WO2018168779A1 - Biomarqueur de la bronchopneumopathie chronique obstructive - Google Patents

Biomarqueur de la bronchopneumopathie chronique obstructive Download PDF

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WO2018168779A1
WO2018168779A1 PCT/JP2018/009529 JP2018009529W WO2018168779A1 WO 2018168779 A1 WO2018168779 A1 WO 2018168779A1 JP 2018009529 W JP2018009529 W JP 2018009529W WO 2018168779 A1 WO2018168779 A1 WO 2018168779A1
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protein
apolipoprotein
chronic obstructive
obstructive pulmonary
pulmonary disease
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PCT/JP2018/009529
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English (en)
Japanese (ja)
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吉人 武田
淳 熊ノ郷
太郎 木庭
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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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • 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
    • 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 a biomarker for chronic obstructive pulmonary disease, use thereof and the like.
  • COPD chronic obstructive pulmonary disease
  • COPD biomarkers In the ECLIPSE study, fibrinogen, CC16, SP-D, CCL-18, and sRAGE are shown to be useful as COPD biomarkers.
  • COPD patients not only increased in inflammatory biomarkers such as CRP, fibrinogen, IL-6, IL-8, etc. in peripheral blood, but also a group of COPD patients (phenotype) showing chronic inflammation was acute It has been shown that there are many exacerbations and poor prognosis.
  • high fibrinogen levels not only reflect the pathology of COPD but also correlate with symptoms and exacerbations, and that fibrinogen increases also during acute exacerbations.
  • Non-Patent Document 1 a recent paper on biomarkers that can distinguish COPD and asthma (Non-Patent Document 1) tries to analyze dozens of patients, but the analysis is based on the old mass spectrometry (MS) technique based on two-dimensional electrophoresis.
  • MS mass spectrometry
  • Extracellular vesicles are membrane vesicles that are secreted from cells, and carry information between cells locally and throughout the body by transporting intracellular proteins and genetic information (mRNA, microRNA, etc.) to the outside of the cell. ing. In recent years, extracellular vesicles have attracted attention as cancer test samples, but their usefulness as test samples for respiratory diseases and further COPD chronic obstructive pulmonary disease is not known.
  • An object of the present invention is to provide a biomarker for chronic obstructive pulmonary disease and a method for using the same.
  • the present invention includes the following aspects.
  • a method for testing chronic obstructive pulmonary disease comprising: (1) Protein group (A) in extracellular vesicles of body fluid collected from the subject: (A) Apolipoprotein (a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G / H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor H-related protein 5, Cytochrome b-245 heavy chain, Oncoprotein-induced transcript 3 protein, EGF-containing fibulin-like extracellular matrix protein 1, Zinc finger protein basonuclin-2, Apolipoprotein E, HLA class I histocompatibility antigen, B-38 alpha chain, CD99 antigen, Complement factor H-related protein 1, Apolipoprotein A-II, Kininogen-1, Serum paraoxonase / arylesterase 1, Fibulin-1, Integrin beta-2, Alpha-lactalbumin
  • Item 3 Item 2.
  • the cut-off value is 1 to 1.5 times the value of the amount or concentration of the corresponding protein in extracellular vesicles of body fluid collected from a subject not suffering from chronic obstructive pulmonary disease Inspection method described in 1.
  • Item 4. The examination method according to any one of Items 1 to 3, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum.
  • the protein (A) group is (A1) Apolipoprotein (a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G / H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor Item 5.
  • the examination method according to any one of Items 1 to 4, which is a group consisting of H-related protein 5 and Cytochrome b-245 heavy chain.
  • the protein (A) group is (A2) Fibulin-1, Integrin beta-2, CD99 antigen, Peroxiredoxin-2, Clusterin, Prostaglandin G / H synthase 1, Vesicle-associated membrane protein 3, Ras-related protein Rab-5C, And EGF-containing fibulin-like extracellular matrix protein 1 Item 5.
  • the inspection method according to any one of Items 1 to 4.
  • Item 7. The examination method according to any one of Items 1 to 6, wherein the subject is a human.
  • Item 11 Prevention of chronic obstructive pulmonary disease when the value of the indicator is lower than the amount or concentration of the corresponding protein in the extracellular vesicles of a body fluid collected from an animal not treated with the test substance Item 11.
  • the screening method according to Item 10 which comprises a step of selecting as an active ingredient of a therapeutic agent.
