Method for checking the precision state of vangalides or the risk of secifying covid-19, test kit, compansion diagnostic reagent, and test marker

Abstract

Provided are: a novel pathological marker which is useful in the examination of the progression of vasculitis and the examination of the risk of severity of COVID-19; and a means for examining the progression of vasculitis and the risk of severity of COVID-19 using the marker. According to one embodiment of the present invention, provided are: a method for checking the afflicted state of vasculitis; and a method for checking the critical risk of COVID-19. The method comprises: detecting or quantifying the following proteins in a blood sample collected from a subject: (1) a first APOA2-like protein having a molecular weight of 23-25 kDa and / or a second APOA2-like protein having a molecular weight of 23-25 kDa and / or a third APOA2-like protein having an antigen-antibody reaction with a single-chain variable fragment containing an amino acid sequence represented by SEQ ID NO: 1; (2) A second APOA2-like protein having a molecular weight of 16-20 kDa, said second APOA2-like protein having an antigen-antibody reaction with a single-chain variable fragment comprising the amino acid sequence represented by SEQ ID NO: 1.

Description

Technical Field

[0001] The present invention relates to a method for examining the state of vasculitis or the risk of severe symptoms of COVID-19, and a test kit, a companion diagnostic reagent, and a test marker used in the method. Background Art

[0002] Autoantibodies against myeloperoxidase (MPO) in the cytoplasm of neutrophils (MPO-ANCA (Anti-Neutrophil Cytoplasmic Antibody: Anti-neutrophil Cytoplasmic Antibody)) are associated with refractory vasculitis such as crescent-forming nephritis (Crescent-forming Nephritis), Microscopic Polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). In addition, the titer of MPO-ANCA in the serum of these patients is related to the activity of the disease, so MPO-ANCA is used as a specific marker for diagnosis and treatment determination of these diseases (for example, refer to Non-Patent Document 1).

[0003] However, even when MPO-ANCA is used as a marker, the titer of MPO-ANCA in serum may not necessarily be consistent with the pathological state. This suggests that MPO-ANCA is not the only factor that acts on neutrophils to induce vasculitis or participates in the onset of vasculitis, but that there are other molecules and mechanisms that induce vasculitis.

[0004] In recent years, in addition to MPO of neutrophils, the possibility of the existence of target molecules of autoantibodies has been discussed. In addition, as a mechanism of the onset and progression of vasculitis, attempts have been made to clarify the activation mechanism of vascular endothelial cells caused by autoantibodies such as MPO-ANCA. So far, it has been reported that anti-proteinase-3 autoantibodies (PR3-ANCA) in patients with granulomatosis with polyangiitis induce the expression of ICAM-1 and VCAM-1 (see non-patent documents 2 and 3). However, there are no reports on the direct effects of MPO-ANCA on vascular endothelial cells.

[0005] Thus, the current situation is that, in the diagnosis of vasculitis, in addition to MPO-ANCA, which is clinically used, there are factors related to the pathological state of vasculitis, but their status is not clear. As shown above, the diagnosis and treatment judgment based on MPO-ANCA may not fully reflect the true pathological state. Therefore, the identification of factors other than MPO-ANCA has the possibility of supplementing the diagnosis based on MPO-ANCA or providing new clinical markers to replace it, and its clinical significance is very significant.

[0006] In addition, although diagnostic criteria have been established for Kawasaki disease, a vasculitis syndrome characterized by vasculitis in medium-sized and small muscular arteries throughout the body, there are currently no specific pathological markers. In addition, in the novel coronavirus infection (COVID-19) that has spread to the world since 2020, direct impairment based on the SARS-CoV-2 virus caused by binding to the ACE2 receptor of vascular endothelial cells has been found, and there are cases where the disease worsens regardless of whether the PCR is negative, and it is also known that vasculitis is caused by the immunological response on the host side (see non-patent documents 4 and 5). However, there are no reports of pathological markers that show involvement in vasculitis as a factor in the aggravation of COVID-19.

[0007] It should be noted that the present inventors have found that a single-chain variable region fragment (ScFv; referred to as "VasSF") identified from a 204-clone IgG gene library of human lymphocyte clones (Patent Document 1) inhibits vasculitis at a low dose in the ANCA-associated vasculitis model mouse SCG / Kj. In addition, it was announced that the target molecule bound by this ScFv is vasculitis-associated apolipoprotein A-II (VAP2) (see Patent Document 2 and Non-Patent Document 6).

[0008] Prior art literature Patent Literature Patent Document 1: Japanese Patent Application Publication No. 2016-160265 Patent Document 2: International Publication No. 2018 / 139608 Non-patent literature Non-patent document 1: Goeken JA, Antineutrophil cytoplasmic antibody-A useful serological marker for vasculitis. J. Clin. Immunol., 1991; 11: 61-74 Non-patent document 2: Johnson PA, et al. Up-regulation of the endothelial celladhesion molecule intercellular adhesion molecule-1 (ICAM-1) by autoantibodies in autoimmune vasculitis. Clin. Exp. Immunol., 1997; 108:234-242 Non-patent document 3: Mayet WJ et al., Antibodies to proteinase 3 mediate expression of vascular cell adhesion molecule-1 (VCAM-1). Clin. Exp. Immunol., 1996; 103: 259-267 Non-patent literature 4: Ackermann M et al. Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19. N. Engl. J. Med., 2020; 383 (2): 120-128 Non-patent literature 5: Nishiga M et al., COVID-19 and cardiovascular disease: from basic mechanisms to clinical perspectives. Nature Reviews, 2020; 17 (9): 543-558 Non-patent literature 6: Kameoka Y et al., Efficacy of a recombinant single-chainfragment variable region, VasSF, as a new drug for vasculitis. DrugDes.Devel.Ther., 2019; 13: 555-568 Summary of the invention

[0009] In view of the above-mentioned prior art, an object of the present invention is to provide a new pathological marker useful in examining the state of vasculitis and the risk of severe symptoms of COVID-19, and a means of examining the state of vasculitis and the risk of severe symptoms of COVID-19 using the same.

