Method of detecting risks associated with myositis and dermatomyositis

Abstract

To clarify true causes of interstitial pneumonia associated with myositis / dermatomyositis and enable highly accurate diagnosis of the risk of developing interstitial pneumonia associated with myositis / dermatomyositis or the risk of progressing to rapidly progressive interstitial pneumonia.SOLUTION: The present invention clears the above challenge by providing methods of identifying true causes of developing interstitial pneumonia associated with myositis / dermatomyositis or progressing to rapidly progressive interstitial pneumonia and detecting risks associated with myositis / dermatomyositis by detecting responsible autoantibodies.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting the risk associated with myositis / dermatomyositis and a kit for carrying out the method. [Background technology]

[0002] Dermatomyositis (DM) is an idiopathic inflammatory muscle disease in which inflammation of the muscles occurs due to an autoimmune mechanism, and is known to present as the main symptoms of muscle weakness and muscle pain associated with inflammation of the skeletal muscles, but is also known to present with typical skin eruptions such as edematous erythema. Interstitial lung disease (ILD) and malignant tumors are important pathological conditions that determine the prognosis of myositis / dermatomyositis, and interstitial lung disease, which occurs in approximately 50% of myositis / dermatomyositis cases, is an important pathological condition.

[0003] Interstitial pneumonia occurring in association with myositis / dermatomyositis can be classified into acute rapidly progressive interstitial pneumonia and chronic interstitial pneumonia, of which rapidly progressive interstitial pneumonia is the pathological condition with the poorest prognosis. Clinically, respiratory distress progresses rapidly within a few days to a few weeks, and death often occurs within a short period of time.

[0004] As a result of the investigation so far, the relationship between the prognosis and the therapeutic response of interstitial pneumonia occurring concomitantly with myositis / dermatomyositis is distinguished by the disease type of myositis, imaging findings (high-resolution chest CT), histopathological findings, the type of autoantibody, etc., among which anti-MDA5 autoantibodies are frequently found in patients with clinical amyopathic dermatomyositis (CADM), and are known to frequently occur concomitantly with rapidly progressive interstitial pneumonia (Non-Patent Document 1). Therefore, it is considered important to perform an antibody test to check the presence or absence of anti-MDA5 antibodies for the diagnosis and treatment of myositis / dermatomyositis occurring concomitantly with rapidly progressive interstitial pneumonia.

[0005] MDA5 is a 140 kDa protein found as a melanoma differentiation associated gene (Melanoma Differentiation Associated gene 5 (MDA5)). It functions as an RNA helicase in cells, and is known to recognize double-stranded RNA derived from the virus during infection with an RNA virus, produce type I interferon, and induce a natural immune response. This protein is mainly localized in the nucleus and cytoplasm in cells, so anti-MDA5 antibodies produced in dermatomyositis patients cannot bind to it. Although anti-MDA5 antibodies are a useful marker for interstitial pneumonia associated with myositis / dermatomyositis, it has not yet been confirmed whether MDA5 is related to the pathogenesis of interstitial pneumonia associated with myositis / dermatomyositis.

[0006] It has also been reported that about 50% of anti-MDA5 antibody-positive myositis / dermatomyositis patients develop rapidly progressive interstitial pneumonia (Non-Patent Document 1). However, it has also been reported that anti-MDA5 antibody is not a marker for rapidly progressive interstitial pneumonia (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Sato et al., Arthritis Rheum., 2005, 52, 1571-1576 [Non-Patent Document 2] Shinji Sato, Respiratory Clinical Journal, October 2017 (Vol. 1, No. 1), Summary of the Invention [Problem to be solved by the invention]

[0008] The objective of the present invention is to clarify the true cause of interstitial pneumonia occurring in conjunction with myositis / dermatomyositis, and to enable more accurate diagnosis of the risk of developing interstitial pneumonia occurring in conjunction with myositis / dermatomyositis or the risk of progressing to rapidly progressive interstitial pneumonia. [Means for solving the problem]

[0009] The present invention has demonstrated that the above-mentioned problems can be solved by providing a method for detecting the risks associated with myositis / dermatomyositis by identifying the true cause of the onset of interstitial pneumonia accompanying myositis / dermatomyositis or the progression to rapidly progressive interstitial pneumonia and detecting the causative autoantibodies.