  • a biomarker for chronic obstructive pulmonary disease can be provided.
  • a test for chronic obstructive pulmonary disease and the like can be performed more simply, more efficiently, and at a lower cost.
  • the use of the biomarker may enable prevention or treatment of chronic obstructive pulmonary disease, screening of an active ingredient of a preventive or therapeutic agent for chronic obstructive pulmonary disease, and the like.
  • the number of particles and the particle size of the extracellular vesicle fraction are shown (Test Example 1).
  • Normal shows the result of the extracellular vesicle fraction obtained from a healthy subject
  • COPD shows the result of the extracellular vesicle fraction obtained from a subject with chronic obstructive pulmonary disease.
  • An electron microscope observation image of the extracellular vesicle fraction is shown (Test Example 1).
  • Normal shows the result of the extracellular vesicle fraction obtained from a healthy subject
  • COPD shows the result of the extracellular vesicle fraction obtained from a subject with chronic obstructive pulmonary disease.
  • the result of the western blotting of an extracellular vesicle fraction is shown (Test Example 1).
  • Normal shows the result of the extracellular vesicle fraction obtained from a healthy subject
  • COPD shows the result of the extracellular vesicle fraction obtained from a subject with chronic obstructive pulmonary disease.
  • the antigen of the primary antibody used is shown on the left side of the band photograph.
  • the result of the Western blot of an extracellular vesicle fraction is shown (Test Example 3). Healthy shows the result of the extracellular vesicle fraction obtained from a healthy subject, and COPD shows the result of the extracellular vesicle fraction obtained from a subject with chronic obstructive pulmonary disease.
  • the antigen of the primary antibody used is shown on the left side of the band photograph.
  • the result of immunostaining of Tissue array is shown (Test Example 4).
  • Control shows the result of Tissue array derived from healthy subjects
  • COPD shows the result of TissueTarray derived from subjects with chronic obstructive pulmonary disease
  • pulmonary fibrosis shows the results of TissueTarray derived from subjects with pulmonary fibrosis.
  • the antigen of the primary antibody used is shown on the left side of the stained photograph.
  • the present invention is a method for testing chronic obstructive pulmonary disease, comprising: (1) A test method (in this specification, including a step (step 1) of detecting at least one protein selected from the protein group (A) in an extracellular vesicle of a body fluid collected from a subject. It may be referred to as “the method for examining chronic obstructive pulmonary disease of the present invention” This will be described below.
  • the “chronic obstructive pulmonary disease” to be examined is not particularly limited, and may be either emphysema type or non-emphysema type.
  • the subject is a target organism of the inspection method of the present invention, and its species is not particularly limited.
  • the biological species of the subject include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, and preferably mice.
  • the state of the subject is not particularly limited.
  • the subject for example, it is unknown whether or not it suffers from chronic obstructive pulmonary disease, it has already been determined by another method that it suffers from acute pulmonary inflammatory disease, but chronic obstructive pulmonary disease and acute Specimens that have already been determined by another method as having pulmonary inflammatory disease, Specimens that have already been determined by another method as not having chronic obstructive pulmonary disease, Treatment of chronic obstructive pulmonary disease Samples in the inside.
  • Body fluid is not particularly limited.
  • the body fluid include whole blood, serum, plasma, spinal fluid, saliva, joint fluid, urine, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal discharge, and preferably whole blood, serum. , Plasma and cerebrospinal fluid, and more preferably whole blood, serum and plasma.
  • Body fluids may be used alone or in combination of two or more.
  • Body fluid can be collected from the subject by methods 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 obtained by removing blood cells and a specific blood coagulation factor from whole blood, and can be obtained, for example, as a supernatant after coagulating whole blood.
  • Plasma is a part obtained by removing blood cells from whole blood, and can be obtained, for example, as a supernatant when subjected to centrifugation under conditions that do not coagulate whole blood.
  • Extracellular vesicles are not particularly limited as long as they are membrane vesicles secreted and released from cells.
  • Extracellular vesicles are usually defined as membrane vesicles that carry information between cells locally and throughout the body by transporting intracellular proteins and genetic information (mRNA, microRNA, etc.) to the outside of the cell.
  • the Examples of extracellular vesicles include exosomes, microvesicles, apoptotic bodies, ectosomes, microparticles, and secretory microvesicles.
