Biomarkers for diagnosing desquamative interstitial pneumonia

Abstract

To provide biomarkers for diagnosing desquamative interstitial pneumonia and methods using them. [Solution] A biomarker for determining exfoliative interstitial pneumonia, containing an anti-annexin A autoantibody.

Description

Technical Field

[0001] The present invention relates to a biomarker for diagnosing desquamative interstitial pneumonia. More specifically, the present invention relates to a biomarker for diagnosing desquamative interstitial pneumonia, which includes anti-annexin A autoantibody, and a method for assisting the diagnosis of desquamative interstitial pneumonia, which includes a step of detecting the biomarker in a subject, and the like.

Background Art

[0002] Desquamative interstitial pneumonia (DIP) is a type of idiopathic interstitial pneumonia (IIPs) of unknown cause, and is considered a disease associated with smoking. According to current guidelines, the diagnosis of DIP requires integrating the medical history, radiology, bronchoalveolar lavage, and pathological findings, and needs to be performed by multiple doctors and the like who specialize in different fields. However, due to insufficient understanding of this disease, good consensus criteria do not yet exist, and there is a risk that DIP is not fully recognized in clinical practice (Non-Patent Document 1). Further, to the knowledge of the present inventors, biomarkers for differentiating DIP have not been reported.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] Accordingly, the present invention aims to provide a biomarker for determining desquamative interstitial pneumonia. More specifically, the biomarker of the present invention includes an anti-annexin A autoantibody. The present invention also aims to provide a method for assisting in the diagnosis of desquamative interstitial pneumonia, which includes a step of detecting the above biomarker in a subject. [Means for solving the problem]

[0005] Based on their experience, the inventors have found that DIP patients also suffer from autoimmune diseases such as systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus. From this experience, they focused on autoantibodies, particularly anti-annexin family protein antibodies. Through diligent research based on this focus, they discovered that anti-annexin family protein antibodies can be used as biomarkers to determine (differentiate) whether a subject has DIP. Based on these findings, the inventors conducted further research, ultimately completing the present invention.

[0006] In other words, the present invention is as follows: [1] A biomarker for diagnosing exfoliative interstitial pneumonia, including anti-annexin A autoantibodies. [2] The biomarker according to [1], wherein the annexin A is at least one selected from the group consisting of annexin A4, annexin A5, and annexin A11. [3] The biomarker according to [2], wherein the annexin A is annexin A4. [4] A method for assisting the diagnosis of exfoliative interstitial pneumonia, comprising the step of detecting a biomarker described in any one of [1] to [3] in the subject. [5] The method according to [4], wherein the subject is suffering from or may be suffering from a lung disease and / or collagen disease. [6] The method according to [5], wherein the subject is suffering from or may be suffering from idiopathic interstitial pneumonia. [7] (1) A step of measuring the amount of a biomarker described in any one of [1] to [3] in the subject, and (2) A step of comparing the value measured in step (1) with a predetermined threshold. A method that includes any one of [4] to [6]. [8] The method according to [7], wherein if the value measured in step (1) exceeds a predetermined threshold, the patient is determined to have desquamative interstitial pneumonia. [9] A kit for determining exfoliative interstitial pneumonia, comprising at least one selected from a protein belonging to annexin A, an antigenic partial peptide thereof, and a peptide containing an epitope recognized by an anti-annexin A autoantibody.

[10] The kit according to [9], wherein the protein belonging to annexin A is at least one selected from the group consisting of annexin A4, annexin A5, and annexin A11.

[11] The kit according to

[10] , wherein the protein belonging to annexin A is annexin A4.

[12] (1) A step of measuring the amount of a biomarker described in any one of [1] to [3] in the subject, (2) A step of comparing the value measured in step (1) with a predetermined threshold, and (3) If the value measured in step (1) exceeds a predetermined threshold, the subject is administered a drug for the treatment or prevention of desquamative interstitial pneumonia. A method for treating or preventing desquamative interstitial pneumonia, including [specific example]. [Effects of the Invention]

