JP2024009472A - Method of detecting risks associated with myositis and dermatomyositis - Google Patents

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

The present invention relates to a method for detecting risks associated with myositis/dermatomyositis, and a kit for carrying out the method.

Myositis/Dermatomyositis (DM) is an idiopathic inflammatory muscle disease that causes muscle inflammation due to an autoimmune mechanism, and the main symptoms are muscle weakness and myalgia associated with skeletal muscle inflammation. However, it is also known to exhibit typical skin eruptions such as edematous erythema. Important pathological conditions that determine the prognosis of myositis/dermatomyositis include interstitial lung disease (ILD) and malignant tumors, of which interstitial lung disease, which occurs in approximately 50% of myositis/dermatomyositis, is important. It is a pathological condition.

Interstitial pneumonia that accompanies myositis/dermatomyositis includes acute rapidly progressive interstitial pneumonia and chronic interstitial pneumonia, of which rapidly progressive interstitial pneumonia has the poorest prognosis. It is a pathological condition. Clinically, respiratory distress progresses rapidly over several days to several weeks, and death frequently occurs within a short period of time.

As a result of previous studies, the relationship between the prognosis and treatment response of interstitial pneumonia accompanying myositis/dermatomyositis is determined by the disease type of myositis, imaging findings (chest high-resolution CT), histopathological findings, and the presence of autoantibodies. Among these, anti-MDA5 autoantibodies are often found in patients with clinical amyopathic DM (CADM), and are frequently accompanied by rapidly progressive interstitial pneumonia. This is known (Non-Patent Document 1). Therefore, performing an antibody test to check for the presence of anti-MDA5 antibodies is considered important for the diagnosis and treatment of myositis/dermatomyositis that accompanies rapidly progressive interstitial pneumonia.

MDA5 is a 140 kDa protein discovered as a melanoma differentiation associated gene 5 (MDA5), which has the function of RNA helicase in cells, and when infected with an RNA virus, it is It is known to have the ability to recognize double-stranded RNA, produce type I interferon, and induce an innate immune response. Since this protein is mainly localized in the nucleus and cytoplasm within cells, anti-MDA5 antibodies produced in patients with dermatomyositis cannot bind to it; Although MDA5 is a powerful marker for interstitial pneumonia associated with myositis/dermatomyositis, it is currently unclear whether MDA5 is related to the disease pathogenesis of interstitial pneumonia associated with myositis/dermatomyositis. has not been confirmed.

Furthermore, it has been reported that rapidly progressive interstitial pneumonia develops in about 50% of anti-MDA5 antibody-positive myositis/dermatomyositis (Non-Patent Document 1). However, there is also a report that anti-MDA5 antibodies are not a marker for rapidly progressive interstitial pneumonia (Non-Patent Document 2).

Sato et al., Arthritis Rheum., 2005, 52, 1571-1576 Shinji Sato, Respiratory Clinic, October 2017 issue (Volume 1, No. 1),

The present invention clarifies the true cause of interstitial pneumonia accompanying myositis/dermatomyositis, and the risk of developing interstitial pneumonia accompanying myositis/dermatomyositis or the risk of transitioning to rapidly progressive interstitial pneumonia. The goal is to enable more accurate diagnosis of

In the present invention, we identify the true cause of the onset of interstitial pneumonia accompanying myositis/dermatomyositis or the transition to rapidly progressive interstitial pneumonia, and detect the autoantibodies that are the cause of myositis. / It has been shown that the above problems can be solved by providing a method for detecting risks associated with dermatomyositis.