[0010] Technical solutions for solving technical problems The present inventors conducted intensive research with the goal of solving the above technical problems. As a result, surprisingly, it was found that VAP2 (molecular weight = about 24 kDa), which is known to be a target molecule in the treatment of vasculitis using VasSF, is present at high levels in the serum of patients during the active stage of diseases accompanied by vasculitis such as ANCA-associated vasculitis, Kawasaki disease, and COVID-19. In addition, it was found that the serum level of VAP2 decreased when these patients were discharged from the hospital and during the remission period of the disease, and the serum level of proteins (molecular weight = about 17-19 kDa) that are bound to VasSF but have a molecular weight smaller than VAP2 increased. Based on these insights, the present inventors completed the present invention.

[0011] That is, according to one embodiment of the present invention, there is provided a method for examining the state of vasculitis and a method for examining the risk of severe symptoms of COVID-19, the method comprising: detecting the following proteins in a blood sample collected from a subject: (1) an apolipoprotein A2-like protein that undergoes an antigen-antibody reaction with a single-chain variable region fragment comprising the amino acid sequence shown in SEQ ID NO: 1 and has a molecular weight of 23 to 25 kDa (the protein is referred to as the "first APOA2-like protein" in this specification), and / or (2) an apolipoprotein A2-like protein that undergoes an antigen-antibody reaction with a single-chain variable region fragment comprising the amino acid sequence shown in SEQ ID NO: 1 and has a molecular weight of 16 to 20 kDa (the protein is referred to as the "second APOA2-like protein" in this specification).

[0012] In addition, according to another embodiment of the present invention, a kit for examining the state of vasculitis or a kit for examining the risk of severe symptoms of COVID-19 is provided, the kit comprising: a single-chain variable region fragment comprising the amino acid sequence shown in sequence number 1 or an anti-apolipoprotein A2 antibody.

[0013] Furthermore, according to another embodiment of the present invention, a companion diagnostic reagent for the suffering state of vasculitis or a companion diagnostic reagent for the risk of severe symptoms of COVID-19 used in the method involved in one embodiment of the present invention is provided, and the companion diagnostic reagent includes: a single-chain variable region fragment comprising the amino acid sequence shown in SEQ ID NO: 1 or an anti-apolipoprotein A2 antibody.

[0014] Furthermore, according to another embodiment of the present invention, a marker for detecting the state of vasculitis and a marker for detecting the risk of severe symptoms of COVID-19 are provided, wherein the marker for detecting the state of vasculitis and ...

[0015] Effects of the Invention According to the present invention, a new pathological marker useful for testing the state of vasculitis and the risk of severe COVID-19 is provided. In addition, based on this knowledge, a method for testing the state of vasculitis and the risk of severe COVID-19 and a test kit are also provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a diagram showing the results of Western blotting analysis of VasSF-binding molecules in Example 3 using serum samples collected from five ANCA-associated vasculitis (AAV) patients and one healthy individual.

[0017] Figure 2 This is a graph showing the results of signal analysis of VasSF binding molecules in Example 4 using serum samples collected from one COVID-19 patient at different time points (at the time of hospitalization, at the time of ICU transfer, and at the time of discharge).

[0018] Figure 3 This is a graph showing the results of studying the relationship between the signal value of the VasSF binding molecule and the pathological state of AAV in 67 samples including 34 paired serum samples in the active and remission phases from 17 ANCA-associated vasculitis (AAV) patients and 10 samples from healthy subjects in Example 5.

[0019] Figure 4 This is a graph showing the results of studying the relationship between the signal value of the VasSF binding molecule and the pathological state of COVID-19 in Example 6, for serum samples collected from 65 COVID-19 patients at different time points (at the time of hospitalization, ICU transfer, and discharge).

[0020] Figure 5 This is a graph showing the results of signal analysis in Example 7 using an anti-APOA2 antibody by the same immunoblotting method as in Example 3.

[0021] Figure 6 This is a graph showing the results of signal analysis in Example 8 using an anti-APOA2 antibody by the same immunoblotting method as in Example 4.

[0022] Figure 7 This is a graph showing the results of studying the relationship between the signal value of the anti-APOA2 antibody binding molecule and the pathological state of COVID-19 in Example 9, using the same method as Example 6, for serum samples collected from 65 COVID-19 patients at different time points (at the time of hospitalization, ICU transfer, and discharge).

[0023] Figure 8 This is a graph showing the results of signal analysis of VasSF-binding molecules in Example 10 using serum samples collected from a child with Kawasaki disease at different time points (sixth day, tenth day, and thirtieth day from the onset of the disease).

[0024] Fig. 9 This is a graph showing the results of signal analysis of VasSF binding molecules performed in Example 11 using serum samples collected from 5 COVID-19 patients at different time points (when hospitalized and when discharged) by the same method as in Example 4.

[0025] Fig. 10A These are photographs showing the results of two-dimensional electrophoresis in Example 12 using serum from patients in the active phase and processed using VasSF.