[0007] The present invention provides a biomarker for determining desquamative interstitial pneumonia, specifically an anti-annexin A autoantibody. The present invention also provides a method for assisting in the diagnosis of desquamative interstitial pneumonia, which includes a step of detecting the biomarker in a subject. It is expected that the biomarker and diagnostic assistance method using the present invention will simplify and facilitate the differentiation (definitive diagnosis) of DIP, which previously required multiple tests. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 shows the international classification of idiopathic interstitial pneumonia. Desquamative interstitial pneumonia is classified as a smoking-related type among the major types of idiopathic (of unknown cause) interstitial pneumonia (adapted from Travis WD, et al. Am J Respir Crit Care Med 2013; 188: 733-748). [Figure 2] Figure 2 shows typical results of serum immunoprecipitation analysis in identifying target proteins recognized by autoantibodies in DIP subjects (patients). [Figure 3] Figure 3 shows the levels of anti-ANXA antibodies in each IIP (specifically, DIP, idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonia (iNSIP), and idiopathic organizing pneumonia (COP)), collagen disease-associated interstitial lung disease (CTD-ILD), lung disease without ILD (Non-ILD), and healthy control (HC) subjects (patients). [Figure 4] Figure 4 shows the results of immunohistochemical staining for ANXA4 antigen in representative lung specimens from DIP, IPF, and iNSIP subjects (patients). [Figure 5] Figure 5 shows a comparison of serum anti-ANXA4 antibody levels in patients with DIP and patients with non-DIP idiopathic interstitial pneumonia (non-DIP IIPs). [Figure 6] Figure 6 shows a comparison of serum anti-ANXA5 antibody levels in DIP patients and non-DIP idiopathic interstitial pneumonia (non-DIP IIPs) patients. [Figure 7] Figure 7 shows the comparison of the values of anti-ANXA11 antibody in serum between DIP subjects (patients) and non-DIP idiopathic interstitial pneumonia (non-DIP IIPs) subjects (patients). [Figure 8] Figure 8 shows the results of creating ROC curves using anti-ANXA4 antibody, anti-ANXA5 antibody, and anti-ANXA11 antibody as markers for differentiating DIP subjects (patients) and non-DIP idiopathic interstitial pneumonia. [Figure 9] Figure 9 shows the measured values of anti-ANXA4 antibody in DIP, non-DIP IIPs, CTD-ILD, collagen diseases without interstitial lung disease (CTD w / o ILD), lung diseases other than interstitial lung disease (PUL; chronic obstructive pulmonary disease, lung cancer, pneumonia), and healthy controls (HV). [Figure 10] Figure 10 shows the measured values of anti-ANXA5 antibody in DIP, non-DIP IIPs, CTD-ILD, collagen diseases without interstitial lung disease (CTD w / o ILD), lung diseases other than interstitial lung disease (PUL; chronic obstructive pulmonary disease, lung cancer, pneumonia), and healthy controls (HV). [Figure 11] Figure 11 shows the measured values of anti-ANXA11 antibody in DIP, non-DIP IIPs, CTD-ILD, collagen diseases without interstitial lung disease (CTD w / o ILD), lung diseases other than interstitial lung disease (PUL; chronic obstructive pulmonary disease, lung cancer, pneumonia), and healthy controls (HV). [Figure 12] Figure 12 shows the positive rates of the diseases shown in Figures 9 to 11 when using the cut-off values in Table 1 described below.

Mode for Carrying Out the Invention

[0009] 1. Biomarker of the present invention The present invention provides a biomarker for determining exfoliative interstitial pneumonia (hereinafter also referred to as "the biomarker of the present invention"), which includes an anti-annexin A autoantibody. In this specification, "annexin A" refers to a protein belonging to the annexin family. In this specification, proteins belonging to the annexin family and proteins belonging to annexin A are used interchangeably. Specific examples of proteins belonging to the annexin family include, for example, annexin A1 (ANXA1), annexin A2 (ANXA2), annexin A3 (ANXA3), annexin A4 (ANXA4), annexin A5 (ANXA5), annexin A6 (ANXA6), annexin A7 (ANXA7), annexin A8 (ANXA8), annexin A9 (ANXA9), annexin A10 (ANXA10), annexin A11 (ANXA11), and annexin A13 (ANXA13).

[0010] In this specification, "anti-annexin A autoantibody" refers to an autoantibody that specifically recognizes proteins belonging to annexin A. Specifically, examples of such autoantibodies include anti-annexin A1 (ANXA1) antibody, anti-annexin A2 (ANXA2) antibody, anti-annexin A3 (ANXA3) antibody, anti-annexin A3 (ANXA3) antibody, anti-annexin A4 (ANXA4) antibody, anti-annexin A5 (ANXA5) antibody, anti-annexin A6 (ANXA6) antibody, anti-annexin A7 (ANXA7) antibody, anti-annexin A8 (ANXA8) antibody, anti-annexin A9 (ANXA9) antibody, anti-annexin A10 (ANXA10) antibody, anti-annexin A11 (ANXA11) antibody, and anti-annexin A13 (ANXA13) antibody. In one embodiment, the anti-annexin A autoantibody is an anti-annexin A4 (ANXA4) antibody, an anti-annexin A5 (ANXA5) antibody, or an anti-annexin A11 (ANXA11) antibody. In another embodiment, the anti-annexin A autoantibody is an anti-annexin A4 (ANXA4) antibody, or an anti-annexin A5 (ANXA5) antibody. In yet another embodiment, the anti-annexin A autoantibody is an anti-annexin A4 (ANXA4) antibody.