More specifically, the present application provides the following aspects in order to solve the above-mentioned problems:
[1-1]: Amino acid sequence of SEQ ID NO: 1, amino acid having deletion, substitution, or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 1 in a sample collected from a human individual. Detects antibodies that bind to Protein A method of detecting the risks associated with myositis/dermatomyositis, including:
[1-2]: The method of [1-1], where the sample is a blood-derived sample;
[1-3]: The risks associated with myositis/dermatomyositis are the risk of developing myositis/dermatomyositis, the risk of developing interstitial pneumonia associated with myositis/dermatomyositis, and the risk of developing interstitial pneumonia associated with myositis/dermatomyositis. The method according to [1-1] or [1-2], which is selected from the group consisting of the risk of transition from pneumonia 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]: If antibodies that bind to protein 2] method described in
[1-6]: Human protein The method according to [1-1] or [1-2], which detects an autoantibody that binds to;
[1-7]: Amino acid sequence of SEQ ID NO: 1, amino acid sequence with deletion, substitution, or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 1, or amino acid of SEQ ID NO: 1 Binds to a solid phase on which Protein X or a variant thereof is immobilized, which is the full length or a part thereof of a protein having 80% or more amino acid sequence identity with the sequence, Protein X or a variant thereof Detecting risks associated with myositis/dermatomyositis by detecting antibodies that bind to protein X or its variants present in samples taken from human individuals, including anti-human antibodies for detecting human antibodies kit for;
[1-8]: The kit of [1-7], where the sample is a blood-derived sample;
[1-9]: A 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 the kit according to [1-7] or [1-8], which is a membrane on which protein X or a variant thereof is immobilized;
[1-10]: The risks associated with myositis/dermatomyositis are the risk of developing myositis/dermatomyositis, the risk of developing interstitial pneumonia associated with myositis/dermatomyositis, and the risk of developing interstitial pneumonia associated with myositis/dermatomyositis. The kit according to [1-7] or [1-8], which is selected from the group consisting of: a risk of transition from pneumonia 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]: If antibodies that bind to protein 8];
[2-1]: Amino acid sequence of SEQ ID NO: 1, amino acid sequence with deletion, substitution, or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 1, or amino acid of SEQ ID NO: 1 myositis/skin due to the presence in the blood of antibodies that bind to protein Non-human model animals that develop myositis and accompanying interstitial pneumonia;
[2-2]: Rapidly progressive interstitial pneumonia in non-human animal models that progresses from myositis/dermatomyositis, interstitial pneumonia associated with myositis/dermatomyositis, or interstitial pneumonia associated with myositis/dermatomyositis. The non-human model animal described in [2-1] that develops pneumonia;
[2-3]: [2-1] or [2], wherein the antibody that binds to protein X or its variant is selected from the group consisting of human antibody, mouse antibody, rat antibody, rabbit antibody, and goat antibody. -2] Method described in
[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]: By administering protein X or its variant into the blood of a non-human animal to produce antibodies that bind to protein X or its variant in the blood of the non-human animal -1] or the non-human model animal described in [2-2].

According to the present invention, by identifying the true cause of the onset of interstitial pneumonia accompanying myositis/dermatomyositis or the transition to rapidly progressive interstitial pneumonia, and detecting the autoantibody that is the cause, / A method for detecting risks associated with dermatomyositis can be provided.

FIG. 1 is a diagram showing a scheme in which the mechanism of onset of interstitial pneumonia accompanying myositis/dermatomyositis was analyzed using anti-MDA5 antibodies or cells producing anti-MDA5 antibodies. FIG. 2 is a diagram showing the results of using protein array CWPA to search for the true target of anti-MDA5 antibodies generated in the body of a subject who developed interstitial pneumonia associated with myositis/dermatomyositis. FIG. 3 is a diagram showing a scheme in which the mechanism of onset of interstitial pneumonia accompanying myositis/dermatomyositis was analyzed by immunizing a model animal with MDA5 protein and producing anti-MDA5 antibodies in the body. FIG. 4 is a diagram showing a lung histology image of interstitial pneumonia that occurred in a non-human animal model immunized with MDA5 protein.

In the present invention, we identify 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, We have shown that the above problems can be solved by providing a method for detecting the risk associated with myositis/dermatomyositis by detecting the autoantibodies that cause it.

Up to now, it has been reported that approximately 50% of cases of myositis/dermatomyositis that are positive for anti-MDA5 antibodies develop rapidly progressive interstitial pneumonia. It has been tested as a marker for interstitial pneumonia. However, although the anti-MDA5 antibody is a powerful marker for rapidly progressive interstitial pneumonia, the inventor of the present invention considered that the MDA5 protein is not involved in the true pathogenesis of myositis/dermatomyositis and the accompanying rapidly progressive interstitial pneumonia.