[0026] Fig. 10B These are photographs showing the results of two-dimensional electrophoresis in Example 12 using serum from patients in remission and processed using VasSF. DETAILED DESCRIPTION

[0027] 《Inspection markers (pathological markers)》 As described above, the present inventors have found that VAP2 (molecular weight = about 24 kDa), which is known to be a target molecule in the treatment of vasculitis using VasSF, is present at high levels in the serum of patients during the active stage of diseases accompanied by vasculitis such as ANCA-associated vasculitis, Kawasaki disease, and COVID-19. In addition, it was found that the serum level of VAP2 decreased when these patients were discharged from the hospital and during the remission period of the disease, and the serum level of a protein (molecular weight = about 17-19 kDa) that binds to VasSF but has a smaller molecular weight than VAP2 increased.

[0028] Based on the above findings, according to the present invention, the following markers are provided as markers for examining the state of vasculitis and the risk of severe symptoms of COVID-19.

[0029] A marker for examining the state of vasculitis, which undergoes an antigen-antibody reaction with a single-chain variable region fragment comprising the amino acid sequence shown in SEQ ID NO: 1 and comprises a first APOA2-like protein having a molecular weight of 23 to 25 kDa; A marker for examining the state of vasculitis, which undergoes an antigen-antibody reaction with a single-chain variable region fragment comprising the amino acid sequence shown in SEQ ID NO: 1 and comprises a second APOA2-like protein having a molecular weight of 16 to 20 kDa; A marker for testing the risk of severe symptoms of COVID-19, which undergoes an antigen-antibody reaction with a single-chain variable region fragment comprising the amino acid sequence shown in SEQ ID NO: 1 and comprises a first APOA2-like protein having a molecular weight of 23 to 25 kDa; and A marker for testing the risk of severe symptoms of COVID-19, which undergoes an antigen-antibody reaction with a single-chain variable region fragment comprising the amino acid sequence shown in SEQ ID NO: 1 and comprises a second APOA2-like protein having a molecular weight of 16 to 20 kDa.

[0030] Here, it is known that human apolipoprotein A2 (hAPOA2) is encoded by the APOA2 gene (RefSeq Accession No. NM_001643) containing 473 bp, contains 100 amino acids (RefSeq Accession No. NP_001634), and has a molecular weight of about 11 kDa. Therefore, the proteins used as the test markers in (1) and (2) above are molecules different from human apolipoprotein A2 (hAPOA2) in terms of their molecular weight. On the other hand, the proteins used as the test markers above are all proteins that undergo antigen-antibody reactions with the single-chain variable region fragment containing the amino acid sequence shown in SEQ ID NO. 1. In addition, it has been confirmed that these proteins all undergo antigen-antibody reactions with anti-human APOA2 antibodies. Therefore, in this specification, these proteins are referred to as "apolipoprotein A2 'like' proteins".

[0031] It should be noted that the isoelectric point (pI) of the first APOA2-like protein and the second APOA2-like protein is not particularly limited. In a preferred embodiment, the isoelectric point (pI) of the first APOA2-like protein is preferably 6.0 to 6.6, more preferably 6.1 to 6.5, and further preferably 6.2 to 6.4. In addition, the isoelectric point (pI) of the second APOA2-like protein is preferably 5.2 to 5.8, more preferably 5.3 to 5.7, and further preferably 5.4 to 5.6. Here, the molecular weight and isoelectric point of the first APOA2-like protein and the second APOA2-like protein can be measured by two-dimensional electrophoresis described in the Example column described later.

[0032] "Methods for checking the status of vasculitis and the risk of severe COVID-19" Using the test marker (pathological marker) involved in one embodiment of the present invention, according to another embodiment of the present invention, a method for testing the state of vasculitis and a method for testing the risk of severe COVID-19 are provided. That is, another embodiment of the present invention is a method for testing the state of vasculitis and a method for testing the risk of severe COVID-19 (hereinafter, also collectively referred to as "the test method of the present invention"). The test method of the present invention includes: detecting or quantifying the following proteins in a blood sample collected from a subject: (1) a first APOA2-like protein having a molecular weight of 23 to 25 kDa that undergoes an antigen-antibody reaction with a single-chain variable region fragment comprising the amino acid sequence of SEQ ID NO: 1, and / or (2) A second APOA2-like protein having an antigen-antibody reaction with the single-chain variable region fragment comprising the amino acid sequence shown in SEQ ID NO: 1 and having a molecular weight of 16 to 20 kDa. Here, in the test method of the present invention, only one of the first APOA2-like protein or the second APOA2-like protein may be detected or quantified. However, from the viewpoint of performing a more accurate test, it is preferred to detect or quantify both.

[0033] According to the inspection method of the present invention, based on the results of detection or quantification of the proteins of (1) and / or (2) above, it is possible to determine, as the state of vasculitis, for example, the presence or absence of vasculitis, the type of vasculitis, the degree of progression of vasculitis, the severity of vasculitis, the state of recovery from vasculitis, etc. Here, examples of the degree of progression of vasculitis include early stage, middle stage, and late stage. In addition, examples of the severity of vasculitis include mild, moderate, and severe. In addition, examples of the risk of severe COVID-19 include mild, moderate, and severe.

[0034] The above-mentioned subject may also be a confirmed patient suffering from vasculitis or COVID-19. In addition, the above-mentioned subject is preferably a subject before severe symptoms of vasculitis or COVID-19 (i.e., a mild or moderate patient), and is particularly preferably a mild patient.

[0035] In the testing method of the present invention, the blood sample is not particularly limited as long as the first APOA2-like protein and the second APOA2-like protein can be detected. Examples of the blood sample include plasma and serum, and serum is preferably used.