[0011] In this specification, "desquamative interstitial pneumonia (DIP)" refers to a type of idiopathic interstitial pneumonia (IIPs) that is associated with smoking, and is classified as, for example, Travis WD, et al. Am J Respir Crit Care Med 2013; 188: 733-748.

[0012] In this specification, "determining desquamative interstitial pneumonia" means determining whether or not a subject has desquamative interstitial pneumonia. Determining whether or not a subject has desquamative interstitial pneumonia includes not only determining whether or not a subject has desquamative interstitial pneumonia, but also evaluating or calculating the possibility or probability of having desquamative interstitial pneumonia or idiopathic interstitial pneumonia of non-DIP, which is useful in diagnosing idiopathic interstitial pneumonia of desquamative or non-diponic interstitial pneumonia (Non-DIP IIPs).

[0013] 2. Diagnostic assistance method of the present invention The present invention provides a method for assisting the diagnosis of exfoliative interstitial pneumonia (hereinafter also referred to as "the diagnostic assistance method of the present invention"), which includes a step of detecting the biomarker of the present invention in a subject. The contents of "1. Biomarker of the present invention" are entirely incorporated herein by reference.

[0014] In the present invention, the subjects are not particularly limited and may be, for example, healthy subjects or non-healthy subjects. Non-healthy subjects include, for example, subjects whose health check results exceeded the standard values, subjects with (specific) chronic diseases, subjects who have or may have lung diseases and / or collagen diseases, subjects who have or may have idiopathic interstitial pneumonia, etc. Examples of lung diseases include idiopathic interstitial pneumonia, collagen disease-associated interstitial lung disease (CTD-ILD), pneumonia, lung cancer, COPD, etc. Examples of collagen diseases include collagen diseases without interstitial lung disease (CTD w / o ILD), etc.

[0015] In this invention, the subject is not limited to humans, but to mammals in general. Examples of such mammals include rodents (e.g., mice, rats, guinea pigs, gerbils, hamsters, etc.), primates (e.g., rhesus monkeys, crab-eating macaques, Japanese macaques, chimpanzees, etc.), laboratory animals such as ferrets, rabbits, dogs, and miniature pigs, pet animals such as dogs and cats, and livestock such as cows, horses, pigs, and sheep.

[0016] The detection of the biomarker of the present invention in a subject can be performed using a biological sample derived from the subject. The biological sample is not particularly limited as long as it is collected from the subject and may contain the anti-annexin A autoantibody, which is the biomarker of the present invention. Examples of biological samples include body fluids such as blood, plasma, serum, lymph, cerebrospinal fluid, ascites, synovial fluid, urine, sweat, saliva, bronchoalveolar lavage fluid, or fractions thereof, and serum is preferred. The sample may undergo known pretreatment (e.g., purification) or storage (including freezing and thawing) before detecting the anti-annexin A autoantibody. Furthermore, the detection of the biomarker of the present invention in a subject may be quantitative or qualitative. In one embodiment, the detection involves measuring the amount of the biomarker present.

[0017] Detection of the anti-annexin A autoantibody, which is the biomarker of the present invention, in the above-mentioned biological sample can be performed by immunological assays (e.g., immunoprecipitation, ELISA, FIA, RIA, Western blotting, etc.) using, for example, a protein belonging to annexin A, an antigenic partial peptide thereof, a peptide containing an epitope recognized by the anti-annexin A autoantibody, or an antibody that binds to the anti-annexin A autoantibody. The protein belonging to annexin A, an antigenic partial peptide thereof, a peptide containing an epitope recognized by the anti-annexin A autoantibody, or an antibody that binds to the anti-annexin A autoantibody used for detection of the anti-annexin A autoantibody may be isolated or purified.

[0018] The proteins belonging to annexin A used for detection, their antigenic partial peptides, peptides containing epitopes recognized by anti-annexin A autoantibodies, and antibodies that bind to anti-annexin A autoantibodies may have tag peptides attached to them. Examples of tag peptides include, but are not limited to, epitope tags (e.g., Flag, polyhistidine, c-Myc tag, HA, AU1, GST, MBP, etc.), fluorescent proteins, and immunoglobulin Fc regions.

[0019] The proteins belonging to annexin A used for detection, the antigenic partial peptides thereof, peptides containing epitopes recognized by anti-annexin A autoantibodies, antibodies that bind to anti-annexin A autoantibodies, etc., may be labeled with appropriate labeling agents (e.g., biotin, enzymes, fluorescent substances, luminescent substances, etc.).