  • Extracellular vesicles can be purified, separated, concentrated, etc. from body fluids according to or according to known methods.
  • methods for purifying, separating and concentrating extracellular vesicles include, for example, ultracentrifugation (eg, pellet down method, sucrose cushion method, density gradient centrifugation method, etc.), a method using an immunoaffinity carrier, gel filtration, Examples thereof include a flow fractionation method and a FACS method.
  • purification, separation, concentration and the like of extracellular vesicles can be performed using a commercially available kit. These methods may be employed singly or in combination of two or more.
  • the detection target in step (1) is at least one protein selected from the protein group (A) (in the present specification, these may be collectively referred to as “target protein”).
  • Protein group (A) consists of (A) Apolipoprotein (a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G / H synthase 1, Apolipoprotein CI, Apolipoprotein C-III, Apolipoprotein C-II, Complement factor H-related protein 5, Cytochrome b-245 heavy chain, Oncoprotein-induced transcript 3 protein, EGF-containing fibulin-like extracellular matrix protein 1, Zinc finger protein basonuclin-2, Apolipoprotein E, HLA class I histocompatibility antigen, B-38 alpha chain, CD99 antigen, Complement factor H-related protein 1, Apolipoprotein A-II, Kininogen-1, Serum paraoxonase / arylesterase 1, Fibulin-1, Integrin beta-2, Alpha-lactalbumin, Clusterin, Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D, 60S ribosom
  • Protein group (A) is a biomarker specific to chronic obstructive pulmonary disease, and it can be used as an index to distinguish chronic obstructive pulmonary disease.
  • Apolipoprotein (a) Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M, Prostaglandin G / H synthase 1, Apolipoprotein CI , Apolipoprotein C-III, Apolipoprotein C-II, Complement factor H-related protein 5, Cytochrome b-245 heavy chain, etc., more preferably Apolipoprotein (a), Elongation factor 1-alpha 2, Apolipoprotein D, Apolipoprotein M , Prostaglandin G / H synthase 1 and the like.
  • Fibulin-1 Fibulin-1, Integrin beta-2, CD99 antigen, Peroxiredoxin-2, Clusterin, Prostaglandin G / H synthase are preferable from the viewpoint of relationship with inflammation and membrane protein.
  • Vesicle-associated membrane protein 3 Ras-related protein Rab-5C, EGF-containing fibulin-like extracellular matrix protein 1 and the like.
  • EGF-containing fibulin-like extracellular matrix protein 1 is preferable from the viewpoint of being more promising as a common biomarker for mice and humans.
  • Proteins of the protein group (A) are proteins specified by UniProtKB accession numbers shown in Table 1 in the examples described later in the case of humans. In the case of other species, it is the ortholog of the protein specified by the UniProtKB accession number shown in Table 1.
  • the number of target proteins may be only one type or a combination of two or more types.
  • more detection targets for example, 2, 5, 10, 20, and 30 or more types
  • Detecting is usually performed by measuring the amount or concentration of the target protein.
  • the “density” is not limited to the absolute density, but may be a relative density, a weight per unit volume, or raw data measured to know the absolute density.
  • the method for detecting the target protein is not particularly limited as long as it is a method capable of specifically detecting a part or all of the target protein.
  • Specific examples of the detection method include a mass spectrometry method for detecting a peptide constituting the target protein, and an immunological measurement method using an antibody that specifically recognizes the target protein.
  • the amino acid sequence information of the target protein can be obtained by searching the EBI (http://www.ebi.ac.uk/IPI/IPIhelp.html) database based on the UniProtKB accession number.
  • an immunohistochemical staining method As the immunological measurement method, an immunohistochemical staining method, ELISA method, sandwich ELISA method, EIA method, RIA method, Western blotting method and the like can be preferably exemplified.
  • a peptide sample is turned into gaseous ions using an ion source (ionization), and the peptide sample ionized by moving it in a vacuum and using electromagnetic force or by a time-of-flight difference in the analysis section is mass-charged.
  • ionization ionization
  • This is a measurement method using a mass spectrometer that can be separated and detected according to the ratio.
  • Ionization using an ion source includes the EI method, CI method, FD method, FAB method, MALDI method, and ESI method.
  • the method of separating ionized peptide samples in the analysis section can be selected as appropriate.
  • a separation method such as an ion cyclotron resonance type can be appropriately selected.
