis. Furthermore, the present invention relates to providing a screening method for therapeutic drugs for rheumatoid arthritis. [Means for solving the problem]

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

[13] . [1] A step of measuring the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from a subject suffering from rheumatoid arthritis or a subject suspected of suffering from rheumatoid arthritis, and A step of comparing the measured value with a threshold corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene, A method for testing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject, including the following: [2] The method according to [1], wherein if the measured value is equal to or greater than the threshold, it indicates that the subject has a high severity of rheumatoid arthritis or a high risk of developing the disease. [3] The method according to [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 a subject who has rheumatoid arthritis, or the risk of a subject suspected of having rheumatoid arthritis developing rheumatoid arthritis, A step of measuring the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from the subject, and A step of providing information relating to the aforementioned measurement values, A method that includes this. [5] The method according to [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 a subject suffering from rheumatoid arthritis, or the risk of a subject suspected of having rheumatoid arthritis developing rheumatoid arthritis, The determination involves comparing a 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. Use including. [7] The use described in [6], wherein if the measured value is equal to or greater than the threshold, it indicates that the subject has a high severity of rheumatoid arthritis or a high risk of developing the disease. [8] The use described in [6] or [7] above, wherein the sample is a body fluid, blood cells, or tissue. [9] A method for evaluating the effectiveness of treatment for rheumatoid arthritis, A 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 investigational drug or treatment method, and A step of comparing the measured value with a measured value derived from the subject before the treatment, An evaluation method that includes this.

[10] The method according to [9], wherein the measured value is lower than the measured value from the subject before the treatment, indicating that the treatment is effective.

[11] The method according to [9] or

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

[12] A step of conferring IGFL2 protein and an investigational drug to cells expressing the IGFL2 receptor, and A step of measuring the amount of physiologically active substances produced by the aforementioned cells, A screening method for rheumatoid arthritis medications, including [specific example].

[13] The screening method according to

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

[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. Furthermore, the present invention relates to providing a screening method for therapeutic drugs for rheumatoid arthritis. [Brief explanation of the drawing]

[0009] [Figure 1]Figure 1 is a scatter plot showing the correlation between the average expression level of the IGFL2 gene in synovial CD4-positive T cells and disease activity (DAS28-ESR or ESR). Each dot in the figure represents an individual patient. [Figure 2] Figure 2 is a graph showing the IGFL2 protein concentration in serum between a healthy control group and a rheumatoid arthritis patient group. In Figure 2, data are shown as dots and mean ± SD, and unpaired two-tailed t-test was performed. [Figure 3] Figure 3 shows ROC curves and AUC values for identifying rheumatoid arthritis patients based on the serum IGFL2 protein concentration in a healthy control group and a rheumatoid arthritis patient group. [Figure 4] Figure 4 is a graph showing the IGFL2 protein concentration in serum between a rheumatoid arthritis patient remission group and a rheumatoid arthritis patient non-remission group. In Figure 4, data are shown as dots and mean ± SD, and unpaired two-tailed t-test was performed. [Figure 5] Figure 5 shows ROC curves and AUC values for identifying rheumatoid arthritis remission based on the serum IGFL2 protein concentration in a rheumatoid arthritis patient remission group and a rheumatoid arthritis patient non-remission group. [Figure 6] Figure 6 is a graph showing the amounts of various physiologically active substances in culture supernatant. A culture supernatant obtained by adding IGFL2 protein is indicated as IGFL2 +, and a culture supernatant obtained without adding the protein is indicated as IGFL2 -. Data are shown as dots and mean ± SD, and unpaired two-tailed t-test was performed. [Figure 7] Figure 7 is a graph showing the amounts of various physiologically active substances in culture supernatant. A culture supernatant obtained by adding IGFL2 protein is indicated as IGFL2 +, and a culture supernatant obtained without adding the protein is indicated as IGFL2 -. Data are shown as dots and mean ± SD, and unpaired two-tailed t-test was performed. [Figure 8] Figure 8 is a graph showing the amounts of various physiologically active substances in culture supernatant. A culture supernatant obtained by adding IGFL2 protein is indicated as IGFL2 +, and a culture supernatant obtained without adding the protein is indicated as IGFL2 -. Data are shown as dots and mean ± SD, and unpaired two-tailed t-test was performed. [Figure 9] Figure 9 shows the effect of anti-IGFLR1 antibody on CXCL10 production by IGFL2 receptor constitutively expressing THP1 cells. In the figure, data are shown as dots and mean ± SD, and an unpaired two-sided t-test was performed. In the figure, the concentration of CXCL10 when human recombinant IGFL2 protein and isotype control antibody are added is shown as IGFL2IgG1, and the concentration of CXCL10 when human recombinant IGFL2 protein and the IGFLR1 neutralizing antibody FLR are added is shown as IGFL2IGFLR1. [Modes for carrying out the invention]