[0020] When applying individual immunological detection or quantification methods to the diagnostic support methods of the present invention, no special conditions or procedures are required. The detection or quantification system for the biomarkers of the present invention can be constructed by adding the usual technical considerations of those skilled in the art to the usual conditions and procedures of each method. Details of these general technical means can be found in reviews, textbooks, etc. For example, "Radioimmunoassay" edited by Hiroshi Irie (Kodansha, published 1974), "Continued Radioimmunoassay" edited by Hiroshi Irie (Kodansha, published 1979), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (Igaku Shoin, published 1978), "Enzyme Immunoassay" (2nd edition) edited by Eiji Ishikawa et al. (Igaku Shoin, published 1982), "Enzyme Immunoassay" (3rd edition) edited by Eiji Ishikawa et al. (Igaku Shoin, published 1987), "Methods in ENZYMOLOGY" Vol. 70 (Immunochemical Techniques (Part A)), Vol. 73 (Immunochemical Techniques (Part B)), Vol. 74 (Immunochemical Techniques (Part C)), Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), Vol. 92 (Immunochemical You can refer to Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods) and Vol. 121 of the same book (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (both published by Academic Press).

[0021] In this specification, "assisting in diagnosis" means providing information that serves as an indicator for determining whether or not the subject has desquamative interstitial pneumonia, and does not include the process of diagnosing whether or not the subject has desquamative interstitial pneumonia, which is a medical act.

[0022] In the diagnostic support method of the present invention, the determination of whether or not a subject has desquamative interstitial pneumonia is typically made by setting a threshold for the amount of the biomarker of the present invention in advance, comparing the measured amount of the biomarker with the threshold, and determining whether or not the subject has desquamative interstitial pneumonia based on whether it is above or below the threshold, or whether the possibility of having desquamative interstitial pneumonia can be evaluated. In one embodiment, if the measured amount of the biomarker exceeds a predetermined threshold, it is determined that the subject has desquamative interstitial pneumonia.

[0023] The threshold (cutoff value) described above is a value that satisfies both high diagnostic sensitivity (accuracy in diagnosing prevalence) and high diagnostic specificity (accuracy in diagnosing absence of disease) when the disease is diagnosed based on that value. For example, a threshold (cutoff value) can be set that shows a high positive rate in the population of individuals with desquamative interstitial pneumonia and a high negative rate in the population of individuals who do not have desquamative interstitial pneumonia (non-desquamative interstitial pneumonia) (for example, a population of individuals with lung disease and / or collagen disease but not desquamative interstitial pneumonia, or a population of individuals with idiopathic interstitial pneumonia but not desquamative interstitial pneumonia (idiopathic interstitial pneumonia that is not desquamative interstitial pneumonia)).

[0024] Methods for calculating cutoff values ​​are well known in this field. For example, the abundance of each biomarker of the present invention in biological samples derived from the aforementioned population can be measured or quantified, and the diagnostic sensitivity and diagnostic specificity at the measured or quantified values ​​can be determined. Based on these values, an ROC (Receiver Operating Characteristic) curve can be created using analysis software (e.g., statistical analysis software JMP, statistical analysis software R, and the scikit-learn package for Python). The value at which the diagnostic sensitivity and diagnostic specificity are as close to 100% as possible can then be determined and set as the threshold value. When using two or more biomarkers of the present invention, or when using one or more biomarkers of the present invention and other biomarkers (e.g., the subject's sex, age, amount of smoking, duration of smoking, etc.), the abundance of each biomarker can be combined to create an ROC curve.

[0025] In the diagnostic support method of the present invention, for example, the threshold (cutoff value) for distinguishing between desquamative interstitial pneumonia and idiopathic interstitial pneumonia that is not desquamative interstitial pneumonia is as shown in Table 1 below. If the value exceeds the cutoff value shown in Table 1, the subject may be determined to have desquamative interstitial pneumonia. Note that the cutoff values ​​in Table 1 may vary depending on the biological sample and the measurement conditions of the biomarker of the present invention, and are not limited to these values.

[0026] [Table 1]

[0027] 3. The treatment method of the present invention If, as a result of the method described in "2. Diagnostic support method of the present invention" above, it is determined that the subject has desquamative interstitial pneumonia (or is likely to have desquamative interstitial pneumonia), then, based on the result of said determination, a therapeutic drug for desquamative interstitial pneumonia to be administered to the subject can be selected or determined, and the subject can be treated by administering a therapeutically effective amount of the therapeutic drug. The present invention also provides such a treatment method (hereinafter also referred to as "the treatment method of the present invention"). In this specification, "treatment" includes alleviation or improvement of symptoms, prevention, delay or cessation of the progression of the disease or symptoms, or the manifestation of symptoms. In this specification, "therapeutic drug" includes not only pharmaceuticals aimed at curative treatment of desquamative interstitial pneumonia, but also, for example, pharmaceuticals aimed at suppressing the progression of the disease or pharmaceuticals aimed at alleviating symptoms.

[0028] The specific treatment method (treatment or preventive method) of the present invention provided is as follows: (1) A step of measuring the amount of the biomarker of the present invention in the subject, (2) A step of comparing the value measured in step (1) with a predetermined threshold, and (3) If the value measured in step (1) exceeds a predetermined threshold, the subject is administered a drug for treating or preventing desquamative interstitial pneumonia. Includes.