[0036] The vasculitis to be the subject of the inspection method of the present invention is not particularly limited, and examples thereof include: ANCA-associated vasculitis such as microscopic polyangiitis (MPA), granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis (EGPA); Kawasaki disease; COVID-19 (Coronavirus Disease 2019; SARS-CoV-2 infectious disease); agammaglobulinemia, Guillain-Barré syndrome, Wegener's granulomatosis, thrombotic thrombocytopenic purpura (TTP), IgA vasculitis, acute progressive glomerulonephritis, idiopathic interstitial pneumonia, idiopathic pulmonary fibrosis, sarcoidosis, diffuse panbronchiolitis, Behcet's disease, systemic lupus erythematosus (SLE), Sjogren's syndrome, Takayasu's arteritis, thromboangiitis obliterans (Buerger's disease), polyarteritis nodosa, malignant rheumatoid arthritis, giant cell arteritis, antiphospholipid antibody syndrome, scleroderma, eosinophilic fasciitis, pemphigus, nephritis, glomerulonephritis and other vasculitis or vasculitis associated with various diseases. Among them, the method for examining the state of vasculitis of the present invention is particularly useful in the examination of vasculitis associated with COVID-19 in addition to ANCA-associated vasculitis and Kawasaki disease.

[0037] Here, as shown in the examples described below, the first APOA2-like protein (VAP2) is present at high levels in the serum of patients in the active stage of diseases accompanied by vasculitis and vascular inflammation, such as ANCA-associated vasculitis, COVID-19, and Kawasaki disease, and disappears after the symptoms improve. In addition, the second APOA2-like protein is not found in the serum of the active stage of the above diseases, but is present at high levels in the serum after the symptoms improve.

[0038] Thus, when the concentration of the first APOA2-like protein (VAP2) in the serum of the subject is above the prescribed value (critical value), it can be judged that the subject is in the active stage of the above-mentioned disease or the possibility thereof is high. In addition, when the subject is a patient with COVID-19, it can be judged that there is a risk of the disease becoming severe or the possibility thereof is high. On the other hand, when the concentration of the first APOA2-like protein (VAP2) in the serum of the subject is less than the prescribed value (critical value), it can be judged that the subject is not in the active stage of the above-mentioned disease or the possibility thereof is low. In addition, when the victim is a patient with COVID-19, it can be judged that there is no risk of the disease becoming severe or the possibility thereof is low.

[0039] Furthermore, when the concentration of the second APOA2-like protein in the serum of the subject is above a predetermined value (critical value), it can be determined that the subject has passed the active phase of the above-mentioned disease and is in a remission phase or a cured state, or the possibility thereof is high. In addition, when the subject is a COVID-19 patient, it can be determined that there is no risk of the disease recurring or the possibility thereof is low.

[0040] It should be noted that since it is difficult to unambiguously define the specific value of the critical value for making these judgments, it can be appropriately determined by the therapist. However, if the critical value is too low, false positives increase, and if the critical value is too high, there is a risk of missing patients suffering from the above-mentioned diseases, for example.

[0041] It should be noted that, in view of the above-mentioned dynamics of the first APOA2-like protein as a disease marker, the subject of the test method of the present invention, i.e., the test subject, is preferably a patient in the early stage after the onset of the disease. Specifically, it is preferably a patient within 10 days from the onset of the disease, more preferably within 7 days, further preferably within 5 days, particularly preferably within 3 days, and most preferably within 1 day.

[0042] From the perspective of the progression of the disease and the monitoring of the process, the above-mentioned detection or quantification is preferably performed multiple times (preferably at least 2 times, more preferably 3 times or more, and further preferably 4 times or more) at intervals of a specified number of days. There is no particular restriction on the upper limit of the number of times of the above-mentioned detection or quantification, and for example, less than 15 times or less than 10 times can be cited. There is no particular restriction on the number of days intervals when the above-mentioned detection or quantification is performed multiple times, and more than 1 day (one quantification) can be cited, preferably more than 2 days, more preferably more than 3 days, and particularly preferably more than 4 days. It should be noted that there is no particular restriction on the upper limit of the number of days intervals when the above-mentioned detection or quantification is performed multiple times, and for example, once every 3 weeks, once every 2 weeks, etc. can be cited.

[0043] The testing method of the present invention may or may not include the step of collecting a serum sample from a subject.

[0044] In the inspection method of the present invention, the method for detecting or quantifying the first APOA2-like protein and the second APOA2-like protein is not particularly limited, and examples thereof include methods using enzyme immunoassay (EIA, ELISA), fluorescent enzyme immunoassay (FLEIA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA), fluorescent antibody method (FA), radioimmunoassay (RIA), immunoblotting (WB), immunoblotting, etc. In any of these assays, the above proteins can be detected or quantified by using VasSF and / or anti-APOA2 antibodies.

[0045] In the inspection method of the present invention, the above-mentioned quantification can be performed by the following method: a standard curve is prepared based on the relationship between the intensity of the measured label (e.g., absorbance, enzyme labeling intensity, fluorescence intensity, ultraviolet intensity, radiation intensity, etc.) and the amount (e.g., concentration) of the first APOA2-like protein and / or the second APOA2-like protein, and quantification is performed based on the above-mentioned standard curve (e.g., comparison).

[0046] Furthermore, according to the present invention, a method for diagnosing vasculitis can be provided, which includes the above-mentioned method for examining the state of suffering from vasculitis.

[0047] In addition, according to the present invention, a method for treating and / or preventing vasculitis or COVID-19 can be provided, the method comprising: the inspection method of the present invention and at least one step selected from the following steps: a step of performing treatment on a subject corresponding to the state of vasculitis or the risk of severe symptoms of COVID-19 determined by the inspection method; and a step of administering to the subject a therapeutic or preventive drug for vasculitis or COVID-19 corresponding to the state of vasculitis or the risk of severe symptoms of COVID-19 determined by the inspection method.