[0010] In this invention, "rheumatoid arthritis (RA)" refers to an autoimmune disease, without any particular limitations, 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 classification criteria for rheumatoid arthritis.

[0011] In the present invention, "suffering from rheumatoid arthritis" means, without any particular limitations, that the subject has the above-described condition of rheumatoid arthritis, and can be determined, for example, by the above-described criteria.

[0012] In this invention, the term "subject" is not particularly limited, but refers to, for example, a human or ape, and includes not only those suffering from rheumatoid arthritis but also those that may be susceptible to it. A human suffering from rheumatoid arthritis may be referred to as a "patient." Subjects suspected of having rheumatoid arthritis include those who subjectively suspect they have the disease, those whose health checkups include tests related to the present invention, those for whom there is some objective evidence, and / or those for whom a physician has determined that there is a reasonable possibility of having the disease based on the results of an examination. In this invention, "risk" refers to the risk that a subject suspected of having rheumatoid arthritis will actually develop rheumatoid arthritis.

[0013] In this invention, "healthy person" refers to a subject in whom all autoimmune diseases have been ruled out, and is determined by a physician's diagnosis based on symptoms such as autoantibodies, joint swelling, joint tenderness, inflammatory response, X-ray findings, and hand stiffness.

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

[0015] In the present invention, the term "sample" is not particularly limited, but is preferably, for example, 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 membrane and ligaments.

[0016] In this invention, "IGFL2 protein" refers to insulin-like growth factor (IGF) family member 2. The sequence information of the human IGFL2 protein and IGFL2 gene is publicly known and can be obtained from publicly 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. Currently, the IGFL2 gene has been found in humans and great apes, as shown in Table 1 below.

[0017] [Table 1]

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

[0019] The method for measuring the expression level of the IGFL2 gene in the present invention is not particularly limited, but examples include RNA sequencing, single-cell RNA sequencing, quantitative PCR, and LAMP. The target of measurement for the IGFL2 gene may be the DNA encoding the entire gene, a part thereof, cDNA, or RNA.

[0020] The measured 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 from a single metric, or it may be a value calculated using multiple metric measurements. This calculation may also include arbitrary coefficients and / or constants as needed.

[0021] This invention was developed 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 this invention are illustrated below.

[0022] <Method for testing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject> In one embodiment, the present invention is A step of measuring the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from a subject suffering from rheumatoid arthritis or a subject suspected of suffering from rheumatoid arthritis, and A step of comparing the measured value with a threshold corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene, A method for testing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in a subject, including the following:

