WO2023153496A1 - Biomarqueur de l'asthme éosinophilique ou bronchique - Google Patents

Biomarqueur de l'asthme éosinophilique ou bronchique Download PDF

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WO2023153496A1
WO2023153496A1 PCT/JP2023/004548 JP2023004548W WO2023153496A1 WO 2023153496 A1 WO2023153496 A1 WO 2023153496A1 JP 2023004548 W JP2023004548 W JP 2023004548W WO 2023153496 A1 WO2023153496 A1 WO 2023153496A1
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protein
domain
group
immunoglobulin
receptor
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吉人 武田
華子 吉村
淳 熊ノ郷
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国立大学法人大阪大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to eosinophilia or bronchial asthma biomarkers and the like.
  • Bronchial asthma is defined as "a disease characterized by clinical symptoms such as fluctuating airway narrowing and coughing, characterized by chronic inflammation of the airways.” According to a recent survey, the total number of asthma patients receiving continuous medical care in Japan is more than 1 million, and the total number of asthma patients in the world is about 300 million. There are no clear diagnostic criteria for asthma, and guidelines for asthma diagnosis are defined in the Asthma Prevention and Management Guidelines. In addition to repeated paroxysmal dyspnea and coughing, it is recommended to refer to the presence of reversible airflow limitation, increased airway hyperresponsiveness, presence of atopic predisposition, and presence of airway inflammation. It is necessary to make a diagnosis by comprehensively judging the indicators. Biomarkers currently used to diagnose asthma include blood and sputum eosinophil counts, exhaled nitric oxide (FeNO), and serum IgE levels. Have difficulty.
  • proteomics which is the omics closest to the phenotype, is of particular interest as a biomarker.
  • proteins cannot be amplified, and a comprehensive analysis system has not been established. Therefore, proteomics research for diseases is currently lagging behind.
  • Serum is considered an ideal test sample because it can be collected non-invasively and repeatedly. Therefore, from the viewpoint of proteomics, its usefulness as test data is low.
  • a recent paper on biomarkers that can distinguish between COPD and asthma attempted to analyze dozens of patients, but the analysis was based on an old mass spectrometry (MS) technique based on two-dimensional electrophoresis. The biomarkers found were not only
  • An object of the present invention is to provide a biomarker for eosinophilia or bronchial asthma and a method for using the same.
  • the present inventors found that extracellular vesicles in body fluids collected from subjects or specific protein groups in body fluids are useful as biomarkers for eosinophilia or bronchial asthma. I found As a result of further research based on this knowledge, the present invention was completed. That is, the present invention includes the following aspects.
  • Section 1 A method of testing for eosinophilia or bronchial asthma, comprising: (1) Protein group (A), protein group (X), protein group (B), protein group (C), protein group (D), protein in extracellular vesicles of body fluid collected from a subject or body fluid Group (E), Protein Group (F), and Protein Group (G): (A) Protein group consisting of Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, and Eosinophil cationic protein, (X) Cholinesterase, Galectin-10, Exportin-2, Eosinophil peroxidase, Coagulation factor XII, Ficolin- 3, a protein group consisting of Immunoglobulin heavy variable 4-28, Immunoglobulin heavy variable 6-1, Immunoglobulin heavy variable 7-43, Immunoglobulin heavy variable 8-61, and Immunoglobulin heavy variable 9-49, (B)
  • Section 4 The examination method according to any one of Items 1 to 3, wherein the disease to be examined is eosinophilic asthma.
  • Item 5 wherein the protein to be detected contains at least one protein selected from the group consisting of protein group (F) and protein group (G).
  • Item 6 When the detected protein contains at least one protein (protein F') selected from the protein group (F), (2F) determining that the subject has eosinophilic asthma when the amount or concentration of protein F' detected in step (1) is equal to or greater than the cutoff value;
  • the inspection method according to Item 4 or 5, comprising:
  • Item 7 When the detected protein contains at least one protein (protein G') selected from the protein group (G), (2G) determining that the subject has eosinophilic asthma when the amount or concentration of protein G' detected in step (1) is equal to or less than a cutoff value; 7.
  • the inspection method according to any one of items 4 to 6.
  • Item 8 The examination method according to any one of items 1 to 3, wherein the disease to be examined is atopic asthma.
  • Item 10 When the detected protein contains at least one protein (protein D') selected from the protein group (D), (2D) determining that the subject has atopic asthma when the amount or concentration of protein D' detected in step (1) is equal to or greater than the cutoff value;
  • the inspection method according to Item 8 or 9, comprising:
  • Item 11 When the detected protein contains at least one protein (protein E') selected from protein group (E), (2E) determining that the subject has atopic asthma when the amount or concentration of protein E′ detected in step (1) is equal to or less than a cutoff value;
  • Item 12 The inspection method according to any one of items 1 to 11, wherein the proteins to be detected include protein group (A).
  • the detected protein contains at least one protein (protein A') selected from protein group (A), (2A) when the amount or concentration of protein A' detected in step (1) is equal to or greater than the cutoff value, the subject suffers from highly severe eosinophilia or bronchial asthma; determining that there is a high possibility of suffering from high-severity eosinophilia or bronchial asthma in the future;
  • the inspection method according to any one of Items 1 to 12, comprising:
  • biomarkers for eosinophilia or bronchial asthma can be provided.
  • the biomarker it is possible to test for eosinophilia or bronchial asthma, prevent or treat eosinophilia or bronchial asthma, and use a preventive or therapeutic agent for eosinophilia or bronchial asthma. Screening of active ingredients, evaluation of inducing or aggravating eosinophilia or bronchial asthma, and the like may become possible.
  • FIG. 1 shows the measurement results of Galectin-10 expression levels in extracellular vesicle fractions (Exosomes) in Test Example 6.
  • FIG. 1 shows the measurement results of Galectin-10 expression level in tissues (inferior nasal turbinate and polyp) of Test Example 6.
  • FIG. Fig. 8 shows measurement results of Galectin-10 amounts in exosomes and cell lysates with and without eosinophil activation treatment (IL-5+PAF (+)/(-)) in Test Example 8.
  • IL-5+PAF (+)/(-) shows the measurement results of proteomics analysis in Test Example 2.
  • FIG. Each graph information shows the protein to be measured. The vertical axis indicates the expression level in serum exosomes.
  • HC indicates healthy subjects
  • ABA indicates atopic asthmatic subjects
  • EBA indicates eosinophilic asthmatic subjects.
  • 2 shows the results of immunoblotting in Test Example 9.
  • FIG. HC indicates healthy subjects
  • COPD indicates COPD subjects
  • BA indicates asthmatic subjects.
  • 2 shows the results of fluorescent immunostaining in Test Example 10.
  • FIG. HC indicates healthy subjects and BA indicates asthmatic subjects.
  • 2 shows the proteomics results of Test Example 11.
  • FIG. HC indicates healthy subjects and BA indicates asthmatic subjects.
  • a method for measuring and calculating the degree of bronchial wall thickening (WA%) is shown.
  • FIG. 12 shows the correlation between the expression level of Galectin-10 in serum exosomes in Test Example 12, the blood eosinophil count, and the degree of bronchial wall thickening (WA%).
  • the expression level of Galectin-10 in serum exosomes of Test Example 12 and the correlation between blood eosinophil count and respiratory function (FEV1%) are shown.
  • FIG. 10 shows the correlation coefficient (horizontal axis) between the extracellular vesicle fraction and the tissue expression level in Test Example 6.
  • a method for testing eosinophilia or bronchial asthma comprising: (1) cells of a body fluid collected from a subject; From protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), protein group (F), and protein group (G) in outer vesicles or body fluids
  • the present invention relates to an inspection method (also referred to herein as the "inspection method of the present invention") comprising the step of detecting at least one protein selected from the group consisting of: This will be explained below.
  • Process (1) The type of "eosinophilia", which is the disease to be examined, is not particularly limited. All classes, grades, and stages of eosinophilia in various classification criteria for progression of eosinophilia can be tested. Specific examples of eosinophilia include allergic diseases, skin diseases, infectious diseases, respiratory diseases, granulomatous diseases, digestive diseases, endocrine diseases, autoimmune diseases, malignant tumors, and the like. mentioned.