[0048] 《Test kit》 According to another embodiment of the present invention, a kit for testing the state of vasculitis and a kit for testing the risk of severe COVID-19 are also provided, wherein the kit comprises: a single-chain variable region fragment or an anti-apolipoprotein A2 antibody comprising the amino acid sequence shown in SEQ ID NO: 1. The kit is preferably used in the above-mentioned testing method of the present invention, and more preferably is a POC (Point of care: timely detection) kit.

[0049] In addition, according to another embodiment of the present invention, a companion diagnostic reagent for the suffering state of vasculitis or a companion diagnostic reagent for the risk of severe symptoms of COVID-19 used in the above-mentioned inspection method of the present invention is provided, and the companion diagnostic reagent includes: a single-chain variable region fragment comprising the amino acid sequence shown in sequence number 1 or an anti-apolipoprotein A2 antibody.

[0050] The test kits and companion diagnostic reagents according to these embodiments may contain, for example, the following substances.

[0051] (1) VasSF or microplate with anti-APOA2 antibody immobilized on it (2) Reaction buffer (3) Enzyme-labeled antibodies (4) Enzyme substrate solution (5) Cleaning agent (any buffer, surfactant, etc.) (6) Reaction stop solution (7) Standard buffer (any buffer, etc.) (8) Standards of the first APOA2-like protein (VAP2) and the second APOA2-like protein.

[0052] Furthermore, the test kit and companion diagnostic reagent according to these aspects preferably include a protein storage buffer containing BSA for the purpose of preventing protein adsorption.

[0053] In this specification, "companion diagnostic reagent" refers to a diagnostic reagent used in an examination performed before actually starting treatment or administering medicine, etc., in order to predict, for example, the effect or risk of treatment to be performed on each patient with vasculitis or COVID-19 according to the state of vasculitis or the risk of severe symptoms of COVID-19 determined by the examination method of the present invention, etc., the effect of the administered pharmaceutical (therapeutic drug, preventive drug, etc.), the risk of side effects, the appropriate dosage, etc. In addition, the above-mentioned companion diagnostic reagent can be used to select treatment corresponding to the state of vasculitis or the risk of severe symptoms of COVID-19 determined by the above-mentioned examination method of the present invention, and can also be used to select a therapeutic drug or preventive drug for vasculitis corresponding to the state of vasculitis, or a therapeutic drug or preventive drug for COVID-19 corresponding to the risk of severe symptoms.

[0054] Example Hereinafter, the present invention will be described in detail using examples, but the present invention is not limited to the following examples. It should be noted that all the following experiments were approved by the Ethics Committee of Chiba University. In addition, the collection of serum samples from humans was all carried out after the informed consent was confirmed by the provider. In addition, the processing of patient samples was analyzed by coding without personal information.

[0055] [Example 1: Preparation of VasSF and anti-APOA2 antibodies] "VasSF" (amino acid sequence (SEQ ID NO. 1) encoded by DNA containing the base sequence (CDS) of SEQ ID NO. 2) as a single-chain variable region fragment (ScFv) was prepared by the method described in Patent Document 1. In addition, as an anti-apolipoprotein A2 antibody, Anti-Apolipoprotein A2, Rabbit antibody was purchased from Sigma (cat. No.: SAB1410671) and used in the following experiments.

[0056] [Example 2: Quantification of Binding Molecules of VasSF and Anti-APOA2 Antibodies] The quantification of the first APOA2-like protein (VAP2) and the second APOA2-like protein, molecules bound to VasSF and anti-APOA2 antibodies, was performed by the following method. That is, albumin and IgG were removed from 5 mL of frozen serum collected from a subject, and binding to VasSF and anti-APOA2 antibodies was detected by immunoblotting. In addition, the bands detected in this way were quantified by the imaging signal method and the scoring method. The molecular weight of the first APOA2-like protein was about 24 kDa, and the molecular weight of the second APOA2-like protein was about 19 / 17 kDa.

[0057] [Example 3: Signal Analysis of VasSF Binding Molecule (First APOA2-Like Protein) in Serum Samples Collected from AAV Patients] Using serum samples collected from five patients with ANCA-associated vasculitis (AAV) and one healthy individual, the signal analysis of VasSF binding molecules was performed by immunoblotting. The results are shown in Figure 1 .

[0058] like Figure 1 As shown, in the active phase of AAV in 5 patients (p1-p5) ( Figure 1 In the serum sample of the patient with AAV remission (shown as “Active”), a strong band of a protein with a molecular weight of about 24 kDa (first APOA2-like protein; VAP2) was confirmed. Figure 1 In the serum sample of "Remission") shown in FIG. 1 , a strong band of a protein (second APOA2-like protein) with a molecular weight of about 19 / 17 kDa different from the first APOA2-like protein was confirmed. Furthermore, in the serum sample of a healthy person (HC), neither the first APOA2-like protein nor the second APOA2-like protein band was confirmed (at 0 level). It should be noted that Figure 1 "M" shown is a molecular weight marker.

[0059] In summary, the first APOA2-like protein (molecular weight of about 24 kDa) and the second APOA2-like protein (molecular weight of about 19 / 17 kDa) are both useful as pathological markers of AAV, and these proteins can be detected using VasSF.

[0060] [Example 4: Signal analysis of VasSF-binding molecules (first APOA2-like protein) in serum samples collected from COVID-19 patients (1)] The signal analysis of VasSF binding molecules was performed using serum samples collected from a COVID-19 patient at different time points (hospitalization, ICU transfer, and discharge). The results are shown in Figure 2 .