[0029] Treatment options for desquamative interstitial pneumonia include corticosteroids and immunomodulatory drugs. Examples of corticosteroids include prednisolone, methylprednisolone, hydrocortisone, betamethasone, and dexamethasone. Examples of immunomodulatory drugs include cyclophosphamide, azathioprine, methotrexate, cyclosporine, and tacrolimus. Other commonly used medications are also acceptable and are not limited to those listed above. These medications may be used in combination as appropriate, depending on the symptoms and other factors.

[0030] The above-mentioned therapeutic agents may be administered orally or parenterally as a pharmaceutical composition in an appropriate dosage form, either by mixing the active ingredient alone or with a pharmaceutically acceptable carrier, excipient, diluent, etc. Compositions for oral administration may be in solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. On the other hand, compositions for parenteral administration may include, for example, injections and suppositories, and injections may include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip infusions. Furthermore, the dosage of the therapeutic agent can be appropriately determined based on various conditions such as the type of compound, the symptoms of the target patient, age, weight, and drug tolerance.

[0031] The therapeutic method of the present invention described above utilizes all the contents of "1. Biomarker of the present invention" and "2. Diagnostic support method of the present invention" described above.

[0032] 4. The present invention kit The present invention provides a kit for determining exfoliative interstitial pneumonia (hereinafter also referred to as "the kit of the present invention"), comprising at least one selected from a protein belonging to annexin A, an antigenic partial peptide thereof, and a peptide containing an epitope recognized by an anti-annexin A autoantibody. The kit of the present invention may also include the biomarker of the present invention as a positive control.

[0033] Specifically, the proteins belonging to annexin A included in the kit of the present invention include, for example, annexin A1 (ANXA1), annexin A2 (ANXA2), annexin A3 (ANXA3), annexin A4 (ANXA4), annexin A5 (ANXA5), annexin A6 (ANXA6), annexin A7 (ANXA7), annexin A8 (ANXA8), annexin A9 (ANXA9), annexin A10 (ANXA10), annexin A11 (ANXA11), and annexin A13 (ANXA13). In one embodiment, the proteins belonging to annexin A included in the kit of the present invention are annexin A4 (ANXA4), annexin A5 (ANXA5), or annexin A11 (ANXA11). In one embodiment, the protein belonging to annexin A included in the kit of the present invention is annexin A4 (ANXA4) or annexin A5 (ANXA5). In a further embodiment, the protein belonging to annexin A is annexin A4 (ANXA4).

[0034] The proteins belonging to annexin A, their antigenic partial peptides, or peptides containing epitopes recognized by anti-annexin A autoantibodies included in the kit of the present invention may or may not be isolated or purified. Furthermore, the proteins belonging to annexin A, their antigenic partial peptides, or peptides containing epitopes recognized by anti-annexin A autoantibodies included in the kit of the present invention may be immobilized on a solid phase. The solid phase that can be used in the kit of the present invention is not particularly limited, but examples include polymers such as polystyrene, glass beads, magnetic particles, microplates, immunochromatographic filter paper, and glass filters, which are insoluble carriers.

[0035] The kit of the present invention may include, in addition to proteins belonging to annexin A, antigenic partial peptides thereof, or epitopes recognized by anti-annexin A autoantibodies, other reagents. These reagents may be pre-mixed with the proteins belonging to annexin A, antigenic partial peptides thereof, or epitopes recognized by anti-annexin A autoantibodies, or they may be stored in separate containers. Other reagents include secondary antibodies for detecting anti-annexin A autoantibodies bound to proteins belonging to annexin A, antigenic partial peptides thereof, or epitopes recognized by anti-annexin A autoantibodies (e.g., anti-human IgG labeled with enzymes such as peroxidase or alkaline phosphatase, or fluorescent dyes), blocking solutions, substrates, solid phases, reaction vessels, as well as processing solutions, buffers, positive controls, negative controls, and instructions describing the protocol. These elements may be pre-mixed as needed.

[0036] The above-described kit of the present invention incorporates all of the contents of "1. Biomarker of the present invention," "2. Diagnostic support method of the present invention," and "3. Treatment method of the present invention" described above.