[0010] More specifically, in order to solve the above-mentioned problems, the present application provides the following aspects: [1-1]: A method for detecting a risk associated with myositis / dermatomyositis, comprising detecting an antibody that binds to protein X, which is the full-length or a portion of the amino acid sequence of SEQ ID NO: 1, an amino acid sequence having one or more amino acid deletions, substitutions or additions in the amino acid sequence of SEQ ID NO: 1, or a protein having 80% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, or a variant thereof, in a sample collected from a human individual; [1-2]: The method of [1-1], wherein the sample is a blood-derived sample; [1-3]: the method according to [1-1] or [1-2], wherein the risk associated with myositis / dermatomyositis is selected from the group consisting of the risk of developing myositis / dermatomyositis, the risk of developing interstitial pneumonia associated with myositis / dermatomyositis, and the risk of transitioning from interstitial pneumonia associated with myositis / dermatomyositis to rapidly progressive interstitial pneumonia; [1-4]: the method according to [1-1] or [1-2], wherein the antibody that binds to protein X or a variant thereof is an IgG antibody or an IgM antibody; [1-5]: the method according to [1-1] or [1-2], in which it is determined that there is a risk associated with myositis / dermatomyositis when an antibody binding to protein X or a variant thereof is detectable in a sample; [1-6]: the method according to [1-1] or [1-2], which comprises detecting an autoantibody that binds to human protein X or a variant thereof in a sample by ELISA, Western blotting, indirect fluorescent antibody testing, double immunodiffusion, chemiluminescent enzyme immunoassay, or chemiluminescent immunoassay; [1-7]: A kit for detecting a risk associated with myositis / dermatomyositis by detecting an antibody that binds to protein X or its variant present in a sample collected from a human individual, the kit comprising: a solid phase on which protein X or a variant thereof is immobilized, the full length or a part of the amino acid sequence of SEQ ID NO: 1, an amino acid sequence having one or more amino acid deletions / substitutions / additions in the amino acid sequence of SEQ ID NO: 1, or a protein having 80% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, and an anti-human antibody for detecting a human antibody that binds to protein X or its variant; [1-8]: the kit according to [1-7], wherein the sample is a blood-derived sample; [1-9]: the kit according to [1-7] or [1-8], wherein the solid phase on which protein X or a variant thereof is immobilized is an ELISA plate, a slide glass, or a particle on which protein X or a variant thereof is immobilized, an agarose gel on which protein X or a variant thereof is dropped, or a membrane on which protein X or a variant thereof is immobilized; [1-10]: the kit according to [1-7] or [1-8], wherein the risks associated with myositis / dermatomyositis are selected from the group consisting of the risk of developing myositis / dermatomyositis, the risk of developing interstitial pneumonia associated with myositis / dermatomyositis, and the risk of transition from interstitial pneumonia associated with myositis / dermatomyositis to rapidly progressive interstitial pneumonia; [1-11]: the kit according to [1-7] or [1-8], wherein the antibody that binds to protein X or a variant thereof is an IgG antibody or an IgM antibody; [1-12]: the kit according to [1-7] or [1-8], which determines that there is a risk associated with myositis / dermatomyositis when an antibody binding to protein X or a variant thereof is detectable in a sample; [2-1]: a non-human animal model that develops myositis / dermatomyositis or interstitial pneumonia associated therewith, by allowing an antibody that binds to protein X or a variant thereof in the blood, which is the full-length or partial length of the amino acid sequence of SEQ ID NO: 1, an amino acid sequence having one or more amino acid deletions / substitutions / additions in the amino acid sequence of SEQ ID NO: 1, or a protein having 80% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1; [2-2]: a non-human model animal according to [2-1], in which the non-human model animal develops myositis / dermatomyositis, interstitial pneumonia associated with myositis / dermatomyositis, or rapidly progressive interstitial pneumonia that progresses from interstitial pneumonia associated with myositis / dermatomyositis; [2-3]: the method according to [2-1] or [2-2], wherein the antibody that binds to protein X or a variant thereof is selected from the group consisting of a human antibody, a mouse antibody, a rat antibody, a rabbit antibody, and a goat antibody; [2-4]: the non-human model animal according to [2-1] or [2-2], wherein the non-human animal is selected from the group consisting of mouse, rat, rabbit, and goat; [2-5]: the non-human model animal according to [2-1] or [2-2], by administering an antibody that binds to protein X or a variant thereof into the blood of the non-human animal; [2-6]: A non-human model animal according to [2-1] or [2-2], wherein protein X or a variant thereof is administered into the blood of a non-human animal to produce an antibody that binds to protein X or a variant thereof in the blood of the non-human animal. Effect of the Invention