  • tandem mass spectrometry (MS / MS) or triple quadrupole mass spectrometry combining two or more mass spectrometry methods can be used.
  • the sample contains a phosphorylated peptide
  • the sample can be concentrated using iron ion-immobilized affinity chromatography (Fe-IMAC) before introducing the sample into the mass spectrometer.
  • the peptide which comprises target protein can be isolate
  • a detection part and a data processing method can also be selected suitably.
  • a peptide labeled with a stable isotope having a known amino acid sequence and having a known concentration is used as an internal standard. can do.
  • Such a stable isotope labeled peptide is a stable isotope labeled peptide in which at least one of the amino acids in the peptide constituting the target protein to be detected contains at least one of 15 N, 13 C, 18 O, and 2 H. If necessary, the type, position, number, etc. of amino acids can be appropriately selected.
  • Such stable isotope-labeled peptides can be obtained by F-moc method (Amblard., Et al. Methods) using amino acids labeled with stable isotopes.
  • Mol Biol. 298: 3-24 (2005) can be chemically synthesized by iTRAQ (registered trademark) reagent, ICAT (registered trademark) reagent, ICPL (registered trademark) reagent, NBS (registered trademark). (Trademark) It can also produce using labeling reagents, such as a reagent.
  • the amount and / or concentration of the target protein that is a detection index of chronic obstructive pulmonary disease can be provided, thereby detecting chronic obstructive pulmonary disease, etc. Can assist.
  • Test results of the test method of the present invention including the step (1) are as follows: therapeutic effect determination, elucidation of the pathology of chronic obstructive pulmonary disease, prognosis prediction of chronic obstructive pulmonary disease (respiratory function decline group, acute aversion), patient group , Selection of treatment method (individualized medicine, treatment responsiveness), intractable chronic obstructive pulmonary disease, evaluation of remodeling, differentiation of chronic obstructive pulmonary disease histology and phenotype, etc. .
  • the inspection method of the present invention further includes, as one aspect, (2) A step of determining that the subject is suffering from chronic obstructive pulmonary disease when the amount or concentration of the protein detected in the step (1) is not less than a cutoff value (step 2) It is preferable to include. According to the test method of the present invention including the step 2, it becomes possible to determine chronic obstructive pulmonary disease.
  • the cut-off value can be appropriately set by those skilled in the art from the viewpoint of sensitivity, specificity, positive predictive value, negative predictive value, and the like, for example, collected from a subject not suffering from chronic obstructive pulmonary disease. Based on the amount and / or concentration of the target protein in the extracellular vesicles of the collected body fluid, a predetermined value or a predetermined value can be obtained.
  • the cut-off value is, for example, the amount and / or concentration of the target protein in the extracellular vesicles of body fluid collected from a subject not suffering from chronic obstructive pulmonary disease (in the case of multiple subjects, the average value, For example, it can be 1 to 1.5 times the median value.
  • the cutoff value is determined, for example, the amount and / or concentration of the protein of interest in a past sample for the same sample.
  • the therapeutic effect can be determined by setting the value based on the above.
  • the test method of the present invention is used. Further, by combining the steps of applying diagnosis by a doctor of chronic obstructive pulmonary disease, it is possible to diagnose chronic obstructive pulmonary disease with higher accuracy. In addition, since the test method of the present invention can more accurately detect chronic obstructive pulmonary disease, the above-described steps are combined with the test method of the present invention more efficiently and more accurately. Can be diagnosed.
  • test method of the present invention including the treatment step (2) of chronic obstructive pulmonary disease is determined to cause the subject to suffer from chronic obstructive pulmonary disease, in addition to the test method of the present invention or the above When it is diagnosed that the patient is suffering from chronic obstructive pulmonary disease as described in "2. Diagnosis with higher accuracy of chronic obstructive pulmonary disease” And (3) subjecting the subject to be diagnosed or diagnosed as suffering from chronic obstructive pulmonary disease, by treating the subject with the disease, Can be treated.
  • the test method of the present invention can detect chronic obstructive pulmonary disease more accurately, the test method of the present invention or a combination of the test method of the present invention and a step of applying a diagnosis by a doctor. By combining step 3, a subject suffering from chronic obstructive pulmonary disease can be treated more efficiently and reliably.
  • the method for treating chronic obstructive pulmonary disease is not particularly limited, but typically includes medication.