[0061] like Figure 2 As shown, the active period of COVID-19 (at admission; Figure 2 A strong band of a protein with a molecular weight of about 24 kDa (first APOA2-like protein; VAP2) was confirmed in the serum sample of the patient with the disease "A" shown in the figure. Figure 2 "U" shown) and at discharge ( Figure 2 In the serum sample of the patient with the disease "D" shown in FIG. 1 ), a strong band of a protein (second APOA2-like protein) with a molecular weight of about 19 / 17 kDa different from the first APOA2-like protein was confirmed. Figure 2 The indicated "M" is a molecular weight marker, and "Std" is a standard sample of VAP2.

[0062] In summary, the above-mentioned first APOA2-like protein (molecular weight of about 24 kDa) and the second APOA2-like protein (molecular weight of about 19 / 17 kDa) are useful as pathological markers of COVID-19, and these proteins can be detected using VasSF.

[0063] [Example 5: Relationship between the signal value of VasSF binding molecule (first APOA2-like protein) in serum samples of AAV patients and the pathological state] The relationship between the signal value of VasSF binding molecules and the pathological state of AAV was studied for 67 samples including 34 paired serum samples in the active and remission phases from 17 ANCA-associated vasculitis (AAV) patients and 10 samples from healthy subjects. The results are shown in Figure 3 .

[0064] like Figure 3As shown, the signal value of the VasSF binding molecule (first APOA2-like protein; VAP2) was high in the serum samples of the active phase of AAV. On the other hand, the signal value of the VasSF binding molecule (first APOA2-like protein; VAP2) was low in the serum samples of the remission phase of AAV and the serum samples of healthy persons.

[0065] In conclusion, the first APOA2-like protein (VAP2) is useful as a pathological marker of AAV.

[0066] [Example 6: Relationship between the signal value of VasSF binding molecule (first APOA2-like protein) in serum samples of COVID-19 patients and the pathological state] Serum samples collected from 65 COVID-19 patients at different time points (hospitalization, ICU transfer, and discharge) were used to study the relationship between the signal value of the VasSF binding molecule and the pathological state of COVID-19. The results are shown in Figure 4 It should be noted that Figure 4 The values ​​and error bars of the bar graphs shown represent the mean ± standard error (SE).

[0067] like Figure 4 As shown, during the active period of COVID-19 (at admission; Figure 4 In the serum sample of the patient with the condition "A"), the signal value of the VasSF binding molecule (first APOA2-like protein; VAP2) showed the highest level (signal fraction 4 / 2 μL serum). On the other hand, at the time of ICU transfer ( Figure 4 The signal value was significantly reduced at discharge ( Figure 4 The signal scores of patients who died and were transferred to other hospitals shortly after hospitalization remained at the same high level as those when they were hospitalized (these results are not included in the results). Figure 4 middle).

[0068] In summary, the above-mentioned first APOA2-like protein (VAP2) is useful as a pathological marker for COVID-19.

[0069] [Example 7: Confirmation of the Presence of Anti-APOA2 Antibody Binding Molecules in AAV Patient Serum Samples] In the experiment using the serum samples collected from the above AAV patients, in order to confirm whether the target molecule (first APOA2-like protein; VAP2) bound by VasSF is the target of the anti-APOA2 antibody, the anti-APOA2 antibody was used to perform signal analysis by the same immunoblotting method as in Example 3. The results are shown in Figure 5.

[0070] like Figure 5 As shown, in the active phase of AAV in 5 patients (p1-p5) ( Figure 5 In the serum sample of the patient with AAV remission (shown as “Active”), a strong band of a protein with a molecular weight of about 24 kDa (first APOA2-like protein; VAP2) was confirmed even when anti-APOA2 antibodies were used. Figure 5 In the serum samples of the healthy subjects (“Remission” shown in the figure) and the serum samples of the healthy subjects (HC), no band of the first APOA2-like protein showing binding to the anti-APOA2 antibody was confirmed (level 0).

[0071] As described above, it was found that the first APOA2-like protein (VAP2), which is useful as a pathological marker of AAV, can be detected even by using an anti-APOA2 antibody.

[0072] [Example 8: Confirmation of the Presence of Anti-APOA2 Antibody Binding Molecules in Serum Samples from COVID-19 Patients] In the experiment using serum samples collected from the above-mentioned COVID-19 patients, in order to confirm whether the target molecule (first APOA2-like protein; VAP2) bound by VasSF becomes the target of the anti-APOA2 antibody, the anti-APOA2 antibody was used to perform signal analysis by the same immunoblotting method as in the above-mentioned Example 4. The results are shown in Figure 6 .

[0073] like Figure 6 As shown, during the active period of COVID-19 (at admission; Figure 6 In the serum samples of patients with ICU (shown as "A"), a strong band of a protein with a molecular weight of about 24 kDa (first APOA2-like protein; VAP2) was confirmed even when anti-APOA2 antibodies were used. Figure 6 "U" shown) and at discharge ( Figure 6 In the serum sample of the patient (shown as "D"), no band of the first APOA2-like protein was confirmed (level 0).

[0074] In summary, it was found that the first APOA2-like protein (VAP2), which is useful as a pathological marker of COVID-19, can be detected even using an anti-APOA2 antibody.

[0075] [Example 9: Relationship between the signal value of anti-APOA2 antibody binding molecules (first APOA2-like protein) in serum samples of COVID-19 patients and the pathological state] The relationship between the signal value of the anti-APOA2 antibody binding molecule and the pathological state of COVID-19 was studied using the same method as in Example 6 above for serum samples collected from 65 COVID-19 patients at different time points (at hospitalization, ICU transfer, and discharge). The results are shown in Figure 7 It should be noted that Figure 7 The values ​​and error bars of the bar graphs shown represent the mean ± standard error (SE).