[0011] The present invention provides a method for identifying the true cause of the development of interstitial pneumonia accompanying myositis / dermatomyositis or the progression to rapidly progressive interstitial pneumonia, and for detecting the causative autoantibodies, thereby detecting the risk associated with myositis / dermatomyositis. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows a scheme for analyzing the mechanism of onset of interstitial pneumonia accompanying myositis / dermatomyositis using anti-MDA5 antibody or cells producing anti-MDA5 antibody. [Diagram 2] FIG. 2 shows the results of searching for the true targets of anti-MDA5 antibodies generated in the bodies of subjects who developed interstitial pneumonia associated with myositis / dermatomyositis, using the protein array CWPA. [Diagram 3] FIG. 3 shows a scheme for analyzing the mechanism of onset of interstitial pneumonia associated with myositis / dermatomyositis by immunizing a model animal with MDA5 protein and causing anti-MDA5 antibody production in the body. [Figure 4] FIG. 4 shows images of lung tissue of interstitial pneumonia that developed in a non-human animal model immunized with MDA5 protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention has demonstrated that the above-mentioned problems can be solved by providing a method for detecting the risks associated with myositis / dermatomyositis by identifying the true cause of the onset of myositis / dermatomyositis, the onset of interstitial pneumonia accompanying myositis / dermatomyositis, or the transition from interstitial pneumonia to rapidly progressive interstitial pneumonia, and detecting the causative autoantibodies.

[0014] It has been reported that about 50% of anti-MDA5 antibody positive myositis / dermatomyositis patients develop rapidly progressive interstitial pneumonia, and the presence or absence of anti-MDA5 antibody has been examined as a marker for rapidly progressive interstitial pneumonia. However, although anti-MDA5 antibody is a powerful marker for rapidly progressive interstitial pneumonia, since MDA5 protein is mainly localized in the nucleus and cytoplasm in cells, the inventors of the present invention believe that MDA5 protein is not involved in the true mechanism of myositis / dermatomyositis or the rapidly progressive interstitial pneumonia that occurs concomitantly therewith.

[0015] The inventors collected blood from anti-MDA5 antibody-positive individuals who developed interstitial pneumonia and added it to a protein array CWPA (comprehensive wet protein array) (Fukuda et al., Genes to Cells, 2021), which presents proteins equivalent to approximately 80% of the human proteome, to search for proteins other than MDA5 protein to which anti-MDA5 antibodies in the blood bind. As a result, it was revealed that anti-MDA5 antibodies bind to protein X, which is known to be a cell membrane-transmitting protein with 12 transmembrane domains. This protein X is a protein having the amino acid sequence of SEQ ID NO: 1, and was predicted to have Na-independent organic anion transporter and thyroid hormone transporter activity.

[0016] Protein X detected by anti-MDA5 antibody is a protein expressed on the cell membrane surface of macrophages and placental cells, and is a protein that can be approached by antibodies present in the blood. When mice were immunized with this protein X and anti-protein X antibodies were produced in the mice, it was revealed that the mice developed interstitial pneumonia. These results revealed that the true cause of myositis / dermatomyositis, interstitial pneumonia accompanying myositis / dermatomyositis, and the rapidly progressive interstitial pneumonia that progresses from these conditions is the binding of antibodies to protein X on the cell membrane surface.

[0017] First aspect: method invention Based on this finding, in one embodiment, the present invention can provide a method for detecting a risk associated with myositis / dermatomyositis, comprising the step of detecting an antibody that binds to protein X or a variant thereof in a sample collected from a human individual. According to the finding of the present invention, since antibodies that bind to protein X or a variant thereof are formed in the blood of patients with myositis / dermatomyositis, interstitial pneumonia accompanying myositis / dermatomyositis, and further with rapidly progressive interstitial pneumonia which progresses therefrom, the risk associated with myositis / dermatomyositis can be detected by the step of detecting the presence of antibodies that bind to protein X or a variant thereof.

[0018] In this embodiment of the present invention, protein X used to detect an antibody binding to protein X or a variant thereof is preferably human protein X, and may be the whole protein X (SEQ ID NO: 1) or a part thereof. In addition, a variant of protein X to which an antibody to be detected binds in the present invention may be a protein having an amino acid sequence having one or more amino acid deletions, substitutions or additions in the amino acid sequence of SEQ ID NO: 1, or a protein having 80% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, or a part of these. When a part of protein X or a variant thereof is used, the extracellular domain of protein X or a variant thereof may be used.

[0019] Here, in the modified form of protein X, "a protein having an amino acid sequence comprising one or more amino acid deletions, substitutions or additions in the amino acid sequence of SEQ ID NO: 1", "one or more" amino acids means, for example, the number of amino acid residues included in the range of 1 to 50, although it varies depending on the position and type of each amino acid residue in the three-dimensional structure of protein X, and preferably means 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. Furthermore, in this term, "deletion, substitution or addition of amino acids" may refer to any mutation as long as the binding of the antibody to be detected in this embodiment of the present invention to protein X is maintained.