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

[0024] In 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," the measured value is compared with the threshold. The test method of the present invention is carried out through these two steps. In the test method of the present invention, if the measured value is above the threshold, it is indicated that the subject has a high severity of rheumatoid arthritis or 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 as appropriate. For example, a predetermined threshold value can be empirically set by accumulating data on the amount of IGFL2 protein or the expression level of the IGFL2 gene from patients with rheumatoid arthritis. More specifically, samples are collected from multiple patients diagnosed with rheumatoid arthritis and multiple healthy individuals, and measurements of the amount of IGFL2 protein or the expression level of the IGFL2 gene are obtained. Then, the value that most accurately distinguishes the patient group from the healthy group is 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 this invention, the "severity of rheumatoid arthritis" can be evaluated, for example, by disease activity. In this invention, "disease activity" refers to a value expressed as, for example, DAS-28-ESR or ESR, where a DAS-28-ESR value 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] <A 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 is A method for providing information for determining and / or monitoring the severity of rheumatoid arthritis in subjects suffering from rheumatoid arthritis, or the risk of developing rheumatoid arthritis in subjects suspected of having rheumatoid arthritis, A step of measuring the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from the subject, and The process of providing information relating to the aforementioned measurement values. This is a method that includes [something].

[0028] In the present invention, "determination" and / or "monitoring" of "severity of rheumatoid arthritis" and "risk of developing rheumatoid arthritis" means, for example, determining which of the above-described severity levels of rheumatoid arthritis a subject falls under ("determination"), 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 the subject, determining the preventive effect of rheumatoid arthritis in the subject, determining the therapeutic effect in patients with rheumatoid arthritis, determining whether or not rheumatoid arthritis has relapsed in the subject, and determining the risk of rheumatoid arthritis relapse in the subject.

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

[0030] In the "step of providing information related to measured values" of the present invention, "providing information" means, for example, providing information related to the measured values ​​obtained in the above measurement step (e.g., measured values, threshold values) to a doctor or the like. The information can be provided to recipients other than physicians, for example, to medical technologists. Physicians can use the provided information, for example, independently of the process of the present invention, to determine and / or monitor the severity of rheumatoid arthritis. The method for providing information according to the present invention is carried out through these two steps.

[0031] <Use as a biomarker for IGFL2 protein or IGFL2 gene in samples> In one embodiment, the present invention is 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 a subject suffering from rheumatoid arthritis, or the risk of a subject suspected of having rheumatoid arthritis developing rheumatoid arthritis, The determination involves comparing a 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. This includes usage.

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

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

[0035] <Methods for evaluating the effectiveness of treatment for rheumatoid arthritis> In one embodiment, the present invention is A method for evaluating the effectiveness of treatment for rheumatoid arthritis, A 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 investigational drug or treatment method, and A step of comparing the measured value with a measured value derived from the subject before the treatment, This is an evaluation method that includes [something].

[0036] In the present invention, "treatment of rheumatoid arthritis" means, for example, reducing, improving, or relieving the severity of rheumatoid arthritis as described above.

[0037] In this invention, "treatment effectiveness" refers, for example, to the reduction, improvement, or remission of the severity of rheumatoid arthritis as described above. Evaluating whether these effects have occurred is referred to, for example, as "evaluation of treatment effectiveness."

[0038] In the present invention, the investigational drug or treatment method is not particularly limited, but examples include steroids, NSAIDs (analgesics), conventional low-molecular-weight antirheumatic drugs (csDMARDs), molecularly targeted antirheumatic drugs (tsDMARDs), LCAP, and synovectomy. Furthermore, a subject who has received 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 investigational drug or treatment" 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 from the subject before the treatment," the measured value obtained in the above step is compared with the measured value from the subject before the treatment. In the present invention, the measured values ​​derived from the subject before treatment refer, for example, to the measured values ​​of IGFL2 protein or gene expression before the above treatment.

[0041] The method for evaluating the effectiveness of 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 from the subject before receiving the treatment, it is indicated that the treatment is effective. Specifically, the effectiveness of the treatment is demonstrated when an unpaired two-sided t-test shows a significantly lower value compared to the control group.