  • allergic diseases include bronchial asthma, atopic dermatitis, urticaria, allergic rhinitis, drug allergy, allergic rhinitis/conjunctivitis, allergic bronchi, allergic granulomatous vasculitis, angioedema/Quincke's edema, Oral mucosal allergy syndrome, allergic purpura, and the like.
  • Skin diseases include, for example, pemphigus vulgaris, eosinophilic pimples, folliculitis, eczema, eosinophilic fasciitis, drug eruption, psoriasis, lymphomatous papules, and erythroderma.
  • Infectious diseases include, for example, parasitism, pulmonary aspergillosis, tuberculosis, syphilis, toxoplasmosis, visceral tick disease, scarlet fever, primary immunodeficiency syndrome, and carinii pneumonia.
  • respiratory diseases include bronchial asthma, eosinophilic lung infiltration, pneumoconiosis/silicosis, hypersensitivity pneumonitis, and idiopathic septic iron.
  • granulomatous diseases include eosinophilic granulomatosis, Wegener's granulomatosis, sarcoidosis, and Kimura disease.
  • Gastrointestinal diseases include, for example, eosinophilic gastroenteritis, allergic gastroenteritis, ulcerative colitis, pancreatitis, and inflammatory bowel disease.
  • Endocrine diseases include, for example, Addison's disease and hyperthyroidism.
  • Autoimmune diseases include, for example, polyarteritis, rheumatoid arthritis, chronic active hepatitis, pemphigus, sarcoidosis, malignant rheumatoid arthritis, polyarteritis nodosa, and Kaplan's syndrome.
  • Malignant tumors include, for example, malignant lymphoma, Hodgkin's lymphoma, adult T-cell leukemia, mycosis fungoides, immunoblastic lymphadenitis, soft tissue sarcoma, carcinomatous lymphangiosis, paramalignant tumor syndrome, heavy chain disease , colon cancer, lung cancer, and the like.
  • bronchial asthma that is the disease to be tested is not particularly limited. All classes, grades, and stages of bronchial asthma in various classification criteria for progression of bronchial asthma can be tested. In addition, all classification categories of bronchial asthma (eg, eosinophilic asthma, atopic asthma, etc.) can be tested.
  • the subject is the target organism of the testing method of the present invention, and its species is not particularly limited.
  • Species of the subject include, for example, various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, preferably humans.
  • Subjects include, for example, subjects with unknown eosinophilia, subjects already determined to have eosinophilia by another method, eosinophilia Examples include specimens that have already been determined by another method to be free of disease, specimens undergoing treatment for eosinophilia, specimens after treatment for eosinophilia, and the like.
  • the sample to be detected in step (1) is preferably extracellular vesicles of body fluid.
  • Body fluids include, for example, whole blood, serum, plasma, cerebrospinal fluid, saliva, synovial fluid, urine, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal discharge, exhaled breath, exhaled breath condensate, etc.
  • Whole blood, serum, plasma and cerebrospinal fluid are preferred, and whole blood, serum and plasma are more preferred.
  • One type of body fluid may be used alone, or two or more types may be used in combination.
  • a body fluid can be collected from a subject by a method known to those skilled in the art.
  • whole blood can be collected by blood collection using a syringe or the like.
  • Serum is a portion of whole blood from which blood cells and specific blood coagulation factors have been removed, and can be obtained, for example, as a supernatant after coagulation of whole blood.
  • Plasma is a portion of whole blood from which blood cells have been removed, and can be obtained, for example, as a supernatant when whole blood is subjected to centrifugation under non-clotting conditions.
  • blood samples blood itself such as whole blood, serum, plasma, etc., or blood-derived samples are referred to as "blood samples”.
  • Extracellular vesicles are not particularly limited as long as they are membrane vesicles that are secreted or released from cells.
  • Extracellular vesicles are usually defined as membrane vesicles that carry intracellular proteins and genetic information (mRNA, microRNA, etc.) to the outside of cells, and are responsible for local and systemic intercellular communication. be.
  • Extracellular vesicles include, for example, exosomes, microvesicles, apoptotic bodies, ectosomes, microparticles, secretory microvesicles and the like.
  • Extracellular vesicles can be purified, separated, concentrated, etc. from body fluids according to or in accordance with known methods.
  • Methods for purifying, separating, and concentrating extracellular vesicles include, for example, ultracentrifugation (e.g., pellet down method, sucrose cushion method, density gradient centrifugation method, etc.), methods using immunoaffinity carriers, gel filtration, field centrifugation, and the like. Flow fractionation method, FACS method and the like can be mentioned. Purification, separation, concentration and the like of extracellular vesicles can also be performed using commercially available kits. These methods may be employed singly or in combination of two or more.
  • Detection targets in step (1) include protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), protein group (F), and protein group (G ) (in the present specification, these are sometimes collectively referred to as the “protein of interest”).
  • Protein group (B) includes (B) HLA class II histocompatibility antibody, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase , Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 members A, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2
  • Protein group (B) is a protein whose expression level in all bronchial asthma subjects is higher than that in healthy subjects.
  • protein group (B) preferably HLA class II histocompatibility antibody, DRB1-9 beta chain, Galectin-10, Eosinophil peroxidase, Protein mono-ADP-ribosyltransferase PARP4, Non-secretory ribonuclease, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase , Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2-29, Immunoglobulin heavy constant gamma 2, Cytochrome P450 4F8, C-type lectin domain family 12 members A, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-9, Immunoglobulin lambda variable 3-21, Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial, 2
  • Protein group (C) includes (C) Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, von Willebrand factor A domain-containing protein 5A, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, Gamma-enolase, LHFPL tetraspan subfamily member 6 protein, FYVE, RhoGEF and PH domain-containing protein 4, Sorcin, Tetraspanin-33, Tight junction protein ZO-2, NucleoLysin Tia-1 IsoForm P40, NucleOlysin Tia-1 Isoform P40, NucleOlysin TIAR, DISINTEGRIN and Metalloproteinase Domain-Contening ProtEin 10, Choline T Ransport-Like Protein 2, Probable UDP-Sugar Transporter
  • Protein group (C) is a protein whose expression level in all bronchial asthma subjects is lower than that in healthy subjects.
  • the protein group (C) preferably Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2, Mitogen-activated protein kinase kinase kinase 7, Methyltransferase-like 26, etc., more preferably Thioredoxin domain-containing protein 12, Bone morphogenetic protein 10, Butyrophilin subfamily 3 member A2.
  • Protein group (D) includes (D) Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III -A, Non-secretory ribonuclease, Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol factor 1B, Neutrophil cytosol factor 1, Neutrophil cytosol factor 4, Matrix metalloproteinase-25, Heme oxygenase 2, Urokinase plasminogen activator surface receptor, Epoxide hydrolase 1, Torsin-1A-interacting protein 1, Arachidonate 5-lipoxygenase, 2'-deoxyn
  • Protein group (D) is a protein whose expression level in atopic asthmatic subjects is higher than that in healthy subjects.
  • the protein group (D) preferably Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A, Low affinity immunoglobulin gamma Fc region receptor III -A, Non-secretory ribonuclease, Cartilage intermediate layer protein 1, Galectin-10, Resistin, Estradiol 17-beta-dehydrogenase 11, Bactericidal permeability-increasing protein, Cell surface glycoprotein CD200 receptor 1, Putative neutrophil cytosol factor 1B, Putative neutrophil cytosol Factor 1B, Neutrophil cytosol factor 1, Neutrophil cytosol factor 4, etc., more preferably Putative inactive carboxylesterase 4, Neutrophil cytosol factor 2, Immunoglobulin heavy constant gamma 2, Prelamin-A/C, C-type lectin domain family 12 member A.
  • Protein group (E) includes (E) HLA class II histocompatibility antibody, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine/threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A -like 1, von Willebrand factor A domain-containing protein 5A, Tight junction protein ZO-2, Proteinase-activated receptor 1, Gamma-enolase, Mitogen-activated protein kinase kinase kinase 7, Cytohesin-2, Immunoglobulin-binding protein 1, Armadillo
  • Protein group (E) is a protein whose expression level in atopic asthmatic subjects is lower than that in healthy subjects.