[0020] Furthermore, in the case of a variant of protein X "having 80% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1", the variant of protein X may be a protein having an amino acid sequence that has, for example, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identity to the amino acid sequence of protein X (SEQ ID NO: 1), so long as the variant maintains the function of protein X.

[0021] In the present invention, a sample collected from a human individual refers to a sample containing an antibody that binds to protein X or a variant thereof, and includes blood-derived samples. As blood-derived samples, plasma or serum can be used as the subject of the method of the present invention.

[0022] Risks associated with myositis / dermatomyositis that can be detected in the present invention include the risk of developing myositis / dermatomyositis, the risk of developing interstitial pneumonia associated with myositis / dermatomyositis, and the risk of transitioning from interstitial pneumonia associated with myositis / dermatomyositis to rapidly progressive interstitial pneumonia. Studies in the present invention have revealed that the stronger the signal intensity derived from the concentration of an antibody that binds to protein X or its variant in a blood sample, the higher the risk associated with myositis / dermatomyositis.

[0023] The antibody to be detected in the present invention may be an IgG antibody or an IgM antibody that binds to protein X or a modified form thereof. Considering the distribution of protein X in the body, it is preferable to use blood as a sample, and in that case, it is preferable to detect an IgG antibody or an IgM antibody. This antibody can be used as a marker for clinical amyopathic dermatomyositis (CADM), particularly as a marker for myositis / dermatomyositis complicated with rapidly progressive interstitial pneumonia, in diagnosis, selection of a treatment method, and prediction of prognosis (e.g., prediction of the onset of myositis / dermatomyositis or interstitial pneumonia, and the worsening of the disease).

[0024] When carrying out the method of the present invention for detecting a risk associated with myositis / dermatomyositis, a step of detecting the presence or absence of an antibody that binds to protein X or a variant thereof in a sample is carried out. If the antibody is detectable in the sample, the sample is positive for an antibody that binds to protein X or a variant thereof, and it can be determined that there is a risk of developing myositis / dermatomyositis, that there is a risk of developing interstitial pneumonia associated with myositis / dermatomyositis, and further that there is a risk that interstitial pneumonia associated with myositis / dermatomyositis will progress to rapidly progressive interstitial pneumonia.

[0025] In the present invention, the method used in the step of detecting an antibody that binds to protein X or a variant thereof in a sample may be an enzyme immunoassay (EIA, ELISA), Western blot, indirect fluorescent antibody technique, double immunodiffusion, chemiluminescent enzyme immunoassay, or chemiluminescent immunoassay. In particular, when the concentration of an antibody that binds to protein X or a variant thereof is to be measured, it is preferable to use an enzyme immunoassay (EIA, ELISA) or Western blot.

[0026] When a step of detecting an antibody that binds to protein X or a variant thereof in a sample by enzyme immunoassay (EIA, ELISA) is carried out, - immobilizing the full length or a part (antigen) of protein X or its variant on a solid phase; reacting the sample with an antigen; A process to detect and quantify the antibody bound to the antigen using an anti-human antibody (corresponding to a secondary antibody) with a detection marker. By carrying out the above steps, it is possible to detect the antibody or measure its concentration.

[0027] When a step of detecting an antibody binding to protein X or a variant thereof in a sample by Western blotting is carried out, A step of subjecting a specified amount of protein X or its modified form (full length or a part thereof (antigen)) to electrophoresis by SDS-PAGE or the like to develop molecules based on differences in molecular weight; Then, transfer the image to a nitrocellulose membrane, etc. Thereafter, the antigen transferred to the membrane is reacted with a sample. A process in which the antibody bound to the antigen on the membrane is detected and quantified using an anti-human antibody (corresponding to a secondary antibody) with a detection marker. By carrying out the above steps, it is possible to detect the antibody or measure its concentration.

[0028] When detecting an antibody that binds to protein X or a variant thereof in a sample by indirect fluorescent antibody technique, A step of adding a sample onto a solid phase such as a slide glass on which a specified amount of protein X or a full-length or a part (antigen) of a variant thereof is immobilized; A step of reacting the antigen on the slide with the antibody in the sample (primary reaction); After washing, a step of adding a labeled anti-human antibody for detection and reacting (secondary reaction); A process to form a complex of antigen, antibody and labeled antibody and detect and quantify the label This makes it possible to detect the antibody in the sample or to measure its concentration.