[0042] <Screening methods for rheumatoid arthritis medications> A further aspect of the present invention is a screening method for therapeutic drugs for rheumatoid arthritis, the method being A process for conferring IGFL2 protein and an investigational drug to cells expressing the IGFL2 receptor, and A step of measuring the amount of physiologically active substances produced by the aforementioned cells, This is a screening method for the treatment of rheumatoid arthritis, including [specific example].

[0043] In the "step of conferring IGFL2 protein and the investigational drug to cells expressing the IGFL2 receptor," it is preferable that the cells expressing the IGFL2 receptor are those that constitutively express the IGFL2 receptor. Such cells can be constitutively expressed by introducing genes from human cells, such as THP1 cells, Jurkat cells, or peripheral blood mononuclear cells from healthy individuals that express the IGFL2 receptor, using known methods, such as lentiviruses. More specifically, they can be produced by the method described in the examples.

[0044] This process can be carried out, for example, by culturing cells expressing the IGFL2 receptor and adding the IGFL2 protein and the investigational drug to the culture medium. Furthermore, known conditions and methods can be used without particular restriction for detailed culture conditions and methods. Furthermore, the concentration, timing, and method of administration of the IGFL2 protein and the investigational drug should be appropriately determined considering the physical properties of the investigational drug used and its toxicity to living organisms.

[0045] An IGFL2 receptor is a structure that has the function of binding to the IGFL2 protein, such as IGFLR1. The sequence information of the IGFLR1 gene is publicly known and can be obtained from publicly available 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 to express fluorescent proteins such as GFP or luciferase proteins such as Renilla Luciferase in response to stimulation by the IGFL2 protein. Measuring the expression levels of these proteins using such modified cells can be used as a substitute for quantifying physiologically active substances.

[0047] In the "step of measuring the amount of physiologically active substances produced by the cells," the physiologically active substances to be measured are preferably soluble proteins such as chemokines or cytokines produced by stimulation of the IGFL2 protein, and physiologically active substances such as small molecule signaling molecules. Specific examples of such physiologically active substances include, for example, CXCL10, CXCL1, CXCL11, TNF, CXCL5, CCL20, GM-CSF, CCL24, CXCL8, CCL2, and CCL15. Stimulation with the IGFL2 protein can be performed, for example, by adding the IGFL2 protein to the culture medium of cells expressing the IGFL2 receptor.

[0048] The method for measuring physiologically active substances is not particularly limited, but examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and Western blotting. The actual values ​​of physiologically active substances to be measured include, for example, quantity and concentration. As the measured value of a physiologically active substance, it is also possible to use, for example, the average of several measurement values. Furthermore, the measured value of a physiologically active substance may be the measurement value of a single physiologically active substance, or it may be a value calculated using multiple physiologically active substances. In this calculation, arbitrary coefficients and / or constants may be used as needed.

[0049] As a criterion for determining whether an investigational drug is a treatment for rheumatoid arthritis, for example, if the "measured values ​​of bioactive substances obtained by conferring IGFL2 protein and the investigational drug" are less than "the mean value of the measured values ​​of bioactive substances obtained by conferring IGFL2 protein but not the investigational drug - 3 standard deviations," then the investigational drug can be determined to be a treatment for rheumatoid arthritis.

[0050] With regard to the embodiments described above, the present invention further discloses the following diagnostic method. [1] A step of measuring the amount of IGFL2 protein or the expression level of the IGFL2 gene in a sample derived from a subject suffering from rheumatoid arthritis or a subject suspected of suffering from rheumatoid arthritis, and A step of comparing the measured value with a threshold corresponding to the amount of IGFL2 protein or the expression level of the IGFL2 gene, A method for diagnosing the severity of rheumatoid arthritis or the risk of developing rheumatoid arthritis in the subject, including the above. [2] The method according to [1], wherein if the measured value is above the threshold, it indicates that the subject has a high severity of rheumatoid arthritis or a high risk of developing the disease. [3] The method according to [1] or [2] above, wherein the sample is body fluid, blood cells, or tissue. [Examples]

[0051] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples described below. Furthermore, the ethical approval for the experiment according to the present invention was obtained from the Ethics Committee of the Graduate School of Medicine and the 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, California, USA) or R (v40.5). A p-value < 0.05 was considered statistically significant.