[0076] like Figure 7 As shown, during the active period of COVID-19 (at admission; Figure 7 In the serum sample of the patient with the disease "A"), the signal value of the anti-APOA2 antibody binding molecule (first APOA2-like protein; VAP2) showed a high level. On the other hand, during the ICU transfer ( Figure 7 The signal value was significantly reduced at discharge ( Figure 7 It should be noted that in this experiment, the signal scores of patients who died at discharge and transferred to other hospitals shortly after hospitalization remained at the same high level as when they were hospitalized (these results are not included in the Figure 7 middle).

[0077] In summary, it was found that the first APOA2-like protein (VAP2), which is useful as a pathological marker of COVID-19, can be detected even using an anti-APOA2 antibody.

[0078] [Example 10: Signal Analysis of VasSF Binding Molecules (First APOA2-Like Protein) in Serum Samples Collected from Kawasaki Disease Patients] Using serum samples collected from a child with Kawasaki disease at different time points (sixth, tenth, and thirtieth days from onset), the signal analysis of VasSF binding molecules was performed. The results are shown in Figure 8 .

[0079] like Figure 8 As shown, a strong band of a protein with a molecular weight of about 24 kDa (first APOA2-like protein; VAP2) was confirmed in serum samples of patients in the active phase of Kawasaki disease (acute phase; sixth day from onset). On the other hand, in serum samples collected after the disease was stabilized by treatment (tenth and thirtieth days from onset), no band of the first APOA2-like protein was confirmed (0 level). Figure 8 "M" shown is a molecular weight marker, "Std" is a standard sample of VAP2, and "HC" is a sample of a healthy person.

[0080] In conclusion, the first APOA2-like protein (VAP2) is useful as a pathological marker for Kawasaki disease, and the protein can be detected using VasSF.

[0081] [Example 11: Signal analysis of VasSF-binding molecules (first APOA2-like protein) in serum samples collected from COVID-19 patients (2)] Using serum samples collected from 5 COVID-19 patients at different time points (at hospitalization and at discharge), the signal analysis of VasSF binding molecules was performed in the same manner as in Example 4. The results are shown in Fig. 9 .

[0082] like Fig. 9 As shown, during the active period of COVID-19 (at admission; Fig. 9 In the serum sample of the patient with the disease "A"), a strong band of a protein with a molecular weight of about 24 kDa (first APOA2-like protein; VAP2) was confirmed as in Example 4. On the other hand, at the time of discharge ( Fig. 9 In the serum sample of the patient "D"), no band of the first APOA2-like protein was confirmed (0 level) as in Example 4. However, at the time of discharge ( Fig. 9 In the serum sample of the patient (shown as "D"), a strong band of a protein (second APOA2-like protein) with a molecular weight of about 19 / 17 kDa, which is different from the first APOA2-like protein, was confirmed.

[0083] In summary, the above-mentioned first APOA2-like protein (molecular weight of about 24 kDa) and the second APOA2-like protein (molecular weight of about 19 / 17 kDa) are both useful as pathological markers of COVID-19, and the proteins can also be detected using VasSF.

[0084] [Example 12: Two-dimensional electrophoresis and two-dimensional immunoblotting using HRP-VasSF monomer and HRP-anti-APOA2 antibody] AG (-) proteins were prepared from the serum of one patient in the active phase of ANCA-associated vasculitis (AAV) and one patient in the remission phase, after albumin and IgG were removed, and were subjected to two-dimensional electrophoresis (~10 μg / lane) under non-reducing conditions. Two-dimensional electrophoresis can separate proteins based on molecular weight and isoelectric point. At this time, Precision Plus stained protein standard (manufactured by Bio-Rad) was used as a molecular weight marker.

[0085] After transferring the proteins after two-dimensional electrophoresis to a PVDF membrane, all proteins on the membrane were visualized using SYPRO Ruby blot stain (manufactured by ThermoFisher Scientific). Then, the membrane was treated with HRP-conjugated VasSF monomer or HRP-conjugated anti-APOA2 antibody and further treated with a chemiluminescent substrate. Chemiluminescent images were captured using LAS-3000 (manufactured by Fujifilm Corporation).

[0086] Fig. 10A This is a photograph showing the results of two-dimensional electrophoresis using serum from patients in the active phase and processed using VasSF. Fig. 10A As shown, a protein (first APOA2-like protein) with a molecular weight of about 24 kDa and an isoelectric point (pI) of about 6.3 was confirmed in the serum of patients in the active stage. It should be noted that the same results were obtained when an anti-APOA2 antibody was used instead of VasSF.

[0087] Fig. 10B This is a photograph showing the results of two-dimensional electrophoresis using serum from patients in remission and processed using VasSF. Fig. 10B As shown, a protein (second APOA2-like protein) with a molecular weight of about 17 kDa and an isoelectric point (pI) of about 5.5 was confirmed in the serum of patients in the remission period. It should be noted that the same results were obtained when an anti-APOA2 antibody was used instead of VasSF.

[0088] In summary, the above-mentioned first APOA2-like protein (molecular weight of about 24 kDa, isoelectric point of about 6.3) and the second APOA2-like protein (molecular weight of about 17 kDa, isoelectric point of about 5.5) are both useful as pathological markers of COVID-19, and these proteins can also be detected using VasSF.

[0089] The above series of results indicate that the first APOA2-like protein (VAP2), which is present at high levels in the serum of patients with active diseases accompanied by vasculitis and vascular inflammation such as ANCA-associated vasculitis, COVID-19, and Kawasaki disease, and disappears after symptoms improve, is useful as a pathological marker for these diseases. In addition, it was shown that the second APOA2-like protein, which is not found in the serum of the active stage of the above diseases but is present at high levels in the serum after symptoms improve, is also useful as a pathological marker for each of the above diseases.