[0029] When detecting an antibody that binds to protein X or a variant thereof in a sample by double immunodiffusion, A step of placing the full-length or a part (antigen) of protein X or its variant, positive serum, and a sample into three holes formed in an agarose gel, respectively; A step of diffusing the antigen and the positive serum, and the antigen and the antibody in the sample, respectively, in agarose; A process to detect and identify the specificity of antibodies from the morphology of the precipitation line resulting from the antigen-antibody reaction. In this case, if the precipitation line morphology is of the fuse type, it indicates that the reference antibody and the antibody in the sample recognize the same antigen, if the precipitation line morphology is of the spur type, it indicates that the reference antibody and the antibody in the sample recognize part of the same antigen, and if the precipitation line morphology is of the cross type, it indicates that the reference antibody and the antibody in the sample recognize two different antigens.

[0030] When detecting an antibody that binds to protein X or a variant thereof in a sample by chemiluminescence enzyme immunoassay (CLEIA), - immobilizing a predetermined amount of protein X or a full-length or part of its variant (antigen) on a solid phase such as magnetic particles; reacting the sample with an antigen; a step of washing to remove unreacted substances other than the antigen-bound antibody, and then reacting with an enzyme-labeled antibody; After further washing to remove unreacted materials, a chemiluminescent enzyme substrate is added, and the amount of luminescence generated by hydrolysis of the chemiluminescent enzyme substrate by the enzyme of the labeled antibody is measured. This makes it possible to detect the antibody in the sample or to measure its concentration.

[0031] When detecting an antibody that binds to protein X or a variant thereof in a sample by chemiluminescence immunoassay (CLIA), the principle is the same as that of the above-mentioned chemiluminescence enzyme immunoassay (CLEIA), and the antibody in the sample or the concentration thereof can be detected by measuring the amount of chemiluminescence generated by using a chemiluminescent compound as a label that is bound to the detection antibody. That is, - immobilizing a predetermined amount of protein X or a full-length or part of its variant (antigen) on a solid phase such as magnetic particles; reacting the sample with an antigen; a step of washing to remove unreacted substances other than the antigen-bound antibody, and then reacting with a chemiluminescent compound-labeled antibody; After further washing to remove unreacted substances, a chemiluminescent enzyme substrate is added and the amount of luminescence generated by the chemiluminescent compound of the labeled antibody is measured. This makes it possible to detect the antibody in the sample or to measure its concentration.

[0032] A labeling substance for detection or concentration measurement can be bound to an anti-human antibody (corresponding to a secondary antibody) for detecting an antibody that binds to protein X or a modified form thereof. Those skilled in the art can appropriately select a labeling substance suitable for the various methods described above, and examples of such labeling substances include those that are generally usable in the art, such as biotin, enzymes (e.g., alkaline phosphatase (AP), horseradish peroxidase (HRP)), fluorescent dyes (e.g., Alexa Fluor (registered trademark), DyLight (registered trademark), FITC, PE, etc.).

[0033] Second aspect: kit invention As another embodiment, the present invention can also provide a kit for detecting an antibody that binds to protein X or a variant thereof in a sample. Specifically, the present invention can provide a kit for detecting an antibody that binds to protein X or a variant thereof present in a sample collected from a human individual, the kit comprising a solid phase on which the full length or a part (antigen) of protein X or a variant thereof is immobilized, and an anti-human antibody for detecting a human antibody that binds to protein X or a variant thereof. This kit can be used to detect the risk associated with myositis / dermatomyositis.

[0034] The kit of the present invention may be a kit for ELISA, a kit for Western blotting, a kit for indirect fluorescent antibody assay, a kit for double immunodiffusion, a kit for chemiluminescent enzyme immunoassay, or a kit for chemiluminescent immunoassay, etc. When preparing a kit for an ELISA method, an ELISA plate on which the full-length or part (antigen) of protein X or its modified form is immobilized is When preparing a kit for Western blotting, the membrane on which the antigen is immobilized is When preparing a kit for indirect fluorescent antibody technique, the solid phase, such as a slide glass on which the antigen is immobilized, In the case of a kit for double immunodiffusion, the following is required: agarose gel with holes for antigen, positive serum, and sample (the antigen, positive serum, and sample are dropped into the respective holes of the agarose gel when used); When preparing a kit for chemiluminescent enzyme immunoassay or chemiluminescent immunoassay, the magnetic particles to which the antigen is immobilized are Each can be used.