  • the drug used for the drug treatment is not particularly limited, but for example, anticholinergic drugs such as Spiriva, Seeburi, Enclasse, Ecrila, Atrovent, Telcigan, etc .; ⁇ 2-stimulating drugs such as Serebent, Ombres, Auxis, Sultanol, Meptin; Ultimolo, Anolo And anti-cholinergic / ⁇ 2-stimulant combination drugs such as sport; steroid drugs such as cuvard, flutide, palmicoat, olvesco, and azmanex; steroid drugs / beta-stimulant combination drugs such as adair, simbicoat, and lerbea.
  • a pharmaceutical can be used 1 type, 2 types, or 3 or more types in combination.
  • test agent for chronic obstructive pulmonary disease in the present specification, a test agent for chronic obstructive pulmonary disease, comprising a detection agent for at least one protein selected from the protein group (A)) , Sometimes referred to as “inspection drug of the present invention”). This will be described below.
  • Protein group (A), chronic obstructive pulmonary disease and the like are the same as defined in “1. Method for examining chronic obstructive pulmonary disease” above.
  • the detection agent is not particularly limited as long as it can specifically detect the target protein.
  • the detection agent include an antibody against the target protein.
  • the detection agent may be labeled. Examples of the label include fluorescent substances, luminescent substances, dyes, enzymes, gold colloids, and radioisotopes. Further, the detection agent (particularly antibody) may be in a state of being adsorbed on a base material (for example, a plastic base material such as a microwell plate).
  • the antibody is not particularly limited as long as it recognizes the target protein selectively (specifically).
  • “selectively (specifically) recognize” means that the protein of interest can be specifically detected, for example, in 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.
  • the antibody includes a part of the above antibody having antigen binding properties such as a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or a Fab fragment or a fragment generated by a Fab expression library.
  • the antibody of the present invention also includes an antibody having an antigen binding property to a polypeptide consisting of at least continuous amino acid sequence of the subject protein, usually 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids.
  • the antibodies of the present invention can also be produced according to these conventional methods (Current protocol in Molecular Biology, Chapter 11.12 to 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 and purified in Escherichia coli or the like according to a conventional method, or according to a conventional method.
  • a non-human animal such as a rabbit and obtain it from the serum of the immunized animal according to a conventional method.
  • spleen cells obtained by immunizing a non-human animal such as a mouse with a target protein expressed and purified in Escherichia coli according to a conventional method, or an oligopeptide having a partial amino acid sequence of the target protein and 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 antibody production is based on known gene sequence information, DNA cloning, construction of each plasmid, transfection into a host, culture of a transformant, and recovery of the protein from the culture. It can obtain by operation of. These operations are based on methods known to those skilled in the art or methods described in the 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 the target protein in a desired host cell is prepared, introduced into the host cell, transformed, and the transformant is cultured.
  • the protein as the immunizing antigen for producing the antibody of the present invention can be obtained by recovering the target protein from the obtained culture.
  • the partial peptide of the target protein can also be produced by a general chemical synthesis method (peptide synthesis) in accordance with known gene sequence information.
  • the antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of the target protein.
  • the oligo (poly) peptide used for the production of such an antibody does not need to have a functional biological activity, but desirably has an immunogenic property similar to that of the target protein.
  • An oligo (poly) peptide preferably having this immunogenic property and consisting of at least 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of the target protein can be exemplified.
  • Such an antibody against an oligo (poly) peptide can also be produced by enhancing the immunological reaction using various adjuvants depending on the host.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfaces such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol.
  • Active substances, human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum are included.
  • the test agent of the present invention may be in the form of a composition.
  • the composition may contain other components as necessary. Examples of other components include a base, a carrier, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, a thickener, a moisturizer, a colorant, and a fragrance. And chelating agents.
  • the test agent of the present invention may be in the form of a kit.
  • the kit may contain a substance that can be used for detection of a target protein in extracellular vesicles of a body fluid of a subject.
  • reagents for example, secondary antibodies, buffers, etc.
  • instruments for example, instruments for purification, separation, and concentration of extracellular vesicles (for example, columns)).
  • the preventive or therapeutic agent for chronic obstructive pulmonary disease comprising an inhibitor of at least one protein selected from the protein group (A) (In the present specification, it may be referred to as “the agent of the present invention”). This will be described below.
  • the protein group (A) and the like are the same as defined in “1. Method for examining chronic obstructive pulmonary disease” above.