[0037] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0038] Example 1: Identification and statistical analysis of autoantibodies Experimental materials and methods (1) Subjects (patients) Nineteen DIP patients who visited Nagasaki University Hospital, Public Tosei Hospital, Oita University Hospital, Miyazaki University Hospital, Fukuoka University Hospital, and Fukuoka University Chikushi Hospital between 2001 and 2020 were retrospectively enrolled. To compare the specificity of anti-ANXA antibodies, 73 other subtypes of idiopathic interstitial pneumonia (IIPs), 51 CTD-ILD cases, 52 non-ILD cases (20 chronic obstructive pulmonary disease, 16 bacterial pneumonia cases, 16 lung cancer cases), and 15 healthy controls (HC) who visited Nagasaki University Hospital between 2008 and 2023 were included. Among the IIPs cases, 23 cases of idiopathic pulmonary fibrosis (IPF), 15 cases of idiopathic nonspecific interstitial pneumonia (iNSIP), and 5 cases of idiopathic organizing pneumonia (COP) were pathologically diagnosed by surgical lung biopsy. Other IPF cases showing an interstitial pneumonia pattern of typical interstitial pneumonia (UIP) on high-resolution CT were diagnosed according to IPF guidelines without surgical lung biopsy, while other COP cases showing typical high-resolution CT findings were pathologically diagnosed by transbronchial lung biopsy according to the IIP classification. All data, including smoking history, dust exposure, interstitial pneumonia markers, bronchoalveolar lavage fluid (BALF) findings, pulmonary function test results, and post-diagnosis treatment, were obtained from medical records. Positive antinuclear antibody (ANA) was defined as ANA with a titer of at least 1:40. The study protocol was approved by the Institutional Review Board of Human Ethics at Nagasaki University Hospital, and all participants provided written informed consent before enrollment.

[0039] (2) Detection of autoantibodies in serum Immunoprecipitation Serum samples from the subjects (patients) were collected at the initial consultation and stored at -20°C until the next use. In the analysis of autoantibodies, 35 K562 cell extracts radiolabeled with S-methionine and cysteine ​​were immunoprecipitated with IgG purified from 8 μl of human serum sample using a method known to the present day (Satoh M et al., J Clin Invest. 1996;97(11):2619-2626 and Tahara M et al., Scientific reports. 2022;12(1):11122). 35Protein A was labeled with S-methionine and cysteine, dissolved in 0.3% octylphenyl polyethylene glycol (IGEPAL CA-630) buffer containing 0.5 M NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris (pH 7.5), 0.5 mM phenylmethylsulfonyl fluoride, and 0.3 trypsin inhibitory units (TIU) / ml aprotinin, and immunoprecipitated using IgG-coated Protein A Sepharose beads. The immunoprecipitated product was then washed with 0.5 M NaCl-NET / IGEPAL CA-630, and the immunoprecipitated protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography.

[0040] (3) Mass spectrometry Autoantigens bound to autoantibodies of the subject (patient) immobilized on Protein A-Sepharose beads were recovered by adding trifluoroacetic acid to dissociate the antibody-antigen bond. The supernatant was transferred to another tube, 100 μl of 10 mM dithiothreitol was added, and the sample was incubated further at 56°C for 45 minutes. Then, iodoacetamide (100 μl, 55 mM) was added to the mixture and incubated at room temperature in the dark for 30 minutes. Trypsin in 0.05% acetic acid was added to a final concentration of 4 mg / l, and the mixture was incubated overnight at 37°C. Next, trifluoroacetic acid was added and mixed to stop degradation. The peptide mixture was analyzed using a custom nano LC-electrospray ionization-MS / MS system (LTQ-XL, Thermo Fisher Scientific). MS / MS data were extracted using Proteome Discoverer v.3.3 (Thermo Fisher Scientific) and searched against the human protein database (UniProt).

[0041] (4) ELISA for anti-annexin antibodies For the detection of anti-annexin A (ANXA) antibodies, recombinant proteins containing human ANXA1, ANXA2, ANXA3, ANXA4, ANXA5, ANXA6, ANXA7, ANXA8, ANXA10, ANXA11, and ANXA13 were purchased from NKMAX (Seongnam-si, Korea). Briefly, 96-well microtiter plates (Immobilizer Amino, Nunc Naperville, IL, USA) were coated with 0.5 μg / ml of recombinant protein and blocked with 0.5% bovine serum albumin (BSA)-NET / IGEPAL CA-630 at room temperature for 1 hour. As the sample and secondary antibody, we used serum (1:250) from the target (patient) and alkaline phosphatase-conjugated goat anti-human IgG (1:1000, γ-chain specific, Jackson Immunoresearch, Hershey, PA, USA) diluted with 0.5% BSA-NET / IGEPAL CA-630, respectively. A standard curve was created using a four-parameter logistic curve fit with serial 1:5 dilutions of high-titer prototype serum. The optical density of the samples, measured at 405 nm, was converted to units based on the standard curve.