The present inventors collected blood from anti-MDA5 antibody-positive individuals who developed interstitial pneumonia and developed a protein array CWPA (comprehensive wet protein array) (Fukuda et al. al., Genes to Cells, 2021) to search for proteins other than MDA5 protein that blood anti-MDA5 antibodies bind to. The results revealed that the anti-MDA5 antibody binds to protein X, which is known to be a cell membrane-spanning protein with 12 transmembrane regions. This protein X is a protein having the amino acid sequence of SEQ ID NO: 1, and was presumed to have Na-independent organic anion transporter and thyroid hormone transporter activity.

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 suggest that antibodies against protein It has become clear that binding is involved.

First Aspect: Invention of a Method Based on this finding, the present invention, in one aspect, comprises the step of detecting an antibody that binds to protein X or a variant thereof in a sample taken from a human individual. , can provide a method for detecting risks associated with myositis/dermatomyositis. According to the findings of the present invention, protein Since antibodies that bind to the variant have been formed, the risk associated with myositis/dermatomyositis can be detected by detecting the presence of antibodies that bind to protein X or its variant.

The protein X used in this aspect of the invention to detect antibodies that bind to protein X or a variant thereof is preferably human protein X, in its entirety (SEQ ID NO: 1) It may be present, or it may be a part of it. In addition, the variant of protein Alternatively, it may be a protein having 80% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, or a portion thereof. When using a portion of protein X or a variant thereof, the extracellular domain of protein X or a variant thereof can be used.

Here, in the variant of protein Amino acids vary depending on the position and type of each amino acid residue in the three-dimensional structure of protein It means ~20 pieces, more preferably 1 to 10 pieces, even more preferably 1 to 5 pieces. In addition, in this terminology, "amino acid deletion/substitution/addition" may be any mutation as long as the binding of the antibody to be detected to protein X is maintained in this aspect of the present invention. .

In addition, in a variant of protein As long as the function is maintained, for example, 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more with respect to the amino acid sequence of protein X (SEQ ID NO: 1). , particularly preferably a protein having an amino acid sequence with an identity of 99% or more.

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 a blood-derived sample. As a blood-derived sample, plasma or serum can be used in the method of the present invention.

The 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 developing interstitial pneumonia associated with myositis/dermatomyositis. The risk of transition from interstitial pneumonia to rapidly progressive interstitial pneumonia can be mentioned. Studies conducted in the present invention have revealed that the stronger the signal intensity derived from the concentration of antibodies that bind to protein X or its variants in blood samples, the higher the risk associated with myositis/dermatomyositis. .

The antibody to be detected in the present invention can be an IgG antibody or an IgM antibody that binds to protein X or a variant thereof. Considering the distribution of protein X in the body, it is preferable to use blood as the sample, and in that case, it is preferable to detect IgG antibodies or IgM antibodies. This antibody is useful as a marker for clinical amyopathic DM (CADM), especially for myositis/dermatomyositis accompanied by rapidly progressive interstitial pneumonia, for diagnosis, treatment selection, and prediction of prognosis. (For example, it can be used to predict the onset of myositis/dermatomyositis or interstitial pneumonia, or the worsening of the disease).

When carrying out the method of detecting risks associated with myositis/dermatomyositis in the present invention, a step of detecting the presence or absence of an antibody that binds to protein X or a variant thereof in a sample is performed. If the antibody is detectable in the sample, it can be determined that the sample is positive for antibodies that bind to protein It can be determined that there is a risk of developing interstitial pneumonia associated with myositis, and furthermore, that there is a risk of interstitial pneumonia associated with myositis/dermatomyositis progressing to rapidly progressive interstitial pneumonia. be able to.

In the present invention, the methods used in the step of detecting antibodies that bind to protein Immunodiffusion, chemiluminescent enzyme immunoassay, or chemiluminescent immunoassay can be used. In particular, when measuring the concentration of an antibody that binds to protein

When performing the step of detecting an antibody that binds to protein X or its variant in a sample by enzyme immunoassay (EIA method, ELISA method),
- Immobilizing the full length of protein X or its variant (antigen) on a solid phase,
・The step of reacting the sample with the antigen;
・By performing the process of detecting and quantifying the antibody bound to the antigen using an anti-human antibody (corresponding to a secondary antibody) attached with a detection marker, it is possible to detect the antibody or measure its concentration. .