  • protein group (E) preferably HLA class II histocompatibility antibody, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase-like 26, LIM domain and actin-binding protein 1, Serine/threonine-protein phosphatase PP1-gamma catalytic subunit, Methionine--tRNA ligase, cytoplasmic, Butyrophilin subfamily 3 member A2, FYVE, RhoGEF and PH domain-containing protein 4, Katanin p60 ATPase-containing subunit A -like 1, von Willebrand factor A domain-containing protein 5A, tight junction protein ZO-2, and more preferably HLA class II histocompatibility antigen, DRB1-4 beta chain, Gasdermin-D, MAP/microtubule affinity-regulating kinase 3, Thioredoxin domain-containing protein 12, Methyltransferase
  • Protein group (F) includes (F) Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2- 29, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, Sialic acid-binding Ig-like lectin 5, Tor
  • Protein group (F) is a protein whose expression level in eosinophilic asthmatic subjects is higher than that in healthy subjects.
  • the protein group (F) preferably Galectin-10, Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, Non-secretory ribonuclease, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2D-29, Immunoglobulin kappa variable 2- 29, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, Voltage-gated hydrogen channel 1, Eosinophil cationic protein, Cytochrome b-245 light chain, Disintegrin and metalloproteinase domain-containing protein 8, Aldehyde dehydrogenase family 3 member B1, Cytidine deaminase, Heme oxygenase 2, Immunoglobulin heavy constant alpha 1, Metalloreductase STEAP4, Type-1 angiotensin II receptor-associated protein, more preferably Galectin-10, Eosinophil peroxida
  • Protein group (G) includes (G) Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bis phosphate 4 -phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, DAZ-associated protein 1, C-type lectin domain family 2 member L, Leucyl-cystinyl aminopeptida
  • the protein group (G) is proteins whose expression level in eosinophilic asthmatic subjects is lower than that in healthy subjects.
  • the protein group (G) preferably Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4, Probable UDP-sugar transporter protein SLC35A5, Golgi integral membrane protein 4, Protein CASC4, Putative tyrosine-protein phosphatase auxilin, Butyrophilin subfamily 3 member A2, C1GALT1-specific chaperone 1, Protein FAM234B, Tetraspanin-33, Canalicular multispecific organic anion transporter 2, Mitogen-activated protein kinase kinase kinase 7, Type 2 phosphatidylinositol 4,5-bis phosphate 4 -phosphatase, Wiskott-Aldrich syndrome protein family member 1, cGMP-inhibited 3',5'-cyclic phosphodiesterase A, etc., more preferably Bone morphogenetic protein 10, Thioredoxin domain-containing protein 12, Intermediate conductance calcium-activated potassium channel protein 4,
  • Protein group (A) is a protein group consisting of Galectin-10, Bone marrow proteoglycan, Eosinophil peroxidase, Non-secretory ribonuclease, and Eosinophil catic protein.
  • Protein group (A) is a protein whose expression level in subjects with bronchial asthma is higher than that in healthy subjects, and the number of blood eosinophils, respiratory function, tissue pathology, tissue eosinophil infiltration, clinical score , EETosis-positive eosinophil count, etc., is a particularly preferable protein as an index of severity.
  • Galectin-10 is particularly preferred.
  • Protein group (X) includes Cholinesterase, Galectin-10, Exportin-2, Eosinophil peroxidase, Coagulation factor XII, Ficolin-3, Immunoglobulin heavy variable 4-28, Immunoglobulin heavy variable 6-1, Immunoglobulin heavy variable 7-43, Immunoglobulin It is a protein group consisting of heavy variable 8-61 and immunoglobulin heavy variable 9-49.
  • the protein group (X) preferably Immunoglobulin heavy variable 8-61, Eosinophil peroxidase, Immunoglobulin heavy variable 9-49, Galectin-10, Immunoglobulin heavy variable 7-43, more preferably Immunoglobulin heavy variable 8-61 , Eosinophil peroxidase, Immunoglobulin heavy variable 9-49, and Galectin-10, more preferably Immunoglobulin heavy variable 8-61.
  • the proteins of the protein groups (A) to (G) are, in the case of humans, proteins specified by the UniProtKB accession numbers shown in Tables 1 to 6 in Examples below. For other species, it is an orthologue of the protein identified by the UniProtKB accession numbers shown in Tables 1-6.
  • the number of target proteins in step (1) may be one, or a combination of two or more. By combining more detection targets (e.g., 2, 5, 10, 20, 30, 40, 50 or more), eosinophilia can be tested more accurately. becomes possible.
  • Detection is usually performed by measuring the amount or concentration of the target protein.
  • “Concentration” is not limited to absolute concentration, but may be relative concentration, weight per unit volume, or raw data measured to know absolute concentration.
  • the method for detecting the target protein is not particularly limited as long as it can specifically detect part or all of the target protein.
  • Specific examples of the detection method include mass spectrometry for detecting peptides constituting the target protein, and immunoassay using an antibody that specifically recognizes the target protein.
  • the amino acid sequence information of the target protein can be obtained by searching the database of EBI (http://www.ebi.ac.uk/IPI/IPIhelp.html) based on the UniProtKB accession number.
  • Immunohistochemical staining methods ELISA methods, EIA methods, RIA methods, Western blotting methods, etc. can be suitably exemplified as immunological measurement methods.
  • a peptide sample is turned into gaseous ions (ionized) using an ion source, and the peptide sample is moved in vacuum in an analysis unit and ionized using electromagnetic force or by time-of-flight difference.
  • a measurement method using a mass spectrometer that can separate and detect according to the ratio.
  • Methods of ionization using an ion source include the EI method, CI method, FD method, FAB method, MALDI method, and ESI method. and other methods can be selected as appropriate, and methods for separating the ionized peptide sample in the analysis unit include magnetic field deflection, quadrupole, ion trap, time-of-flight (TOF), and Fourier transform.
  • a separation method such as an ion cyclotron resonance type can be appropriately selected.
  • tandem mass spectrometry combining two or more mass spectrometry methods and triple quadrupole mass spectrometry can be used.
  • the sample if the sample contains phosphorylated peptides, the sample can be concentrated using iron ion immobilization affinity chromatography (Fe-IMAC) prior to sample introduction into the mass spectrometer.
  • Fe-IMAC iron ion immobilization affinity chromatography
  • peptides constituting the target protein can be separated and purified and used as a sample by liquid chromatography (LC) or HPLC.
  • the detection unit and data processing method can be selected as appropriate.
  • a peptide that has the same amino acid sequence as the peptide and is labeled with a stable isotope of known concentration is used as an internal standard. can do.
  • a stable isotope-labeled peptide one or more of the amino acids in the peptide constituting the target protein to be detected is a stable isotope-labeled peptide containing any one or more of 15N, 13C, 18O, and 2H. can be selected as appropriate, and such stable isotope-labeled peptides are produced by the F-moc method (Amblard., et al. Methods Mol Biol.
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  • ICPL registered trademark
  • NBS registered trademark
  • the amount and/or concentration of the target protein which is a detection indicator for eosinophilia, can be provided, thereby detecting eosinophilia. It can aid in detection, assessment of severity, and the like.
  • test results obtained by the test method of the present invention including step (1) can be used as a therapeutic marker for eosinophilia therapeutic agents, elucidation of the pathology of eosinophilia, prediction of prognosis of eosinophilia, It can be used for stratification of treatment, selection of treatment method (personalized medicine, treatment response), refractory in eosinophilia, differentiation of histological type and phenotype of eosinophilia, etc. .
  • Process (2) As one aspect of the inspection method of the present invention, When the detected protein contains at least one protein (protein ABDF') selected from the group consisting of protein group (A), protein group (B), protein group (D), and protein group (F), moreover, (2ABDF) if the amount or concentration of the protein ABDF' detected in step (1) is equal to or higher than the cutoff value, the subject suffers from eosinophilia or bronchial asthma, or has high determining that the patient has severe eosinophilia or bronchial asthma or is likely to have severe eosinophilia or bronchial asthma in the future; is preferably included. According to the testing method of the present invention including the step 2ABDF, it is possible to determine and evaluate the pathology of eosinophilia or bronchial asthma.