[0035] The risks associated with myositis / dermatomyositis that can be detected by the kit of the present invention include the risk of developing myositis / dermatomyositis, the risk of developing interstitial pneumonia associated with myositis / dermatomyositis, and the risk of myositis / dermatomyositis-associated interstitial pneumonia progressing to rapidly progressive interstitial pneumonia. Studies in the present invention have revealed that the stronger the signal intensity derived from the concentration of protein X or its variant in a blood sample, the higher the risk associated with myositis / dermatomyositis.

[0036] When the concentration of an antibody that binds to protein X or a variant thereof in a sample is quantified using the kit of the present invention, if the antibody is detectable in the sample, it can be determined that the sample is positive for an antibody that binds to protein X or a variant thereof and that the subject is at risk of developing myositis / dermatomyositis, it can be determined that the subject is at risk of developing interstitial pneumonia associated with myositis / dermatomyositis, and it can be further determined that the subject is at risk of the interstitial pneumonia associated with myositis / dermatomyositis progressing to rapidly progressive interstitial pneumonia.

[0037] Third aspect: model animals In the present invention, as described later in the Examples, in the course of elucidating the mechanism of onset of myositis / dermatomyositis and the associated interstitial pneumonia, it was revealed that when an anti-human MDA5 antibody or a cell producing an anti-human MDA5 antibody is administered to an MDA5 knockout mouse in which MDA5 is deleted, the MDA5 knockout mouse develops interstitial pneumonia, and when an individual mouse is immunized with the protein X or a variant thereof specified in the present invention, the individual mouse develops interstitial pneumonia. It was revealed that the lung tissues of these mice that developed interstitial pneumonia were histologically very similar to the lung tissue images of patients with interstitial pneumonia associated with myositis / dermatomyositis in humans.

[0038] Based on this result, in yet another embodiment, the present invention can provide a non-human model animal that develops myositis / dermatomyositis or concomitant interstitial pneumonia by causing an antibody that binds to protein X or a variant thereof to be present in the blood. Here, "causing an antibody that binds to protein X or a variant thereof to be present in the blood" means that it is necessary to eventually create a state in which an antibody that binds to protein X or a variant thereof is present in the blood, and for example, A method of exogenously administering an antibody that binds to protein X or a variant thereof into the blood of a non-human animal, A method of exogenously administering cells producing an antibody that binds to protein X or a variant thereof to a non-human animal by intravenous injection, and causing the cells to produce an antibody that binds to protein X or a variant thereof; A method for producing antibodies that bind to protein X or its variants by immunizing a non-human animal by administering protein X or its variant into the blood of the animal and generating cells in the animal's internal blood that produce antibodies that bind to protein X or its variants; Any of the above methods may be adopted.

[0039] The non-human model animal of the present invention is characterized by developing myositis / dermatomyositis, myositis / dermatomyositis-associated interstitial pneumonia, or rapidly progressive interstitial pneumonia progressing from myositis / dermatomyositis-associated interstitial pneumonia. When lung tissue sections of mice that developed the pathology were examined, they showed histological images similar to those of human interstitial pneumonia, indicating that the animal of the present invention functions as a model animal for myositis / dermatomyositis-associated interstitial pneumonia that develops in association with protein X or a variant thereof. This non-human model animal is useful for developing a method for treating interstitial pneumonia associated with myositis / dermatomyositis in humans and developing a therapeutic agent.

[0040] When producing the non-human model animal of the present invention, the antibody that binds to protein X or its variant present in the blood can be one that binds to the full-length sequence of protein X (SEQ ID NO: 1) or its variant, or a part of either. Here, when referring to protein X or a part of its variant, the extracellular domain of protein X or its variant can be used.

[0041] When producing the non-human model animal of the present invention, the antibody that binds to protein X or its variant present in the blood may be any of human, mouse, rat, and goat antibodies. For example, in the case of a method in which an antibody that binds to protein X or its variant is administered from the outside, or in the case of a method in which cells that produce an antibody that binds to protein X or its variant are administered from the outside and the cells are allowed to produce an antibody that binds to protein X or its variant, the origin of the antibody present in the body of the non-human model animal may be any animal species. On the other hand, in the case of a method in which a non-human model animal is immunized with protein X or its variant to generate cells that produce an antibody that binds to protein X or its variant in the body and produce an antibody that binds to protein X or its variant, the antibody is newly produced within the individual of the non-human model animal, and therefore the antibody of the animal is present in the blood.

[0042] The non-human model animal of the present invention may be any animal species other than humans, and may be produced using animals such as mice, rats, rabbits, goats, etc. The user can decide which animal species to use based on the purpose of use, ease of handling, etc.