When performing the step of detecting an antibody that binds to protein X or its variant in a sample by Western blotting,
・The step of electrophoresing a specified amount of the entire length of Protein
・Then, the process of transferring to a nitrocellulose membrane, etc.
・Then, a step of reacting the antigen transferred to the membrane with the sample,
・Detection of the antibody or measurement of its concentration is carried out by performing the process of detecting and quantifying the antibody bound to the antigen on the membrane using an anti-human antibody (equivalent to a secondary antibody) attached with a detection marker. be able to.

When detecting antibodies that bind to protein X or its variants in a sample by indirect fluorescent antibody method,
・Adding the sample onto a solid phase such as a glass slide on which a specified amount of protein X or its variant (antigen) is immobilized,
・The step of reacting between the antigen on the slide glass and the antibody in the sample (primary reaction),
・After washing, a step of adding and reacting a labeled anti-human antibody for detection (secondary reaction),
- The antibody in the sample can be detected or its concentration can be measured by forming a complex of antigen, antibody, and labeled antibody, and detecting and quantifying the label.

When detecting antibodies that bind to protein X or its variants in a sample by double immunodiffusion method,
・Injecting the full length of protein X or its variant (antigen), positive serum, and sample into three holes in the agarose gel, respectively;
・The process of diffusing the antigen and positive serum, and the antigen and antibody in the sample, in agarose,
- This method can be carried out by detecting and identifying the specificity of the antibody from the shape of the sedimentation line as a result of the antigen-antibody reaction. In this case, if the shape of the sedimentation line is a fusion type, it indicates that the reference antibody and the antibody in the sample recognize the same antigen, and if the shape of the sedimentation line is a spur type, If the reference antibody and the antibody in the sample partially recognize the same antigen, and the shape of the sedimentation line is a cross type, then the reference antibody and the antibody in the sample are two types of antibodies. Shows recognition of different antigens.

When detecting antibodies that bind to protein X or its variants in a sample by chemiluminescent enzyme immunoassay (CLEIA),
・The step of immobilizing a specified amount of the entire length of protein X or its variant (antigen) on a solid phase such as magnetic particles,
・The step of reacting the sample with the antigen;
・After washing to remove unreacted substances other than the antigen-bound antibody, a step of reacting with the enzyme-labeled antibody,
・After further washing to remove unreacted substances, a chemiluminescent enzyme substrate is added, and the chemiluminescent enzyme substrate is hydrolyzed by the enzyme of the labeled antibody, and the amount of luminescence generated is measured. Detection or measurement of its concentration can be performed.

When detecting antibodies that bind to protein By measuring the amount of chemiluminescence generated by using a chemiluminescent compound as a label bound to the antibody, it is possible to detect the antibody in the sample or measure its concentration. That is,
・The step of immobilizing a specified amount of the entire length of protein X or its variant (antigen) on a solid phase such as magnetic particles,
・The step of reacting the sample with the antigen;
・After washing to remove unreacted substances other than the antigen-bound antibody, a step of reacting with a chemiluminescent compound-labeled antibody,
・After further washing to remove unreacted substances, a chemiluminescent enzyme substrate is added and the amount of light emitted from the chemiluminescent compound of the labeled antibody is measured, thereby detecting the antibody in the sample or measuring its concentration. It can be performed.

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 variant thereof. Those skilled in the art can appropriately select substances that can be used as labeling substances that are suitable for the various methods described above. Examples of such labeling substances include biotin, enzymes (e.g., alkaline Phosphatase (AP), horseradish peroxidase (HRP)), fluorescent dyes (e.g. Alexa Fluor®, DyLight®, FITC, PE, etc.), etc., commonly available in the art. can be mentioned.

Second Aspect: Invention of a Kit As another aspect, 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 provides a solid phase immobilized with the full length of protein X or a variant thereof or a part thereof (antigen), an antibody for detecting a human antibody that binds to protein Kits can be provided for detecting antibodies that bind to protein X or a variant thereof present in a sample taken from a human individual, including human antibodies. This kit can be used to detect risks associated with myositis/dermatomyositis.