  • the inhibitor is preferably an expression inhibitor of the target protein.
  • the target protein expression inhibitor is not particularly limited as long as it can suppress the expression level of the target protein, its mRNA, etc.
  • gene-specific small interfering RNA (siRNA) of the target protein gene-specificity of the target protein MicroRNA (miRNA), gene-specific antisense nucleic acid of target protein, expression vectors thereof; gene-specific ribozyme of target protein; gene gene editing agent of target protein by CRISPR / Cas system.
  • the expression suppression refers to the expression level of the target protein, its mRNA, etc., for example, 1/2, 1/3, 1/5, 1/10, 1/20, 1/30, 1/50, 1/100 , 1/200, 1/300, 1/500, 1/1000, 1 / 10,000 or less, and the expression level of these is set to 0.
  • 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 18 or more bases, 19 or more bases, 20 or more bases, or 21 or more bases in length, for example.
  • it is preferable that siRNA is 25 bases or less, 24 bases or less, 23 bases or less, or 22 bases or less, for example. It is assumed that the upper limit value and the lower limit value of the siRNA length described here are arbitrarily combined.
  • the lower limit is 18 bases and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases; the lower limit is 19 bases, and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases A certain length; a lower limit of 20 bases and an upper limit of 25 bases, 24 bases, 23 bases, or 22 bases; a lower limit of 21 bases and an upper limit of 25 bases, 24 bases, 23 bases, or 22 Combinations of lengths that are bases are envisioned.
  • the siRNA may be shRNA (small hairpin RNA).
  • shRNA can be designed so that a part thereof forms a stem-loop structure. For example, if the sequence of a certain region is a sequence a and the complementary strand to the sequence a is a sequence b, these sequences are present in a single RNA strand in the order of sequence a, spacer, and sequence b. And can be designed to be 45-60 bases in total.
  • the sequence a is a sequence of a partial region of the base sequence encoding the target protein to be targeted, and the target region is not particularly limited, and any region can be a candidate.
  • the length of the sequence a is 19 to 25 bases, preferably 19 to 21 bases.
  • the gene-specific siRNA of the target protein may have an additional base at the 5 'or 3' end.
  • the length of the additional base is usually about 2 to 4 bases.
  • the additional base may be DNA or RNA, but the use of DNA may improve the stability of the nucleic acid. Examples of such additional base sequences include ug-3 ', uu-3', tg-3 ', tt-3', ggg-3 ', guuu-3', gttt-3 ', ttttt-3 Examples include, but are not limited to, ', uuuuuu-3'.
  • SiRNA may have a protruding portion sequence (overhang) at the 3 ′ end, and specifically includes those to which dTdT (dT represents deoxythymidine) is added. Further, it may be a blunt end (blunt end) without end addition.
  • the siRNA may have a different number of bases in the sense strand and the antisense strand. For example, the “asymmetrical interfering RNA” in which the antisense strand has an overhang at the 3 ′ end and the 5 ′ end ( aiRNA) ".
  • a typical aiRNA has an antisense strand consisting of 21 bases, a sense strand consisting of 15 bases, and has an overhang structure of 3 bases 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 is from the 5′-UTR and the start codon to about 50 bases, and from a region other than the 3′-UTR. It is desirable to select the sequence.
  • the selected target sequence candidate group whether or not there is homology in the 16-17 base sequence in the non-target mRNA is determined by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ It is preferable to check using a homology search software such as) to confirm the specificity of the selected target sequence.
  • a sense strand having a 3 'end overhang of TT or UU 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 3 ′ end overhang of UU may be designed as an siRNA.
  • an arbitrary linker sequence for example, about 5-25 bases
  • the sense strand and the antisense strand are connected via the linker sequence. It can be designed by connecting.
  • siRNA and / or shRNA 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/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).
  • the siRNA is synthesized by synthesizing a sense strand and an antisense strand of a target sequence on mRNA with a DNA / RNA automatic synthesizer, denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, 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 an RNA-cleaving protein dicer.
  • the gene-specific miRNA of the target protein is arbitrary as long as the translation of the gene encoding the target protein is inhibited.
  • an miRNA may pair with the 3 'untranslated region (UTR) of the target and inhibit its translation, rather than cleaving the target mRNA like an siRNA.
  • the miRNA may be any of pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA), and mature miRNA.