[0042] (5) Immunohistochemical analysis Formalin-fixed, paraffin-embedded sections were prepared from surgical specimens of DIP, IPF, and iNSIP patients. After deparaffinization and dehydration, the sections were placed in pH 9.0 ethylenediaminetetraacetic acid buffer and autoclaved at 95°C for 30 minutes to restore epitopes. The sections were incubated with 3% hydrogen peroxide for 20 minutes to block endogenous peroxidase activity. After incubation with the blocking solution, the sections were washed twice with phosphate-buffered saline (PBS) and incubated overnight at 4°C with anti-ANXA4 antibody (diluted 1:500, HPA007393, Atlas Antibodies, Stockholm, Sweden) in a 0.1% bovine serum albumin PBS solution. Normal rabbit IgG (200 μl, Santa Cruz Biotechnology Inc., Santa Cruz, CA) was used as a negative control. Sections were washed with PBS and incubated with peroxidase-labeled anti-rabbit antibody (Histofine Simplestain Max PO; Nichirei Co., Tokyo, Japan) at room temperature for 30 minutes. Peroxidase activity was detected with diaminobenzidine (Sigma-Aldrich, St. Louis, MO, USA). Sections were counterstained with hematoxylin and dehydrated.

[0043] (6) Statistical analysis All values ​​are expressed as median and interquartile range (IQR). Differences between two groups were determined using the Mann-Whitney U test, and differences between three or more groups were determined using the Kruskal-Wallis test. Bonferroni post-hoc adjustment tests were then performed for multiple comparisons. For categorical variables, the chi-square test or Fisher's exact test was performed. Correlations between parameters were determined using Spearman's rank correlation coefficient. The upper left corner coordinate point of the receiver operating characteristic curve (ROC curve) was used to determine the optimal cutoff level for differentiating between DIP and non-DIP idiopathic interstitial pneumonia. The significance level was set at p < 0.05. Furthermore, we measured the levels of anti-ANXA antibodies in patients with DIP, non-DIP idiopathic interstitial pneumonia (non-DIP IIPs), collagen disease-associated interstitial lung disease (CTD-ILD), collagen disease without interstitial lung disease (CTD w / o ILD), and lung diseases other than interstitial lung disease (PUL; chronic obstructive pulmonary disease, lung cancer, pneumonia), and examined the positive rates for each disease using the cutoff levels mentioned above.

[0044] result (1) Characteristics of the subject (patient) The characteristics of patients with IIPs in this study are shown in Table 2 below.

[0045] [Table 2]

[0046] A to F in Table 2 represent the following: ap < 0.05 (IPF, DIP, iNSIP) bp < 0.05 (DIP, IPF, iNSIP, COP) cp < 0.05 (DIP and IPF, COP) dp < 0.05 (DIP and iNSIP) ep < 0.05 (DIP and COP) fp < 0.05 (DIP and COP, IPF and iNSIP, COP, iNSIP and COP)

[0047] As shown in Table 2, most of the DIP subjects (patients) were male. Furthermore, all DIP subjects (patients) were currently or have a history of smoking, and their smoking index (Pack-years) was significantly higher than that of IPF and COP. The age of DIP subjects (patients) was younger compared to IPF subjects (patients). More than half of DIP cases had occupational exposure, such as welding or exposure to fumes, which was significantly higher in DIP compared to iNSIP. The ANA positivity rate in DIP subjects (patients) appeared higher than in IPF, iNSIP, and COP, but it was not statistically significant. KL-6 levels in DIP subjects (patients) were significantly higher than in COP subjects (patients). The classification of IIPs is shown in Figure 1.

[0048] (2) Identification of target proteins recognized by autoantibodies in DIP subjects (patients) Representative results of serum immunoprecipitation analysis are shown in Figure 2. The lanes in Figure 2 represent DIP (lanes 1-7), IPF (lanes 8-10), and healthy control (HC) (lanes 11, 12). Several proteins between approximately 33kD and 70kD were immunoprecipitated in the serum of DIP cases, but not in the serum of IPF and healthy individuals. Using mass spectrometry, five possible proteins were identified, all belonging to the annexin protein family (proteins belonging to annexin A). Specifically, the bands around 33kD, 36kD, and 40kD are annexin A4 and A5 (35.9kD), annexin A3 (36.4kD), and annexin A1 (38.7kD). The band around 54kD is annexin A11 (54.4kD).

[0049] (3) Reactivity of anti-annexin antibodies in the serum of the subject (patient) In a preliminary study, the reactivity of serum antibodies against recombinant ANXA1, ANXA2, ANXA3, ANXA4, ANXA5, ANXA6, ANXA7, ANXA8, ANXA10, ANXA11, and ANXA13 was examined using ELISA. Of these, the reactivity of serum antibodies against recombinant ANXA1, ANXA3, ANXA4, ANXA5, and ANXA11 was detected in the serum of DIP patients (data not shown).

[0050] Figure 3 shows the levels of each anti-ANXA antibody (anti-ANXA4 antibody, anti-ANXA5 antibody, and anti-ANXA11 antibody) in each group (patient) of IIPs, CTD-ILD, Non-ILD, and HC. When the cutoff value was set to 2.5 U / ml for each antibody, the positivity rates for anti-ANXA4 antibody, anti-ANXA5 antibody, and anti-ANXA11 antibody differed among the groups (Table 3).