The kit of the present invention includes a kit for ELISA method, a kit for Western blotting method, a kit for indirect fluorescent antibody method, a kit for double immunodiffusion method, and a kit for chemiluminescent enzyme immunoassay method. , or a kit for chemiluminescent immunoassay, etc.
・When using a kit for ELISA method, an ELISA plate on which the full length of protein X or its variant (antigen) is immobilized,
・When using a kit for Western blotting, use the antigen-immobilized membrane as a kit for Western blotting.
・When using a kit for indirect fluorescent antibody method, use a solid phase such as a slide glass on which the antigen is immobilized.
・When using a kit for double immunodiffusion method, use an agarose gel with holes for the antigen, positive serum, and sample (when used, place the antigen, positive serum, and sample into each hole of the agarose gel). (used by dripping)
・When used as a kit for chemiluminescent enzyme immunoassay or chemiluminescent immunoassay, magnetic particles with immobilized antigens are
Each can be used.

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 associated with myositis/dermatomyositis. This includes the risk of transition from interstitial pneumonia 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.

When the kit of the present invention is used to quantify the concentration of an antibody that binds to protein It can be determined that the antibody that binds to the body is positive and there is a risk of developing myositis/dermatomyositis, and it can be determined that there is a risk of developing interstitial pneumonia associated with myositis/dermatomyositis. Furthermore, it can be determined that there is a risk that interstitial pneumonia associated with myositis/dermatomyositis will progress to rapidly progressive interstitial pneumonia.

Third Embodiment: Model Animal In the present invention, as described later in the Examples, in the process of elucidating the mechanism of developing myositis/dermatomyositis and interstitial pneumonia accompanying it, MDA5 knockout mice lacking MDA5 were used. MDA5 knockout mice develop interstitial pneumonia when anti-human MDA5 antibodies or cells that produce anti-human MDA5 antibodies are administered to mice; It has been shown that interstitial pneumonia develops in individuals who are immunized with the following. It was revealed that the lung tissue of these mice that developed interstitial pneumonia was histologically very similar to the lung histology of patients with interstitial pneumonia associated with myositis/dermatomyositis in humans.

Based on this result, the present invention, as a further aspect, aims to treat myositis/dermatomyositis and interstitial pneumonia accompanying it by making antibodies that bind to protein X or its variants present in the blood. A non-human model animal in which the disease develops can be provided. Here, "having antibodies that bind to protein X or its variants exist in the blood" means that antibodies that bind to protein X or its variants are present in the blood. For example,
・A method of externally administering an antibody that binds to protein X or a variant thereof into the blood of a non-human animal,
・Cells that produce antibodies that bind to protein How to make
- Protein a method of producing an antibody that binds to the variant;
Any method may be adopted.

The non-human model animal of the present invention develops rapidly progressive interstitial pneumonia that transitions from myositis/dermatomyositis, interstitial pneumonia associated with myositis/dermatomyositis, or interstitial pneumonia associated with myositis/dermatomyositis. It is characterized by When examining lung tissue sections from mice that had developed the disease, they showed a histology similar to that of interstitial pneumonia in humans. It was shown to function as an animal model for interstitial pneumonia associated with myositis/dermatomyositis. This non-human model animal is useful in developing therapeutic methods and therapeutic agents for interstitial pneumonia associated with myositis/dermatomyositis in humans.

When creating the non-human model animal of the present invention, the antibody that binds to protein You can use something that binds to some parts. Here, when referring to a part of protein X or a variant thereof, the extracellular domain of protein X or a variant thereof can be used.

When producing the non-human model animal of the present invention, the antibody that binds to protein X or its variant that is present in the blood may be any of human antibodies, mouse antibodies, rat antibodies, 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 externally, or a method in which a cell that produces an antibody that binds to protein In the case of a method for producing an antibody that binds to X or a variant thereof, the origin of the antibody present in a non-human model animal may be any animal species. On the other hand, a method for producing antibodies that bind to protein X or its variants by immunizing with protein X or its variants and generating cells that produce antibodies that bind to protein X or its variants in the body. In this case, since antibodies are newly produced within the individual non-human model animal, antibodies from that animal will be present in the blood.

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

Hereinafter, the present invention will be specifically illustrated by giving Examples. The examples presented below are not intended to limit the invention in any way.