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

[0054] [Patients and healthy individuals] Excess tissue generated during joint surgery or at outpatient medical facilities was used as joint samples from rheumatoid arthritis patients. Among rheumatoid arthritis patients, those with a DAS28-ESR score of less than 2.6 were defined as being in remission, and those with a DAS28-ESR score of 2.6 or higher were defined as being in non-remission. A healthy control group was selected from the group in which autoimmune diseases were ruled out by a doctor's diagnosis after examination at a medical institution.

[0055] [Isolation of CD4-positive T cells] Synovial tissue from rheumatoid arthritis patients (including those in remission) and healthy individuals was finely dissected and treated with 100 μg / mL Liberase TL (Roche) and 100 μg / mL DNase I (Roche) at 37°C for 30 minutes. Subsequently, peripheral blood monocytes or synovial monocytes were collected using Lymphocyte Separation Solution 1.077 (Nacalai Tesque). Furthermore, CD4+ T cells derived from synovial tissue and serum were isolated from rheumatoid arthritis patients and healthy individuals, respectively, using the CD4+ T Cell Isolation Kit (Miltenny).

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

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

[0058] Recombinant IGFL2 protein with 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 recombinant IGFL2 protein was measured 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 ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript).

[0059] The amino acid sequence of the human IGFL2 protein is as follows: PAGSEPWLCQPAPRCGDKIYNPLEQCCYNDAIVSLSETRQCGPPCTFWPCFELCCLDSFGLTNDFVVKLKVQGVNSQCHSSPISSKCESRRRFP(Sequence ID:1)

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

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

[0062] To detect the IGFL2 protein, anti-IGFL2 rabbit polyclonal antibodies were produced at Eurofins Genomics (Tokyo, Japan). Maxisorp 96-well plates (Thermo Scientific) were coated with 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 four-fold with 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 to the plates in sequence and reacted, and then the reaction was terminated with 2N sulfuric acid (Nacalai Tesque). The captured IGFL2 protein was detected by measuring the absorbance at 450 nm using Spectra Max iD3 (Molecular Devices).

[0063] [Quantitative PCR for IGFL2] mRNA was extracted using MyOne Silane (Thermo). cDNA was synthesized using SuperPrep (registered trademark) RT Kit for qPCR (TOYOBO). qRT-PCR was performed on the following two genes using THUNDERBIRD (trademark) SYBR (registered trademark) qPCR Mix (TOYOBO) on a C1000 Touch (Bio-Rad).

[0064] The following primers were used: For human 18srRNA gene (h18srRNA) 5'-AACTTTCGATGGTAGTCGCCG-3'(Sequence ID: 2) 5'-CCTTGGATGTGGTAGCCGTTT-3'(Sequence ID: 3) For the human IGFL2 gene (hIGFL2) 5'-GACTACCCCAGGAGTGTGCT-3'(Sequence ID: 4) 5'-CAGCGGGAGCGATGACT-3'(Sequence ID: 5) mRNA expression was standardized by h18srRNA expression.

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

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

[0067] <Example 2> [Expression of IGFL2 protein in the serum of rheumatoid arthritis patients] The inventors investigated the expression of the IGFL2 protein in peripheral blood. (1) The expression level (concentration) of IGFL2 protein in peripheral blood serum obtained from age- and sex-matched healthy control groups and rheumatoid arthritis patient groups was quantified by ELISA. As a result, it was found that the concentration of IGFL2 protein in serum was significantly higher in rheumatoid arthritis patients (RA) than in the healthy control group (Figure 2).