  • protein ABDF' protein selected from the group consisting of protein group (A), protein group (B), protein group (D), and protein group (F)
  • (2ABDF) if the amount
  • the detected protein contains at least one protein (protein CEG') selected from the group consisting of protein group (C), protein group (E), and protein group (G), (2CEG) if the amount or concentration of the protein CEG' detected in step (1) is less than or equal to the cutoff value, the subject suffers from eosinophilia or bronchial asthma, or has high determining that the patient has severe eosinophilia or bronchial asthma or is likely to have severe eosinophilia or bronchial asthma in the future; is preferably included. According to the testing method of the present invention including step 2CEG, it is possible to determine eosinophilia.
  • the examination method of the present invention comprises, as one aspect, When the detected protein contains at least one protein (protein D') selected from the protein group (D), (2D) determining that the subject has atopic asthma when the amount or concentration of protein D' detected in step (1) is equal to or greater than the cutoff value; is preferably included.
  • protein D' protein selected from the protein group (D)
  • (2D) determining that the subject has atopic asthma when the amount or concentration of protein D' detected in step (1) is equal to or greater than the cutoff value; is preferably included.
  • the examination method of the present invention comprises, as one aspect, When the detected protein contains at least one protein (protein E') selected from protein group (E), (2E) determining that the subject has atopic asthma when the amount or concentration of protein E′ detected in step (1) is equal to or less than a cutoff value; It is preferred to include
  • the testing method of the present invention comprises, as one aspect, When the detected protein contains at least one protein (protein F') selected from the protein group (F), (2F) determining that the subject has eosinophilic asthma when the amount or concentration of protein F' detected in step (1) is equal to or greater than the cutoff value; is preferably included.
  • protein F' protein selected from the protein group (F)
  • (2F) determining that the subject has eosinophilic asthma when the amount or concentration of protein F' detected in step (1) is equal to or greater than the cutoff value; is preferably included.
  • the testing method of the present invention comprises, as one aspect, When the detected protein contains at least one protein (protein G') selected from the protein group (G), (2G) determining that the subject has eosinophilic asthma when the amount or concentration of protein G' detected in step (1) is equal to or less than a cutoff value; is preferably included.
  • protein G' protein selected from the protein group (G)
  • (2G) determining that the subject has eosinophilic asthma when the amount or concentration of protein G' detected in step (1) is equal to or less than a cutoff value; is preferably included.
  • the testing method of the present invention when determining the presence or absence of morbidity of high-severity eosinophilia and the possibility of morbidity in the future, the testing method of the present invention, as an aspect,
  • the detected protein contains at least one protein (protein A') selected from protein group (A), (2A) when the amount or concentration of protein A' detected in step (1) is equal to or greater than the cutoff value, the subject suffers from highly severe eosinophilia or bronchial asthma; determining that there is a high possibility of suffering from high-severity eosinophilia or bronchial asthma in the future; It is preferred to include
  • the cutoff value can be appropriately set by those skilled in the art from the viewpoint of sensitivity, specificity, positive predictive value, negative predictive value, etc.
  • control subjects e.g., those suffering from eosinophilia subjects not suffering from or suffering from bronchial asthma, subjects not suffering from or suffering from atopic asthma, subjects not suffering from eosinophilic asthma
  • the amount and/or concentration of the protein of interest in extracellular vesicles or body fluids of body fluids collected from diseased subjects, subjects with low or high index values for the severity of eosinophilia, etc. can be a value determined each time or a predetermined value based on .
  • the cutoff value is, for example, the amount and / or concentration of the target protein in the extracellular vesicles or body fluid of the body fluid collected from the reference subject or subject group (when the subject is multiple, the average value, median value), for example, 0.7 to 1.5 times. It can also be set by performing statistical analysis (more specifically, a method using Youden index is exemplified) based on Receiver Operating Characteristic (ROC) curve analysis, etc. can.
  • ROC Receiver Operating Characteristic
  • the cutoff value is, for example, the amount of the protein of interest in a past sample of the same subject. and/or concentration-based values can be used to determine therapeutic efficacy.
  • the subject was determined to be suffering from eosinophilia or bronchial asthma by the test method of the present invention including the step (2) of diagnosing eosinophilia or bronchial asthma with higher accuracy.
  • eosinophilia or bronchial asthma can be diagnosed with higher accuracy by combining the testing method of the present invention with the step of applying diagnosis of eosinophilia or bronchial asthma by a doctor. can.
  • testing method of the present invention can more accurately detect eosinophilia or bronchial asthma, combining the above steps with the testing method of the present invention can more efficiently and accurately detect "eosinophilia.” It can be diagnosed as suffering from polymorphism or bronchial asthma.
  • the test of the present invention is performed. Diagnosed as suffering from eosinophilia or bronchial asthma further to the method or as described above in "2. Diagnosis of eosinophilia or bronchial asthma with greater accuracy".
  • the test method of the present invention and the step of applying diagnosis by a doctor if On the other hand, by performing the step of treating the disease, it is possible to treat the disease in the subject.
  • the testing method of the present invention can more accurately detect eosinophilia or bronchial asthma
  • the testing method of the present invention or the step of applying the testing method of the present invention and a diagnosis by a doctor can be applied.
  • step 3 subjects suffering from eosinophilia or bronchial asthma can be treated more effectively and reliably.
  • the treatment method for eosinophilia or bronchial asthma is not particularly limited, but typically includes drug treatment.
  • Pharmaceuticals used for drug treatment are not particularly limited, but for example, ⁇ -agonists such as adrenaline and salbutamol; anticholinergics such as ipratropium and tiotropium; leukotriene modifiers such as montelukast, zafirlukast and zileuton; obesity such as cromoglycate and nedocromil cell stabilizers; corticosteroids; immunomodulators (antibody drugs) such as omalizumab, mepolizumab, reslizumab; methylxanthines such as theophylline; lidocaine; One, two, or a combination of three or more medicaments can be used.
  • Eosinophilia or bronchial asthma test agent and test kit A test agent for eosinophilia or bronchial asthma, containing at least one protein detection agent selected from the group consisting of (E), protein group (F), and protein group (G) , sometimes referred to as "the test agent of the present invention"). This will be explained below.
  • Protein Group (A), Protein Group (B), Protein Group (C), Protein Group (D), Protein Group (E), Protein Group (F), Protein Group (G), Eosinophilia or Bronchial Asthma and the like are defined in the same manner as in the above "1. Test method for eosinophilia or bronchial asthma".
  • the detection agent is not particularly limited as long as it can specifically detect the target protein.
  • the detection agent includes, for example, an antibody against the target protein.
  • the detection agent may be modified as long as its function is not significantly impaired. Modifications include, for example, addition or introduction of labels such as fluorescent dyes, luminescent substances, dyes, enzymes, proteins, radioisotopes, chemiluminescent substances, colloidal gold, biotin, and the like.
  • the detection agent can also be used by immobilizing it on any solid phase.
  • the test agent of the present invention can be provided in the form of a substrate on which a detection agent is immobilized (for example, a microarray chip on which probes are immobilized, etc. Another example is an ELISA plate on which antibodies are immobilized, etc.).
  • the solid phase used for immobilization is not particularly limited as long as it can immobilize antibodies and the like. Examples include glass plates, nylon membranes, microbeads, silicon chips, capillaries and other substrates. can. Immobilization of the detection agent to the solid phase is not particularly limited.
  • the antibody is not particularly limited as long as it selectively (specifically) recognizes the target protein.
  • selectively (specifically) recognize means that the target protein can be specifically detected in, for example, Western blotting or ELISA, but is not limited thereto. Any substance can be used as long as it can be determined that the detected substance is derived from the target protein.
  • Antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, or portions of the above antibodies that have antigen-binding properties, such as Fab fragments and fragments generated by Fab expression libraries.
  • the antibody of the present invention also includes an antibody that has antigen-binding to a polypeptide consisting of at least 8 consecutive amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of the target protein.