Example 1: Analysis of the effect of anti-human MDA5 antibody in MDA5 knockout mice This example aims to analyze the effect of anti-human MDA5 antibody in MDA5 knockout mice and elucidate the significance of anti-MDA5 antibody in blood. I conducted an experiment for the purpose.

MDA5 knockout mice (n = 5 to 7) in which MDA5 was deleted in the body were used as recipient mice for the anti-MDA5 antibody (B6.Cg-Ifih1 ^tm1.1Cln , The Jackson Laboratory). Although this mouse does not have MDA5 in its body, it has been revealed that it does not have the tendency to develop interstitial pneumonia during its natural history (Figure 1).

On the other hand, wild-type mice (C57/BL6) were immunized with human MDA5 to produce antibodies. After mixing 100μg of MDA5 protein with adjuvant titer max gold (Funakoshi) in a 1:1 ratio and making it soft, it was subcutaneously injected into the back 5 times every week (days 0, 7, 14, 21, 28), and then on day 35. An analysis was conducted to determine whether interstitial pneumonia had developed. The results showed that mice immunized with human MDA5 developed interstitial pneumonia.

Next, B cells were collected from the spleen of a mouse immunized with human MDA5, and 10 ⁶ of these B cells were intravenously administered to the aforementioned MDA5 knockout mice (day 0). Analysis of whether the MDA5 knockout mice developed interstitial pneumonia showed that the MDA5 knockout mice developed interstitial pneumonia (Figure 1).

This suggests that interstitial pneumonia develops even when MDA5 is not present in the body, and that anti-MDA5 antibodies produced in mice immunized with human MDA5 bind to unknown targets other than MDA5. The results suggested that interstitial pneumonia had developed.

Example 2: Search for the true target substance of anti-MDA5 antibody In this example, we investigated the mechanism by which anti-MDA5 antibody causes interstitial pneumonia by using a target substance other than MDA5 protein of anti-MDA5 antibody in serum. An experiment was conducted with the aim of identifying this.

In Example 1, antibodies in the serum of mice that developed interstitial pneumonia were prepared using a wheat germ cell-free expression system using 19,446 types of human proteins, and [FLAG-tag]-[ was added to the N-terminus of each protein. GST-tag]- was added to the protein array CWPA for detection.

In this protein array, we used the affinity of GST-tags attached to the N-terminus of each human protein and glutathione modified on the array substrate to create CWPA in which human proteins were bound to the substrate surface.

Mouse serum was diluted 333 times using antibody diluent, added to CWPA, and reacted at room temperature for 1 hour. After discarding the reaction liquid and washing the CWPA, add the secondary antibody (goat anti-mouse IgG (H+L) Alexa Flour® 647 complex) diluted 1000 times using antibody diluent to the CWPA. and reacted at room temperature for 1 hour. After discarding the post-reaction liquid, washing the CWPA, and air-drying it, a fluorescence image derived from Alexa Flour® 647 was acquired using a fluorescence imager.

The protein bound to the secondary antibody was analyzed from the fluorescence image, and the antigen of the antibody contained in the mouse serum was identified. The results revealed that the anti-MDA5 antibody binds to transmembrane protein X in addition to MDA5 (Figure 2). This protein is different from the intracellular protein MDA5 protein, and has the amino acid sequence of SEQ ID NO: 1:

It was shown that it is a cell membrane-penetrating protein that can be approached with anti-MDA5 antibodies even in vivo.

This suggests that the anti-MDA5 antibodies previously detected in individuals with interstitial pneumonia associated with myositis/dermatomyositis are actually antibodies that bind to protein X on the cell surface; Due to its presence in the body, interstitial pneumonia associated with myositis/dermatomyositis develops based on the binding between protein X and the antibody against it (previously called anti-MDA5 antibody). It has been suggested.

Next, serum antibodies in dermatomyositis patients whose serum anti-MDA5 antibodies were positive were analyzed as follows. First, human proteins that were positive in the analysis using CWPA (namely, MDA5 and transmembrane protein The above human protein, MDA5, or transmembrane protein X was bound to each of the ELISA plates whose surfaces had been modified with glutathione.

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

Measure the absorbance with a plate reader, subtract the value measured at 570 nm, the reference wavelength (OD (570 nm)), from the value measured at 450 nm, which is the absorption wavelength of TMB (OD (450 nm)), and We identified the antigen that anti-human MDA5 antibody contained in serum binds to.