[0090] In addition, it can be said that VasSF and anti-APOA2 antibodies that can detect and quantify the first APOA2-like protein and the second APOA2-like protein that show increases and decreases with the course of the above-mentioned diseases are also useful as concomitant diagnostic reagents for the state of vasculitis used in a method for examining the state of vasculitis using the above-mentioned proteins as markers.

[0091] Furthermore, there are reports that vasculitis is involved in the aggravation of COVID-19, so it can be said that the first APOA2-like protein (VAP2) and the second APOA2-like protein are the same as vasculitis such as Kawasaki disease mentioned above, and are also useful as markers for examining the risk of aggravation of COVID-19.

[0092] [unordered list text] The sequence numbers in the sequence listing of the present application indicate the following sequences.

[0093] [Serial No. 1] The figure shows the amino acid sequence of one single-chain variable region fragment (ScFv) (VasSF) that binds to the first APOA2-like protein and the second APOA2-like protein.

[0094] [Serial No. 2] This figure shows the base sequence of the DNA encoding VasSF (CDS; including the stop codon).

Claims

1. A method for examining the state of vasculitis, characterized in that: include: The following proteins are detected or quantified in a blood sample collected from a subject: (1) a first APOA2-like protein having a molecular weight of 23 to 25 kDa that undergoes an antigen-antibody reaction with a single-chain variable region fragment comprising the amino acid sequence of SEQ ID NO: 1, and / or (2) A second APOA2-like protein that undergoes an antigen-antibody reaction with the single-chain variable region fragment comprising the amino acid sequence shown in SEQ ID NO: 1 and has a molecular weight of 16 to 20 kDa.

2. The method according to claim 1, wherein: The vasculitis is vasculitis associated with COVID-19, ANCA-associated vasculitis or Kawasaki disease.

3. The method according to claim 1 or 2, wherein: The isoelectric point pI of the first APOA2-like protein is 6.0-6.6, and the isoelectric point pI of the second APOA2-like protein is 5.2-5.

8.

4. A method for examining the risk of severe symptoms of COVID-19, characterized in that: include: The following proteins are detected or quantified in a blood sample collected from a subject: (1) a first APOA2-like protein having a molecular weight of 23 to 25 kDa that undergoes an antigen-antibody reaction with a single-chain variable region fragment comprising the amino acid sequence of SEQ ID NO: 1, and / or (2) A second APOA2-like protein that undergoes an antigen-antibody reaction with the single-chain variable region fragment comprising the amino acid sequence shown in SEQ ID NO: 1 and has a molecular weight of 16 to 20 kDa.

5. The method according to claim 4, wherein: The isoelectric point pI of the first APOA2-like protein is 6.0-6.6, and the isoelectric point pI of the second APOA2-like protein is 5.2-5.

8.

6. The method according to claim 1 or 4, wherein: The method includes detecting or quantifying the first APOA2-like protein and the second APOA2-like protein.

7. The method according to claim 1 or 4, wherein: The blood sample is plasma or serum.

8. A kit for testing the state of suffering from vasculitis, characterized in that: include: A single-chain variable region fragment or an anti-apolipoprotein A2 antibody comprising the amino acid sequence shown in SEQ ID NO:

1.

9. The test kit according to claim 8, wherein: The test kit is used for the method according to claim 1.

10. A kit for testing the risk of severe symptoms of COVID-19, characterized in that: include: A single-chain variable region fragment or an anti-apolipoprotein A2 antibody comprising the amino acid sequence shown in SEQ ID NO:

1.

11. The test kit according to claim 10, wherein: The test kit is used for the method according to claim 4.

12. A companion diagnostic reagent, characterized in that: A companion diagnostic reagent for the vasculitis status used in the method of claim 1, wherein the companion diagnostic reagent comprises: a single-chain variable region fragment comprising the amino acid sequence shown in sequence number 1 or an anti-apolipoprotein A2 antibody.

13. The companion diagnostic reagent according to claim 12, wherein: The companion diagnostic reagent is used to select a treatment corresponding to the state of vasculitis determined using the method of claim 1, and / or the companion diagnostic reagent is used to select a therapeutic or preventive drug for vasculitis corresponding to the state of vasculitis.

14. A companion diagnostic reagent, characterized in that: A companion diagnostic reagent for the risk of severe COVID-19 used in the method of claim 4, wherein the companion diagnostic reagent comprises: a single-chain variable region fragment comprising the amino acid sequence shown in sequence number 1 or an anti-apolipoprotein A2 antibody.

15. The companion diagnostic reagent according to claim 14, wherein: The companion diagnostic reagent is used to select a treatment corresponding to the risk of severe COVID-19 determined using the method of claim 4, and / or the companion diagnostic reagent is used to select a therapeutic or preventive drug for vasculitis corresponding to the condition.

16. A marker for testing the risk of severe symptoms of COVID-19, characterized in that: The invention comprises: (1) a first APOA2-like protein which undergoes an antigen-antibody reaction with a single-chain variable region fragment comprising the amino acid sequence shown in SEQ ID NO: 1 and has a molecular weight of 23 to 25 kDa.

17. The inspection marker according to claim 16, wherein: The isoelectric point pI of the first APOA2-like protein is 6.0-6.

6.

18. A marker for testing the risk of severe symptoms of COVID-19, characterized in that: The invention comprises: (2) a second APOA2-like protein which undergoes an antigen-antibody reaction with the single-chain variable region fragment comprising the amino acid sequence shown in SEQ ID NO: 1 and has a molecular weight of 16 to 20 kDa.

19. The inspection marker according to claim 18, wherein The isoelectric point pI of the second APOA2-like protein is 5.2-5.8.

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