  • the antibodies of the present invention can also be produced according to these conventional methods (Current protocols in Molecular Biology, Chapter 11.12-11.13 (2000)).
  • the antibody of the present invention is a polyclonal antibody
  • an oligopeptide having a partial amino acid sequence of the target protein is synthesized using a target protein expressed in Escherichia coli or the like and purified according to a standard method. Then, it is possible to immunize a non-human animal such as a rabbit and obtain the serum from the immunized animal according to a conventional method.
  • a non-human animal such as a mouse is immunized with a target protein expressed and purified in Escherichia coli or the like according to a conventional method, or an oligopeptide having a partial amino acid sequence of the target protein, and spleen cells obtained by immunizing a non-human animal such as a mouse are used. It can be obtained from hybridoma cells prepared by cell fusion with myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11).
  • the target protein used as an immunizing antigen for the production of antibodies is based on the known gene sequence information, DNA cloning, construction of each plasmid, transfection into the host, culture of the transformant, and recovery of the protein from the culture. can be obtained by the operation of These manipulations are performed according to methods known to those skilled in the art or methods described in literature (Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). can be done.
  • a recombinant DNA capable of expressing a gene encoding a target protein in a desired host cell is prepared, introduced into the host cell for transformation, and the transformant is cultured.
  • the protein By recovering the target protein from the resulting culture, the protein can be obtained as an immunizing antigen for producing the antibody of the present invention.
  • a partial peptide of the target protein can also be produced by a general chemical synthesis method (peptide synthesis) according to known gene sequence information.
  • the antibody of the present invention may also be prepared using an oligopeptide having a partial amino acid sequence of the target protein.
  • the oligo(poly)peptides used for the production of such antibodies need not have functional biological activity, but desirably have immunogenic properties similar to the protein of interest.
  • An oligo(poly)peptide preferably having this immunogenic property and consisting of at least 8 consecutive amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of the target protein can be exemplified.
  • Antibodies against such oligo(poly)peptides can also be produced by enhancing the immunological response using various adjuvants depending on the host.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surface agents such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol.
  • Active substances human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
  • the test agent of the present invention may be in the form of a composition.
  • the composition may contain other ingredients as needed.
  • Other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, and perfumes. , chelating agents and the like.
  • the test agent of the present invention may be in the form of a kit.
  • the kit may contain, in addition to the detection agent or the composition containing the same, those that can be used to detect the target protein in extracellular vesicles of body fluids or blood samples of a subject.
  • Specific examples of such materials include various reagents (eg, secondary antibodies, buffer solutions, etc.), instruments (eg, extracellular vesicle purification, separation, and concentration instruments (eg, columns, etc.)), and the like.
  • Prophylactic or therapeutic agent for eosinophilia or bronchial asthma a group consisting of protein group (A), protein group (B), protein group (D), and protein group (F) and at least one protein enhancer selected from the group consisting of protein group (C), protein group (E), and protein group (G). It relates to a prophylactic or therapeutic agent for eosinophilia or bronchial asthma (in this specification, sometimes referred to as "the drug of the present invention") containing at least one drug selected from the above. This will be explained below.
  • Protein Group (A), Protein Group (B), Protein Group (C), Protein Group (D), Protein Group (E), Protein Group (F), Protein Group (G), Eosinophilia or Bronchial Asthma and the like are defined in the same manner as in the above "1. Test method for eosinophilia or bronchial asthma".
  • Inhibitors include, for example, antibodies against target proteins.
  • the antibody the same antibody as explained in the above “4. Test agent and test kit for eosinophilia or bronchial asthma” can be used.
  • Another example of an inhibitor is a target protein expression inhibitor.
  • the agent for suppressing the expression of the target protein is not particularly limited as long as it can suppress the expression level of the target protein, its mRNA, its precursor, etc.
  • examples include gene-specific small interfering RNA (siRNA) of the target protein, target Gene-specific microRNA (miRNA) for proteins, gene-specific antisense nucleic acids for target proteins, expression vectors for these; gene-specific ribozymes for target proteins; gene editing agents for target proteins by CRISPR/Cas system, etc.
  • the expression suppression means that the expression level of the target protein, its mRNA, etc. , 1/200, 1/300, 1/500, 1/1000, 1/10000 or less, and also includes setting the expression level to zero.
  • Enhancers include, for example, target protein expression enhancers.
  • the agent for enhancing the expression of the target protein is not particularly limited as long as it can enhance the expression level of the target protein, its mRNA, its precursor, etc.
  • Examples include expression vectors for the target protein.
  • expression enhancement refers to enhancing the expression level of the target protein, its mRNA, etc., for example, 2, 3, 5, 10, 20, 30, 50, 100, 200, 300, 500, 1000, 10000 times. means.
  • the gene siRNA of the target protein is not particularly limited as long as it is a double-stranded RNA molecule that specifically suppresses the expression of the gene encoding the target protein.
  • the siRNA is preferably, for example, 18 bases or longer, 19 bases or longer, 20 bases or longer, or 21 bases or longer.
  • the siRNA preferably has a length of, for example, 25 bases or less, 24 bases or less, 23 bases or less, or 22 bases or less. Any combination of the upper and lower limits of the siRNA length described herein is assumed.
  • siRNA may be shRNA (small hairpin RNA).
  • a portion of shRNA can be designed to form a stem-loop structure.
  • sequence a if the sequence of a certain region is sequence a, and the complementary strand to sequence a is sequence b, these sequences are present in one RNA strand in the order of sequence a, spacer, and sequence b. and can be designed to be 45-60 bases in length overall.
  • Sequence a is a sequence of a partial region of the base sequence encoding the target protein of interest, and the target region is not particularly limited, and any region can be a candidate.
  • the length of sequence a is 19-25 bases, preferably 19-21 bases.
  • the gene-specific siRNA of the target protein may have additional bases at the 5' or 3' end.
  • the length of the additional bases is usually about 2-4 bases.
  • the additional base may be either DNA or RNA, but using DNA may improve the stability of the nucleic acid.
  • Such additional base sequences include, for example, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', tttt-3 ', uuuuuu-3', and the like, but are not limited to these.
  • the siRNA may have an overhang sequence (overhang) at the 3' end, and specifically includes those with dTdT (dT represents deoxythymidine) added. It may also be a blunt end (blunt end) without terminal addition.
  • the siRNA may have a different number of bases in the sense strand and the antisense strand.
  • aiRNA asymmetrical interfering RNA
  • a typical aiRNA consists of an antisense strand of 21 nucleotides, a sense strand of 15 nucleotides, and an overhang structure of 3 nucleotides at each end of the antisense strand.
  • the position of the target sequence of the gene-specific siRNA of the protein of interest is not particularly limited, but in one embodiment, the target sequence is from the 5'-UTR and the start codon to about 50 bases and from a region other than the 3'-UTR. Sequence selection is preferred. BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ ) to confirm the specificity of the selected target sequence.
  • a sense strand having a TT or UU 3' overhang at 19-21 bases after AA (or NA), a sequence complementary to the 19-21 bases and TT or A double-stranded RNA consisting of an antisense strand having a UU 3'-terminal overhang may be designed as an siRNA.
  • siRNA which is a precursor of siRNA
  • an arbitrary linker sequence (for example, about 5 to 25 bases) capable of forming a loop structure is appropriately selected, and the sense strand and antisense strand are connected via the linker sequence. It can be designed by concatenating.
  • siRNA and/or shRNA sequences can be searched using search software provided free of charge on various websites. Examples of such sites include the following. siRNA Target Finder provided by Ambion (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) Insert design tool for pSilencer® Expression Vector (http://www.ambion.com/ jp/techlib/misc/psilencer_converter.html) GeneSeer provided by RNAi Codex (http://codex.cshl.edu/scripts/newsearchhairpin.cgi).
  • siRNA is prepared by synthesizing the sense strand and antisense strand of the target sequence on mRNA with an automatic DNA/RNA synthesizer, and denaturing them in an appropriate annealing buffer at about 90 to about 95°C for about 1 minute. It can be prepared by annealing at about 30 to about 70° C. for about 1 to about 8 hours. It can also be prepared by synthesizing shRNA, which is a precursor of siRNA, and cleaving it with the RNA-cleaving protein dicer.