Each protein was measured with n=8, and significant differences from the negative control (tag only) were evaluated using the Mann-Whitney U test.

The analysis results showed that the anti-MDA5 antibody in the patient's serum binds to MDA5 (positive control), but also to transmembrane protein X (Table 1).

Example 3: Effect of protein X on interstitial pneumonia associated with myositis/dermatomyositis In this example, protein We conducted an experiment with the aim of clarifying whether or not it is involved in the onset of the disease.

100 μg of Protein

As a negative control, only the tag that was combined for the purpose of producing transmembrane protein X during synthesis was similarly administered (n = 6).

As a result, all cases in the Protein

These results showed that, as suggested in Example 2, protein X actually plays a role in the development of interstitial pneumonia associated with myositis/dermatomyositis.

Furthermore, a lung tissue section from an individual mouse that developed interstitial pneumonia as a result of protein X administration is shown in FIG. These histological images showed that the lungs of mice that developed interstitial pneumonia were accompanied by inflammatory cell infiltration and thickening of alveolar septa, similar to the histological images of lungs that developed interstitial pneumonia in humans. . In contrast, the lungs of mice immunized only with the tag did not exhibit any of the above characteristics, indicating that they had normal alveolar structure.

According to the present invention, by identifying the true cause of the onset of interstitial pneumonia accompanying myositis/dermatomyositis or the transition to rapidly progressive interstitial pneumonia, and detecting the autoantibody that is the cause, / A method for detecting risks associated with dermatomyositis can be provided.

Claims (12)

In a sample collected from a human individual, the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 1 with deletion, substitution, or addition of one or more amino acids, or SEQ ID NO: : myositis, comprising the step of detecting an antibody that binds to protein X or a variant thereof, which is the full length or a part thereof of a protein having an amino acid sequence identity of 80% or more with the amino acid sequence of X. / How to detect the risks associated with dermatomyositis. 2. The method of claim 1, wherein the sample is a blood-derived sample. The risks associated with myositis/dermatomyositis include the risk of developing myositis/dermatomyositis, the risk of developing interstitial pneumonia associated with myositis/dermatomyositis, and the risk of rapidly progressive interstitial pneumonia associated with myositis/dermatomyositis. 3. The method according to claim 1 or 2, wherein the method is selected from the group consisting of: a risk of transitioning to qualitative pneumonia. 3. The method according to claim 1, wherein the antibody that binds to protein X or a variant thereof is an IgG antibody or an IgM antibody. 3. The method according to claim 1, wherein the risk associated with myositis/dermatomyositis is determined when an antibody that binds to protein X or a variant thereof is detectable in the sample. Autoantibodies that bind to human protein 3. The method according to claim 1 or 2, wherein the method detects. Amino acid sequence of SEQ ID NO: 1, amino acid sequence with one or more amino acid deletions, substitutions, or additions in the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence of 80 between SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 1. A solid phase on which protein X or a variant thereof is immobilized, which is the full length or a part thereof of a protein having an amino acid sequence identity of % or more, for detecting human antibodies that bind to protein X or a variant thereof. A kit for detecting risks associated with myositis/dermatomyositis by detecting antibodies that bind to protein X or a variant thereof present in a sample collected from a human individual, including anti-human antibodies. 8. The kit of claim 7, wherein the sample is a blood-derived sample. The solid phase on which Protein X or its variant is immobilized is an ELISA plate, slide glass, particle, agarose gel on which Protein The kit according to claim 7 or 8, which is a membrane with a body immobilized thereon. The risks associated with myositis/dermatomyositis include the risk of developing myositis/dermatomyositis, the risk of developing interstitial pneumonia associated with myositis/dermatomyositis, and the risk of rapidly progressive interstitial pneumonia associated with myositis/dermatomyositis. 9. The kit according to claim 7 or 8, wherein the kit is selected from the group consisting of: a risk of transition to qualitative pneumonia. 9. The kit according to claim 7 or 8, wherein the antibody that binds to protein X or a variant thereof is an IgG antibody or an IgM antibody. 9. The kit according to claim 7, which determines that there is a risk associated with myositis/dermatomyositis when an antibody that binds to protein X or a variant thereof is detectable in the sample.