[0068] (2) Based on the concentrations of IGFL2 protein in the serum of the healthy control group and the rheumatoid arthritis patient group obtained in (1) above, the optimal threshold for distinguishing between the rheumatoid arthritis patient group and the healthy control group 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 obtained ROC curve is shown in Figure 3. This diagnostic method can distinguish between rheumatoid arthritis patients 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 diagnostic method.

[0069] [Table 2]

[0070] The results indicate that the AUC value of 0.92 is sufficiently high when using serum IGFL2 protein concentration as a basis for determination, suggesting that serum IGFL2 protein concentration can be used as a biomarker to distinguish between rheumatoid arthritis patients and healthy individuals.

[0071] <Example 3> [Expression of IGFL2 protein in the serum of rheumatoid arthritis patients in remission] In the same manner as in Example 2, the inventors investigated the expression of IGFL2 protein in peripheral blood. (1) The rheumatoid arthritis patient group was divided into a rheumatoid arthritis remission group and a rheumatoid arthritis non-remission group. The expression level (concentration) of IGFL2 protein in peripheral blood serum obtained from the age and sex-matched rheumatoid arthritis remission group and rheumatoid arthritis non-remission group was quantified by ELISA. As a result, it was found 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).

[0072] (2) Based on the IGFL2 protein concentrations in the serum of rheumatoid arthritis remission patients and non-rheumatoid arthritis patients obtained in (1) above, the optimal threshold for distinguishing between the rheumatoid arthritis remission group and the non-rheumatoid arthritis group was determined. The sensitivity, specificity, and area under the curve (AUC) of the determination using the set threshold (989 pg / mL) were calculated. The obtained ROC curve is shown in Figure 5. This diagnostic method can distinguish between rheumatoid arthritis patients in remission and those not 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 diagnostic method.

[0073] [Table 3]

[0074] The results indicate that, based on the concentration of IGFL2 protein in serum, the AUC value was sufficiently high at 0.8, suggesting that serum IGFL2 protein concentration can be used as a biomarker to distinguish between individuals with and without rheumatoid arthritis remission.

[0075] The results from Examples 2 and 3 revealed a strong correlation between serum IGFL2 protein concentration and the severity of rheumatoid arthritis. Therefore, by measuring the concentration of IGFL2 protein in serum, (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. (2) A method for providing information for determining and / or monitoring the severity of rheumatoid arthritis in patients with rheumatoid arthritis, or a method for providing information for determining and / or monitoring the risk of developing rheumatoid arthritis in patients suspected of having rheumatoid arthritis. ) (3) It can be used to determine whether a subject is suspected of having rheumatoid arthritis, to diagnose rheumatoid arthritis, and to assess the effectiveness of treatments such as drugs on rheumatoid arthritis patients.

[0076] <Example 4> [Investigation of screening methods for rheumatoid arthritis treatment drugs] (1) IGFL2 receptor constitutively expressing THP1 cells were cultured in 10% bovine serum RPMI-1640 with 100 ng / mL of human recombinant IGFL2 protein. After 48 hours, the culture medium was centrifuged at 400 × g for 5 minutes to obtain the culture supernatant. 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 multiplex ELISA. The results are shown as IGFL2+ in the graphs in Figures 6 to 8.

[0077] (2) On the other hand, cells that were cultured using IGFL2 receptor constitutively expressing THP1 cells but without the addition of IGFL2 protein were cultured in the same manner as in (1) above to obtain the culture supernatant, and the amounts of various chemokines 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 adding the IGFL2 protein significantly increased the expression levels of various physiologically active substances.