  • the gene-specific miRNA for the target protein is optional as long as it inhibits translation of the gene encoding the target protein.
  • miRNAs may bind to the 3' untranslated region (UTR) of the target and inhibit its translation, rather than cleaving the target mRNA as siRNAs do.
  • miRNA may be pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA), or mature miRNA.
  • the length of miRNA is not particularly limited, and the length of pri-miRNA is usually several hundred to several thousand bases, the length of pre-miRNA is usually 50-80 bases, and the length of mature miRNA is usually 18 bases. ⁇ 30 bases.
  • the gene-specific miRNA of the protein of interest is preferably pre-miRNA or mature miRNA, more preferably mature miRNA.
  • Such gene-specific miRNAs of target proteins may be synthesized by known methods or purchased from companies that provide synthetic RNAs.
  • a gene-specific antisense nucleic acid for a target protein is a nucleic acid comprising a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the mRNA of the gene encoding the target protein, or a part thereof, and is specific to the mRNA. It is a nucleic acid that has the function of inhibiting target protein synthesis by forming and binding to a stable double strand.
  • Antisense nucleic acids can be DNA, RNA, or DNA/RNA chimeras. When the antisense nucleic acid is DNA, an RNA:DNA hybrid formed by the target RNA and the antisense DNA is recognized by endogenous ribonuclease H (RNase H) to cause selective degradation of the target RNA.
  • RNase H endogenous ribonuclease H
  • the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of the gene of interest protein.
  • the intron sequence can be determined by comparing the genomic sequence and the cDNA nucleotide sequence of the gene of the target protein using homology search programs such as BLAST and FASTA.
  • the target region of the gene-specific antisense nucleic acid of the target protein is not limited in length as long as the hybridization of the antisense nucleic acid results in inhibition of translation into the target protein.
  • a gene-specific antisense nucleic acid for a protein of interest may be the entire sequence or a partial sequence of the mRNA encoding the protein of interest. Oligonucleotides of about 10 to about 40 bases, particularly about 15 to about 30 bases, are preferred in consideration of ease of synthesis, antigenicity, intracellular translocation, etc., but are not limited to these.
  • the 3' end hairpin loop or the like can be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.
  • the gene-specific antisense nucleic acid of the target protein not only hybridizes with the mRNA or initial transcript of the target protein gene to inhibit translation into protein, but also binds to these genes, which are double-stranded DNA. It may be one that can form a triplex and inhibit transcription to RNA (antigene).
  • nucleotide molecules that make up the gene-specific siRNA of the protein of interest, the gene-specific miRNA of the protein of interest, and the gene-specific antisense nucleic acid of the protein of interest described above have stability (chemical and/or enzymatic) and specific activity ( Various chemical modifications may be included in order to improve affinity with RNA).
  • the phosphate residue (phosphate) of each nucleotide that constitutes the antisense nucleic acid is replaced with, for example, phosphorothioate (PS), methylphosphonate, phosphorodithioate. can be substituted with chemically modified phosphate residues such as phosphorodithioates.
  • the base moiety pyrimidine, purine
  • part of the nucleotide molecules that constitute siRNA or miRNA may be replaced with natural DNA.
  • Gene-specific siRNAs for proteins of interest, gene-specific miRNAs for proteins of interest, and gene-specific antisense nucleic acids for proteins of interest, etc. target mRNA or early transcripts based on the cDNA or genomic DNA sequence of the gene for the protein of interest. It can be prepared by determining the sequence and synthesizing a complementary sequence using a commercially available DNA/RNA automatic synthesizer. In addition, antisense nucleic acids containing the various modifications described above can also be chemically synthesized by known techniques.
  • the expression vector comprises a promoter sequence and a gene-specific siRNA for the protein of interest, a gene-specific miRNA for the protein of interest, a gene-specific antisense nucleic acid for the protein of interest, or a coding sequence for the protein of interest (optionally and a transcription termination signal sequence), optionally other sequences.
  • the promoter is not particularly limited, for example, RNA polymerase II (polII) promoters such as CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, ⁇ -actin promoter, CAG promoter; mouse and human U6-snRNA promoters; Examples thereof include RNA polymerase III (polIII) promoters such as human H1-RNase P RNA promoter and human valine-tRNA promoter.
  • Other sequences are not particularly limited, and various known sequences that can be contained in an expression vector can be employed. Examples of such sequences include origins of replication, drug resistance genes, and the like.
  • the types of drug-resistant genes and the types of vectors can be exemplified by those described above.
  • ribozyme in the narrow sense means RNA having enzymatic activity to cleave nucleic acid, but in the present application, it also includes DNA as long as it has sequence-specific nucleic acid cleaving activity.
  • the most versatile ribozyme nucleic acids are self-splicing RNAs found in infectious RNAs such as viroids and virusoids, and hammerhead and hairpin types are known.
  • the hammerhead type exhibits enzymatic activity at about 40 bases, and a few bases at each end (about 10 bases in total) adjacent to the part that forms the hammerhead structure are linked to the sequence complementary to the desired cleavage site of the mRNA. By doing so, it is possible to specifically cleave only the target mRNA.
  • This type of ribozyme nucleic acid has the advantage that it does not attack genomic DNA because it uses only RNA as a substrate.
  • the target sequence is converted to a single strand by using a hybrid ribozyme that ligates an RNA motif derived from a viral nucleic acid that can specifically bind to RNA helicase.
  • a hybrid ribozyme that ligates an RNA motif derived from a viral nucleic acid that can specifically bind to RNA helicase.
  • the application target of the agent of the present invention is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer. .
  • the form of the drug of the present invention is not particularly limited, and can take any form commonly used for each application depending on the use of the drug of the present invention.
  • Forms include, for example, tablets (orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly-like drops, etc.) when the application is pharmaceuticals, health-promoting agents, nutritional supplements (supplements, etc.), etc. ), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), jelly preparations suitable for oral intake forms (oral formulations), formulations suitable for parenteral intake such as nasal drops, inhalants, rectal suppositories, inserts, enemas, jelly, injections, patches, lotions, cream oral dosage forms).
  • liquid, gel or solid food such as juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, furikake, powdered milk for infants , cake mixes, powdered or liquid dairy products, breads, cookies, etc.
  • liquid for example, liquid (solution, milky lotion, suspension, etc.), semisolid (gel, cream, paste, etc.), solid (tablet, granule, capsule, Film agent, kneaded product, molten solid, waxy solid, elastic solid, etc.), more specifically, dentifrice (toothpaste, liquid dentifrice, liquid dentifrice, toothpaste, etc.), mouthwash, Coating agents, patches, mouth fresheners, foods (eg, chewing gum, tablet candy, candy, gummies, films, lozenges, etc.) and the like.
  • the drug of the present invention may further contain other ingredients as necessary.
  • Other ingredients are not particularly limited as long as they are ingredients that can be blended in pharmaceuticals, food compositions, oral compositions, health-promoting agents, nutritional supplements (supplements, etc.). , carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, perfumes, chelating agents and the like.
  • the total content of the suppressor and enhancer for the target protein of the drug of the present invention depends on the type of suppressor and enhancer, application, mode of use, application target, condition of application target, etc., and the limitation is However, it can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.
  • the amount of application (for example, administration, ingestion, inoculation, etc.) of the composition of the present invention is not particularly limited as long as it is an effective amount that exhibits efficacy. mg/kg body weight.
  • the above dosage is preferably administered once or divided into 2 to 3 times a day, and can be adjusted appropriately according to age, condition and symptoms.
  • a screening method for an active ingredient of a preventive or therapeutic agent for eosinophilia or bronchial asthma Protein Group (A), Protein Group (B), Protein Group (C), Protein Group (D), Protein Group (E), Protein Group (F), and Protein Group (G)
  • a screening method for an active ingredient (or a candidate substance thereof) of a prophylactic or therapeutic agent for eosinophilia or bronchial asthma using the amount or concentration of at least one protein as an indicator (herein referred to as "the (also referred to as “active ingredient screening method”). This will be explained below.