[0043] The present invention will be described in more detail below with reference to examples, but the following examples are not intended to limit the present invention in any way. EXAMPLES

[0044] Example 1: Analysis of the effect of anti-human MDA5 antibody in MDA5 knockout mice In this example, an experiment was carried out with the objective of analyzing the action of anti-human MDA5 antibody in MDA5 knockout mice and elucidating the significance of anti-MDA5 antibody in the blood.

[0045] MDA5 knockout mice (n = 5-7) that lacked MDA5 in the body were used as recipient mice for the anti-MDA5 antibody (B6.Cg-Ifih1 tm1.1Cln, The Jackson Laboratory). Although these mice lack MDA5 in their bodies, it was revealed that they do not naturally develop interstitial pneumonia (Fig. 1).

[0046] On the other hand, wild-type mice (C57 / BL6) were immunized with human MDA5 to produce antibodies. 100 μg of MDA5 protein was mixed with the adjuvant Titer Max Gold (Funakoshi) in a 1:1 ratio, and after maceration, the mice were subcutaneously injected into the back every week for a total of five times (days 0, 7, 14, 21, and 28). Analysis was performed on day 35 to see whether interstitial pneumonia had developed. The results showed that mice immunized with human MDA5 developed interstitial pneumonia.

[0047] Next, B cells were collected from the spleens of mice immunized with human MDA5, and these B cells were then injected into the MDA5 knockout mice described above at 10 6 The mice were administered the drug intravenously (day 0) and analyzed for the development of interstitial pneumonia on day 28 after intravenous administration. This showed that MDA5 knockout mice developed interstitial pneumonia (Figure 1).

[0048] This suggests that interstitial pneumonia develops even in the absence of MDA5 in the body, and that anti-MDA5 antibodies produced in mice immunized with human MDA5 bind to an unknown target other than MDA5, resulting in the development of interstitial pneumonia.

[0049] Example 2: Search for the true target substance of anti-MDA5 antibody In this example, an experiment was carried out with the aim of identifying a target substance, other than MDA5 protein, of anti-MDA5 antibody in serum as a mechanism by which anti-MDA5 antibody causes interstitial pneumonia.

[0050] In Example 1, antibodies in the serum of mice that had developed interstitial pneumonia were detected using a protein array CWPA, which was composed of 19,446 types of human proteins prepared in a wheat germ cell-free expression system and each protein was tagged with a [FLAG-tag]-[GST-tag] at its N-terminus.

[0051] In this protein array, the affinity between the GST tag added to the N-terminus of each human protein and the glutathione modified on the array substrate was utilized to create a CWPA in which human proteins were bound to the substrate surface.

[0052] Mouse serum was diluted 333-fold using antibody diluent, added to CWPA, and reacted at room temperature for 1 hour. After discarding the liquid after the reaction and washing the CWPA, a secondary antibody (goat anti-mouse IgG (H+L) Alexa Flour® 647 complex) diluted 1000-fold using antibody diluent was added to the CWPA and reacted at room temperature for 1 hour. After discarding the liquid after the reaction, the CWPA was washed and air-dried, and then a fluorescent image derived from Alexa Flour® 647 was obtained using a fluorescent imager.

[0053] The proteins bound by the secondary antibody were analyzed from the fluorescence images, and the antigen of the antibody contained in the mouse serum was identified. As a result, it was revealed that the anti-MDA5 antibody binds to a transmembrane protein X in addition to MDA5 (Figure 2). This protein is different from the MDA5 protein, which is an intracellular protein, and has the amino acid sequence of SEQ ID NO: 1:

[0054] [ka]

[0055] It has been shown that MDA5 is a transmembrane protein that can be approached by anti-MDA5 antibodies in vivo.

[0056] This suggests that the anti-MDA5 antibodies previously detected in individuals with myositis / dermatomyositis-associated interstitial pneumonia are in fact antibodies that bind to cell surface protein X, and that the presence of these antibodies in the body leads to the development of myositis / dermatomyositis-associated interstitial pneumonia through the binding of protein X with an antibody against it (previously known as anti-MDA5 antibody).

[0057] Next, the serum antibodies of dermatomyositis patients who tested positive for anti-MDA5 antibodies were analyzed as follows. First, each human protein that tested positive in the analysis using CWPA (i.e., MDA5 and transmembrane protein X) was prepared by fusing a GST tag to the N-terminus. The above-mentioned human protein, MDA5, or transmembrane protein X was bound to each of the ELISA plates whose surface was modified with glutathione.