[0051] [Table 3]

[0052] a, b, and c in Table 3 represent the following: ap<0.05 (DIP and IPF, iNSIP, COP, CTD-ILD, Non-ILD, HC) bp < 0.05 (DIP, CTD-ILD, Non-ILD) cp<0.05 (DIP and IPF, iNSIP, COP, Non-ILD, HC)

[0053] Furthermore, antibody levels against each ANXA were significantly correlated (Table 4). In Table 4, Spearman's rank correlation * p<0.05; ANXA, annexin A.

[0054] [Table 4]

[0055] In particular, the prevalence of anti-ANXA4 and anti-ANXA11 antibodies was higher in the DIP group than in other groups.

[0056] (4) Immunohistochemical staining of annexin A4 antigen Figure 4 shows immunohistochemical staining of ANXA4 antigen in representative lung specimens from DIP, IPF, and iNSIP patients. ANXA4 is expressed in the cytoplasm of epithelial cells and macrophages in each disease. It was found that accumulated macrophages showed higher levels of ANXA4 antigen expression in DIP specimens.

[0057] (5) Statistical analysis results 1 Anti-ANXA4 antibodies in the serum of DIP patients were significantly higher than in non-DIP idiopathic interstitial pneumonia (non-DIP IIPs) (Figure 5). Anti-ANXA5 antibodies in the serum of DIP patients were significantly higher than in non-DIP idiopathic interstitial pneumonia (non-DIP IIPs) (Figure 6). Anti-ANXA11 antibodies in the serum of DIP subjects (patients) were significantly higher than in non-DIP idiopathic interstitial pneumonia (non-DIP IIPs) (Figure 7).

[0058] (6) Statistical analysis results 2 ROC curves were created using anti-ANXA4, anti-ANXA5, and anti-ANXA11 antibodies as markers to differentiate between DIP-targeted (DIP) patients and non-DIP idiopathic interstitial pneumonia (Figure 8). As can be seen from Figure 8, the areas under the ROC curves for anti-ANXA4, anti-ANXA5, and anti-ANXA11 antibodies were high and statistically significant, at 0.735, 0.749, and 0.766, respectively. The cutoff values ​​for these antibodies are shown in Table 1.

[0059] (7) Statistical analysis results 3 Figures 9, 10, and 11 show the measured values ​​of anti-ANXA4, anti-ANXA5, and anti-ANXA11 antibodies in DIP, non-DIP idiopathic interstitial pneumonia (non-DIP IIPs), collagen disease-associated interstitial lung disease (CTD-ILD), collagen disease without interstitial lung disease (CTD w / o ILD), lung diseases other than interstitial lung disease (PUL; chronic obstructive pulmonary disease, lung cancer, pneumonia), and healthy controls (HV). The positive rate using the cutoff values ​​shown in Table 1 was high in DIP cases (Figure 12). [Industrial applicability]

[0060] The present invention provides a biomarker for determining desquamative interstitial pneumonia, specifically an anti-annexin A autoantibody. Furthermore, the present invention provides a method for assisting the diagnosis of desquamative interstitial pneumonia, which includes a step of detecting the biomarker in a subject. The biomarker and diagnostic assistance method using the present invention are expected to be useful because they will simplify and facilitate the differentiation (definitive diagnosis) of DIP, which previously required multiple tests.

Claims

1. A biomarker for diagnosing exfoliative interstitial pneumonia, including anti-annexin A autoantibodies.

2. The biomarker according to claim 1, wherein the annexin A is at least one selected from the group consisting of annexin A4, annexin A5, and annexin A11.

3. The biomarker according to claim 2, wherein the annexin A is annexin A4.

4. A method for assisting the diagnosis of exfoliative interstitial pneumonia, comprising the step of detecting a biomarker according to any one of claims 1 to 3 in a subject.

5. The method according to claim 4, wherein the subject is suffering from or may suffer from a lung disease and / or collagen disease.

6. The method according to claim 5, wherein the subject is a subject who is suffering from or may be suffering from idiopathic interstitial pneumonia.

7. (1) A step of measuring the amount of biomarker described in any one of claims 1 to 3 in the subject, (2) A step of comparing the value measured in step (1) with a predetermined threshold. The method according to any one of claims 4 to 6, including the method described in any one of claims 4 to 6.

8. The method according to claim 7, wherein if the value measured in step (1) exceeds a predetermined threshold, it is determined to be desquamative interstitial pneumonia.

9. A kit for determining exfoliative interstitial pneumonia, comprising at least one selected from a protein belonging to annexin A, an antigenic partial peptide thereof, and a peptide containing an epitope recognized by an anti-annexin A autoantibody.

10. The kit according to claim 9, wherein the protein belonging to annexin A is at least one selected from the group consisting of annexin A4, annexin A5, and annexin A11.

11. The kit according to claim 10, wherein the protein belonging to annexin A is annexin A4.

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