[0079] (3) Furthermore, IGFL2 receptor constitutively expressing THP1 cells were cultured in 10% bovine serum RPMI-1640. During the culture, one of the following components (a) to (b) was added. (a) 100 ng / mL human recombinant IGFL2 protein and 20 μg / mL isotype control antibody (R&D Systems) (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 adding anti-human IGFLR1 antibody, which has neutralizing activity, to IGFL2 protein was found to be statistically significantly lower than the concentration of CXCL10 produced by adding isotype control antibody, which does not have neutralizing activity, to IGFL2 protein. Since anti-human IGFLR1 antibody is a neutralizing antibody for IGFLR1, it has inhibitory activity on the IGFL2 / IGFL2 receptor pathway. Therefore, it can be seen that substances that reduce the production of physiologically active substances such as CXCL10, similar to anti-human IGFLR1 antibody, are useful as therapeutic agents for rheumatoid arthritis. Therefore, it has been found that the method of the present invention can provide a screening method for therapeutic drugs for rheumatoid arthritis. [Industrial applicability]

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

Claims

1. A step of measuring the amount of IGFL2 protein in body fluids derived from a subject suffering from rheumatoid arthritis, or a subject suspected of suffering from rheumatoid arthritis, and obtaining a measurement value, and A step of comparing the measured value with a threshold corresponding to the amount of IGFL2 protein, A method for testing the severity of rheumatoid arthritis in a subject suffering from rheumatoid arthritis, whether the subject suffering from rheumatoid arthritis is in remission or not, or whether a subject suspected of having rheumatoid arthritis is affected by the disease.

2. The method according to claim 1, wherein if the measured value is equal to or greater than the threshold, it indicates that the subject suffering from rheumatoid arthritis has a high degree of rheumatoid arthritis, that the subject suffering from rheumatoid arthritis is not in remission, or that a subject suspected of having rheumatoid arthritis is suffering from the disease.

3. A method for providing information for determining the severity of rheumatoid arthritis in subjects suffering from rheumatoid arthritis, whether subjects suffering from rheumatoid arthritis are in remission or not, or for determining whether subjects suspected of having rheumatoid arthritis are affected, and / or for monitoring, A step of measuring the amount of IGFL2 protein in the body fluid derived from the subject to obtain a measurement value, and A step of providing information relating to the aforementioned measurement values, A method that includes this.

4. The use of IGFL2 protein in bodily fluids derived from a subject as a biomarker for determining the severity of rheumatoid arthritis in a subject suffering from rheumatoid arthritis, whether a subject suffering from rheumatoid arthritis is in remission or not, or whether a subject suspected of having rheumatoid arthritis is affected or not, The determination involves comparing the measured amount of IGFL2 protein in the body fluid derived from the subject with a threshold corresponding to the amount of IGFL2 protein. Use including.

5. The use according to claim 4, wherein if the measured value is above the threshold, it indicates that the subject has a high degree of rheumatoid arthritis, that the subject suffering from rheumatoid arthritis is not in remission, or that the subject suspected of having rheumatoid arthritis is suffering from the disease.

6. A method for evaluating the effectiveness of treatment for rheumatoid arthritis, A step of measuring the amount of IGFL2 protein in body fluids derived from a subject after treatment with the investigational drug or treatment method to obtain a measurement value, and A step of comparing the measured value with a measured value derived from the subject before the treatment, An evaluation method that includes this.

7. The method according to claim 6, wherein the measurement value is lower than the measurement value from the subject before the treatment, indicating that the treatment is effective.

8. A process for conferring IGFL2 protein and an investigational drug to cells expressing the IGFL2 receptor, and A step of measuring the amount of physiologically active substances produced by the aforementioned cells, A screening method for rheumatoid arthritis medications, including [specific example].

9. The screening method according to claim 8, wherein the physiologically active substance is a physiologically active substance produced by stimulation of the IGFL2 protein.

10. An IGFL2 protein detection component for testing the severity of rheumatoid arthritis in a subject suffering from rheumatoid arthritis, whether the subject suffering from rheumatoid arthritis is in remission or not, or whether a subject suspected of having rheumatoid arthritis is affected by measuring the amount of IGFL2 protein in the body fluids of a subject suffering from rheumatoid arthritis or a subject suspected of having rheumatoid arthritis.

11. The member according to claim 10, wherein the detection member includes a member coated with an anti-IGFL2 capture antibody.

Images