  • Hyperacidophilia or bronchial asthma measurement of the amount or concentration of the target protein, etc. are defined in the above "1. Test method for eosinophilia or bronchial asthma”.
  • animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.
  • test substances can be used, regardless of whether they are naturally occurring compounds or artificially created compounds.
  • purified compounds but also compositions in which various compounds are mixed, and extracts of animals and plants can be used.
  • Compounds include not only low-molecular-weight compounds, but also high-molecular-weight compounds such as proteins, nucleic acids, and polysaccharides.
  • the active ingredient screening method of the present invention comprises at least one selected from the group consisting of protein group (A), protein group (B), protein group (D), and protein group (F).
  • a test substance is eosinophilic if the value of the indicator for the protein is lower than the amount or concentration of the corresponding protein in extracellular vesicles of bodily fluids or blood samples taken from animals not treated with the test substance.
  • the test substance is eosinophilic if the value of the indicator for the protein is higher than the amount or concentration of the corresponding protein in extracellular vesicles of bodily fluids or blood samples taken from animals not treated with the test substance. It comprises at least one step selected from the group consisting of the steps of selecting an active ingredient for a prophylactic or therapeutic agent for bloating or bronchial asthma.
  • a corresponding protein means the same protein as the target protein used as an index.
  • Low means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.
  • High means, for example, that the index value is 2, 5, 10, 20, 50, or 100 times the control value.
  • a method for evaluating the induction or exacerbation of eosinophilia or bronchial asthma is characterized by: At least selected from the group consisting of protein group (A), protein group (B), protein group (C), protein group (D), protein group (E), protein group (F), and protein group (G)
  • a method for evaluating the induction or exacerbation of eosinophilia or bronchial asthma using the amount or concentration of one kind of protein as an index in the present specification, it may be referred to as the "toxicity evaluation method of the present invention" .
  • the toxicity evaluation method of the present invention includes at least one protein selected from the group consisting of protein group (A), protein group (B), protein group (D), and protein group (F). is greater than the amount or concentration of the corresponding protein in extracellular vesicles of bodily fluids or blood samples taken from animals not treated with the test substance Determining proliferative disease or bronchial asthma provocation or exacerbation, and at least one protein selected from the group consisting of protein group (C), protein group (E), and protein group (G) is lower than the amount or concentration of the corresponding protein in extracellular vesicles of bodily fluids or blood samples taken from animals not treated with the test substance It comprises at least one step selected from the group consisting of the steps of judging proliferative or exacerbating polycythemia or bronchial asthma.
  • a corresponding protein means the same protein as the target protein used as an index.
  • High means, for example, that the index value is 2, 5, 10, 20, 50, or 100 times the control value.
  • Low means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.
  • Test example 1 Preparation of extracellular vesicle fraction Serum from 8 human subjects diagnosed with bronchial asthma (4 with eosinophilic asthma, 4 with atopic asthma) and 4 healthy human subjects ) Extracellular vesicle fractions were prepared from each serum. The extracellular vesicle fraction was prepared using an extracellular vesicle preparation kit (MagCapturTM Exosome Isolation Kit PS, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) with the same volume of serum for each sample. Ta.
  • MagCapturTM Exosome Isolation Kit PS manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
  • the extracellular vesicle particle count and particle size were measured for the obtained extracellular vesicle fraction. Specifically, it was measured using Nanosite (Nippon Quantum Design Co., Ltd., Nanoparticle Tracking Analysis (NTA) Version 2.3 Build 0025). This is an analysis based on the difference in Brownian motion speed for each particle size. The particle size (hydrodynamic diameter) in can be calculated.
  • extracellular vesicles were observed by immunoelectron microscopy. Specifically, it was carried out as follows. 5 to 8 ⁇ L of the fixed extracellular vesicle solution was placed on the grid and allowed to stand for 15 minutes to allow the extracellular vesicles to settle on the formvar of the grid. After washing with PBS three times, a blocking reaction (1% BSA/PBS, 10 minutes) was performed, followed by a primary antibody reaction (Invitrogen AHS0902 Mouse (monoclonal) Anti-Human Leukemia and Platelet Associated Antigen CD9 Clone: MM2/57, 100-fold dilution, 2.5 hours, room temperature).
  • a blocking reaction 1% BSA/PBS, 10 minutes
  • extracellular vesicle fractions were Western blotted using antibodies against exosome markers (anti-Flotillin-1 antibody, anti-CD9 antibody, and anti-CD81 antibody).
  • Test example 2 Proteomics analysis (DIA proteome analysis) Proteins in the extracellular vesicle fraction obtained in Test Example 1 were quantified by DIA proteome analysis. Specifically, it was carried out as follows.
  • Digested peptides were precipitated, acidified with 30 ⁇ L of 5% trifluoroacetic acid to remove detergent, and samples were centrifuged at 17,400 ⁇ g for 15 minutes. The supernatant was desalted using a C18-StageTip, dried using a centrifugal evaporator, and the dried peptide was dissolved in 3% ACN and 0.1% FA.
  • Peptides were injected directly into a 75 ⁇ m ⁇ 20 cm PicoFrit emitter (New Objective) packed with C18 core-shell particles (CAPCELL CORE MP 2.7 ⁇ m, 160 ⁇ material; Osaka Soda Co., Ltd.) at 50 °C and analyzed using an UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific) at a flow rate of 100 nl/min with a gradient of 90 minutes.
  • MS1 spectra were collected at a resolution of 30,000 over the range 495-785 m/z to set an automatic gain control target of 3e6 and a maximum injection time of 55.
  • MS2 collected spectra over 200 m/z at a resolution of 15,000 to set the automatic gain control target to 3e6, the maximum injection time to "auto", and the stepped normalized collision energies to 24, 26, and 28%.
  • the MS2 isolation width was set to 4 m/z and the 500-780 m/z overlapping window pattern used the optimized window placement in Skyline (PMID: 20147306).
  • MS files were searched against the mouse spectral library using Scaffold DIA (Proteome Software, Inc., Portland, OR).
  • Mouse spectral libraries were generated from the human protein sequence database (UniProt id UP000005640, reviewed, canonical) by Prosit (PMID: 32214105, PMID: 31133760).
  • the search parameters for Scaffold DIA were: experimental data search enzyme, trypsin, maximum deletion cleavage site, 1, precursor mass tolerance, 8 ppm, fragment mass tolerance, 10 ppm, static modification, cysteine carbamidomethylation.
  • the protein identification threshold was set to less than 1% for both peptide and protein false discovery rates.
  • Peptide quantification was calculated with Scaffold DIA's EncyclopeDIA algorithm (PMID: 30510204). For each peptide, the four highest quality fragment ions were selected and quantified. Protein quantification was estimated from summed peptide quantification.
  • Proteins serving as biomarkers for eosinophilia or bronchial asthma were extracted from this according to the following criteria.
  • Table 2 shows proteins whose expression levels in all bronchial asthma subjects were lower than in healthy subjects.
  • "Fold change” is the value obtained by dividing the average expression level of healthy subjects by the average expression level of all subjects with bronchial asthma (average expression level of healthy subjects / expression level of all subjects with bronchial asthma). amount average value).
  • Table 5 shows proteins whose expression levels in subjects with eosinophilic asthma were higher than those in healthy subjects, among the extracted biomarkers for eosinophilia or bronchial asthma.
  • "Fold change” is the value obtained by dividing the average expression level of subjects with eosinophilic asthma by the average expression level of healthy subjects (mean expression level of subjects with eosinophilic asthma/ The expression level average value of the subject) is shown.
  • Galectin-10 Eosinophil peroxidase, Bone marrow proteoglycan, Arachidonate 15-lipoxygenase, and Non-secretory ribonuclease could be used to differentiate from atopic asthma (Fig. 4). .
  • Test example 3 Evaluation of eosinophilia or bronchial asthma biomarkers1 Galectin-10, bone marrow proteoglycan, eosinophil peroxidase, non-secretory ribonuclease, and eosinophil cationic protein expression levels of eosinophilia or bronchial asthma biomarkers and blood eosinophils in each sample of Test Example 1 Based on the numbers, correlation coefficients between the expression levels of eosinophilia or bronchial asthma biomarkers and the number of eosinophils were calculated.