[0058] Patient serum diluted approximately 100-fold with antibody diluent was added to this ELISA plate and allowed to react at room temperature for 1 hour. After discarding the liquid after the reaction and washing, a secondary antibody (HRP-labeled anti-human IgG antibody) diluted with antibody diluent was added and allowed to react at room temperature for 1 hour. After discarding the liquid after the reaction and washing the ELISA plate, a chromogenic substrate (3,3',5,5'-tetramethylbenzidine; TMB) was added and allowed to react at room temperature for about 30 minutes, and the color reaction was stopped by adding a reaction stop solution.

[0059] Absorbance measurements were performed using a plate reader, and the measurement value at the reference wavelength of 570 nm (OD (570 nm)) was subtracted from the measurement value at the TMB absorption wavelength of 450 nm (OD (450 nm)) to identify the antigen to which the anti-human MDA5 antibody in the patient's serum binds.

[0060] Each protein was measured in eight replicates, and the significance of the difference from the negative control (tag only) was evaluated by the Mann-Whitney U test.

[0061] The analysis showed that anti-MDA5 antibodies in the patient sera bound to MDA5 (positive control) but also to transmembrane protein X (Table 1).

[0062] [Table 1]

[0063] Example 3: Effect of Protein X on Interstitial Pneumonia Associated with Myositis / Dermatomyositis In this example, an experiment was conducted to clarify whether protein X identified in Example 2 is actually involved in the development of interstitial pneumonia associated with myositis / dermatomyositis.

[0064] 100 μg of protein X prepared in Example 2 was subcutaneously administered together with the adjuvant titer max gold (Funakoshi) to wild-type mice (C57 / BL6) (n=6).

[0065] As a negative control, only the tag that was combined for the purpose of synthesis of transmembrane protein X was administered in the same manner (n=6).

[0066] As a result, all mice in the Protein X-administered group developed interstitial pneumonia, whereas none of the mice in the negative control group immunized with the tag alone developed interstitial pneumonia (Figure 3).

[0067] This result demonstrated that, as suggested in Example 2, protein X is indeed responsible for the development of interstitial pneumonia associated with myositis / dermatomyositis.

[0068] Furthermore, Fig. 4 shows a lung tissue section from a mouse that developed interstitial pneumonia as a result of administration of protein X. The histological images show that the lungs of the mouse with interstitial pneumonia were accompanied by inflammatory cell infiltration and thickening of the alveolar septa, similar to the histological images of human lungs with interstitial pneumonia. In contrast, the lungs of the mouse immunized with only the tag did not show any of the above characteristics, and were shown to have a normal alveolar structure. [Industrial Applicability]

[0069] The present invention provides a method for identifying the true cause of the development of interstitial pneumonia accompanying myositis / dermatomyositis or the progression to rapidly progressive interstitial pneumonia, and for detecting the causative autoantibodies, thereby detecting the risk associated with myositis / dermatomyositis.

Claims

1. (1) A step of reacting a sample collected from a human individual with the full length or a part of Protein X consisting of the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein the variant is (a) an amino acid sequence having a deletion, substitution, or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 1, or (b) an amino acid sequence having an amino acid sequence identity of 80% or more with the amino acid sequence of SEQ ID NO: 1 and, (2) A step of detecting an antibody that binds to the Protein X or its variant A method for detecting the risk associated with myositis / dermatomyositis, comprising the above steps.

2. The method according to Claim 1, wherein the sample is a sample derived from blood.

3. The risk associated with myositis / dermatomyositis according to Claim 1 or 2, wherein the risk is selected from the group consisting of the risk of developing myositis / dermatomyositis, the risk of developing interstitial pneumonia associated with myositis / dermatomyositis, and the risk of transitioning from interstitial pneumonia associated with myositis / dermatomyositis to rapidly progressive interstitial pneumonia.

4. The method according to Claim 1 or 2, wherein the antibody is an IgG antibody or an IgM antibody.

5. The method according to Claim 1 or 2, which is performed by ELISA, Western blotting, indirect fluorescent antibody method, double immunodiffusion method, chemiluminescent enzyme immunoassay, or chemiluminescent immunoassay.

6. A kit for detecting the risk associated with myositis / dermatomyositis, comprising a solid phase immobilized with the full length or a part of Protein X consisting of the amino acid sequence of SEQ ID NO: 1 or a variant thereof, wherein the variant is (a) an amino acid sequence having a deletion, substitution, or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 1, or (b) an amino acid sequence having an amino acid sequence identity of 80% or more with the amino acid sequence of SEQ ID NO: 1 The kit comprising the above.

7. The kit according to Claim 6, wherein the solid phase is an ELISA plate, a slide glass, particles, an agarose gel, or a membrane.

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