  • the correlation coefficient between the expression level of Galectin-10 and the eosinophil count was 0.95
  • the correlation coefficient between the expression level of bone marrow proteoglycan and the eosinophil count was 0.93
  • the expression level of eosinophil peroxidase was 0.93
  • the eosinophil count was 0.88
  • the correlation coefficient between the expression level of non-secretory ribonuclease and the eosinophil count was 0.88
  • the correlation coefficient between the expression level of eosinophil catic protein and the eosinophil count was 0.88.
  • the correlation coefficient was 0.44.
  • Test example 4 Evaluation of eosinophilia or bronchial asthma biomarkers2 Based on the quantitative results of Test Example 2, an ROC curve was prepared when Galectin-10 was used alone as a biomarker for bronchial asthma. Also, as a positive control, an ROC curve was prepared for the same subject using the blood eosinophil count as a bronchial asthma biomarker. Specifically, an ROC curve with sensitivity (positive rate) on the vertical axis and a value obtained by subtracting specificity from 1 (1 - specificity) (false positive rate) on the horizontal axis was created using the statistical software JMP. .
  • the AUC was 0.96875 when Galectin-10 was used as a biomarker, and the AUC was 0.93750 when the blood eosinophil count was used as a biomarker. From this, it was found that galectin-10 has a higher diagnostic ability for asthma than the eosinophil count.
  • Test example 5 Evaluation of eosinophilia or bronchial asthma biomarkers3
  • the correlation coefficient between the expression level of biomarkers for eosinophilia or bronchial asthma such as Galectin-10, blood eosinophil count, and respiratory function (FEV1%) was calculated for each sample in Test Example 1. .
  • the correlation coefficient between the expression level of Galectin-10 and FEV1% was -0.8344 (p-value 0.0014), and the correlation coefficient between eosinophil count and FEV1% was -0.8073 (p-value 0.0027).
  • Ta Other eosinophilia or bronchial asthma biomarkers were also correlated with FEV1%.
  • Test example 6 Evaluation of eosinophilia or bronchial asthma biomarkers4 Refractory asthma may be complicated by eosinophilic sinusitis. Eosinophilic sinusitis is classified into chronic rhinosinusitis with nasal polyp (CRSwNP), which has a poor prognosis, and chronic rhinosinusitis without nasal polyp (CRSwNP), which has a favorable prognosis. polyp: CRSsNP). Therefore, we investigated whether it is possible to distinguish between these by eosinophilia or bronchial asthma biomarkers.
  • Extracellular vesicle fractions (Exosomes) and tissues (inferior turbinate and polyp) were collected from 7 CRSwNP human subjects and 6 CRSsNP human subjects in the same manner as in Test Example 1, and Galectin- Expression levels of eosinophilia or bronchial asthma biomarkers such as 10 were measured. The results are shown in FIGS. 1 and 2.
  • FIG. Galectin-10 was found to be increased in exosome and tissue therapy of CRSwNP.
  • the correlation coefficient between the number of blood eosinophils and the Galectin-10 expression level in tissues, and the correlation coefficient between the Galectin-10 expression level in exosomes and the Galectin-10 expression level in tissues were calculated.
  • the correlation coefficient between blood eosinophil count and Galectin-10 expression level in tissues was 0.5409 (p-value 0.0858), indicating that Galectin-10 expression level in exosomes and Galectin-10 expression level in tissues was 0.6709 (p-value 0.0337). From this, it was found that the Galectin-10 expression level in exosomes is strongly correlated with the Galectin-10 expression level in tissues rather than the blood eosinophil count.
  • the correlation coefficient between blood eosinophil count and tissue eosinophil infiltration (/HPF), and the correlation coefficient between Galectin-10 expression level in exosomes and tissue eosinophil infiltration (/HPF) Calculated.
  • the correlation coefficient between the number of blood eosinophils and tissue eosinophil infiltration (/HPF) was 0.6053 (p-value 0.0284)
  • the expression level of Galectin-10 in exosomes and tissue eosinophil infiltration (/ HPF) was 0.7534 (p-value 0.0119). From this, it was found that the Galectin-10 expression level in exosomes is strongly correlated with tissue eosinophil infiltration rather than the blood eosinophil count.
  • Test example 7 Evaluation of eosinophilia or bronchial asthma biomarkers5 It has been reported that suppression of EETosis suppresses exacerbation of asthma in a mouse asthma model (Nature Cell Biology volume 23, pages 1060-1072 (2021)). Therefore, for the subject of Test Example 6, the number of EETosis-positive eosinophils (/HPF) in the tissue was measured according to the method described in the above document, and the number of blood eosinophils and the expression level of Galectin-10 in exosomes The correlation coefficient of was calculated.
  • the correlation coefficient between the number of blood eosinophils and the number of EETosis-positive eosinophils in tissues was 0.5522 (p value 0.0504).
  • the correlation coefficient with EETosis-positive eosinophil count (/HPF) was 0.7886 (p-value 0.0067). From this, it was found that the Galectin-10 expression level in exosomes correlated more strongly with the number of EETosis-positive eosinophils than the number of blood eosinophils.
  • Test example 8 Evaluation of eosinophilia or bronchial asthma biomarkers6
  • eosinophil activation increases Galectin-10 in exosomes Specifically, according to previous reports (Nat Protoc. 2006; 1(6): 2613-20., Blood. 2013 Mar 14; 121(11): 2074-83.), eosinophils were isolated and EETosis was induced. After that, Western blotting of supernatant-derived exosomes was performed.
  • Galectin-10 was also detectable by immunoblotting, and it was found that asthma, normal, and COPD could be determined.
  • Galectin-10 was found to increase in asthmatic lung tissue.

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Abstract

La présente invention aborde le problème consistant à fournir un biomarqueur de l'asthme éosinophilique ou bronchique, et un procédé d'utilisation du biomarqueur. Ledit problème est résolu par un procédé de test de l'asthme éosinophilique ou bronchique, le procédé comprenant : (1) une étape de détection d'au moins une protéine sélectionnée dans le groupe constitué par un groupe de protéines (A), un groupe de protéines (B), un groupe de protéines (C), un groupe de protéines (D), un groupe de protéines (E), un groupe de protéines (F) et un groupe de protéines (G) dans des vésicules extracellulaires d'un fluide corporel ou dans le fluide corporel recueilli à partir d'un sujet.
PCT/JP2023/004548 2022-02-10 2023-02-10 Biomarqueur de l'asthme éosinophilique ou bronchique WO2023153496A1 (fr)

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JP2005531754A (ja) * 2002-05-15 2005-10-20 ナン、ドン−ホ 気管支喘息及び慢性鼻炎患者におけるサイトケラチン１８蛋白質に対する自己抗体の検出、及び哺乳動物のサイトケラチン１８蛋白質を含む気管支喘息及び慢性鼻炎診断用キットを含む用途
JP2017038553A (ja) * 2015-08-20 2017-02-23 国立大学法人 千葉大学 喘息の診断用マイクロｒｎａ
WO2020004557A1 (fr) * 2018-06-27 2020-01-02 国立大学法人大阪大学 Biomarqueurs de maladies pulmonaires obstructives
JP2021520825A (ja) * 2018-04-13 2021-08-26 アルゲン−エックス ビーブイビーエー ガレクチン−１０抗体

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JP2005531754A (ja) * 2002-05-15 2005-10-20 ナン、ドン−ホ 気管支喘息及び慢性鼻炎患者におけるサイトケラチン１８蛋白質に対する自己抗体の検出、及び哺乳動物のサイトケラチン１８蛋白質を含む気管支喘息及び慢性鼻炎診断用キットを含む用途
JP2017038553A (ja) * 2015-08-20 2017-02-23 国立大学法人 千葉大学 喘息の診断用マイクロｒｎａ
JP2021520825A (ja) * 2018-04-13 2021-08-26 アルゲン−エックス ビーブイビーエー ガレクチン−１０抗体
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