  • the length of miRNA is not particularly limited, the length of pri-miRNA is usually several hundred to several thousand bases, the length of pre-miRNA is usually 50 to 80 bases, and the length of mature miRNA is usually 18 ⁇ 30 bases.
  • the gene-specific miRNA of the protein of interest is preferably a pre-miRNA or a mature miRNA, more preferably a mature miRNA.
  • a gene-specific miRNA of the target protein may be synthesized by a known technique or purchased from a company that provides synthetic RNA.
  • the gene-specific antisense nucleic acid of the target protein is a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of the mRNA encoding the target protein, or a part thereof, and specific to the mRNA. It is a nucleic acid having a function of suppressing target protein synthesis by forming and binding an ideal and stable duplex.
  • the antisense nucleic acid may be any of DNA, RNA, and DNA / RNA chimera.
  • the antisense nucleic acid is DNA
  • the RNA DNA hybrid formed by the target RNA and the antisense DNA is recognized by endogenous ribonuclease H (RNase H) and causes selective degradation of the target RNA.
  • 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 with the cDNA base sequence of the target protein gene using a homology search program such as BLAST or FASTA.
  • the length of the target region of the gene-specific antisense nucleic acid of the target protein is not limited as long as the antisense nucleic acid is hybridized and consequently translation into the target protein is inhibited.
  • the gene-specific antisense nucleic acid of the target protein may be the entire sequence or a partial sequence of mRNA encoding the target protein.
  • an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferable, but is not limited thereto.
  • a 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 transcription product of the target protein gene and inhibits translation into the protein, but also binds to these genes that are double-stranded DNA. It may be a substance capable of forming a triplex and inhibiting transcription to RNA (antigene).
  • the nucleotide molecules constituting the gene-specific siRNA of the target protein, the gene-specific miRNA of the target protein, and the gene-specific antisense nucleic acid of the target protein have stability (chemical and / or anti-enzyme) and specific activity ( Various chemical modifications may be included in order to improve (affinity with RNA).
  • a phosphate residue (phosphate) of each nucleotide constituting an antisense nucleic acid is selected from, for example, phosphorothioate (PS), methylphosphonate, phosphorodithio. It can be substituted with a chemically modified phosphate residue such as a phosphorodithioate.
  • the 2′-position hydroxyl group of the sugar (ribose) of each nucleotide is represented by —OR (R ⁇ CH 3 (2′-O-Me), CH 2 CH 2 OCH 3 (2′-O-MOE), CH 2 CH 2 NHC (NH) NH 2 , CH 2 CONHCH 3 , or CH 2 CH 2 CN) may be substituted.
  • the base moiety pyrimidine, purine
  • pyrimidine, purine may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. May be applied.
  • a part of nucleotide molecules constituting siRNA or miRNA may be replaced with natural DNA.
  • Gene-specific siRNA of the target protein, gene-specific miRNA of the target protein, and gene-specific antisense nucleic acid of the target protein are targets of mRNA or early transcript based on the cDNA sequence or genomic DNA sequence of the target protein gene It can be prepared by determining the sequence and synthesizing a complementary sequence using a commercially available DNA / RNA automatic synthesizer.
  • any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a known method.
  • the expression vector comprises a promoter sequence and a gene-specific siRNA of the protein of interest, a gene-specific miRNA of the protein of interest, or a coding sequence of a gene-specific antisense nucleic acid of the protein of interest (optionally further A polynucleotide containing a transcription termination signal sequence) and optionally other sequences.
  • the promoter is not particularly limited, and examples thereof include 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 include human H1-RNase P ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ RNA promoter, human valine-tRNA promoter, and other RNA polymerase III (polIII) promoters. Among these, polIII promoters can be used for accurate transcription of short RNAs. preferable.
  • Other sequences are not particularly limited, and various known sequences that can be included in the expression vector can be employed. Examples of such sequences include the origin of replication and drug resistance genes. Examples of the drug resistance gene and the vector include those described above.
  • RNA-specific ribozyme of the target protein is a gene-specific ribozyme of the target protein.
  • “Ribozyme” in a narrow sense means RNA having an enzyme activity for cleaving nucleic acid, but in the present application, it includes DNA as long as it has sequence-specific nucleic acid cleaving activity.
  • the most versatile ribozyme nucleic acid is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammerhead type and hairpin type are known.
  • the hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends (about 10 bases in total) adjacent to the part having the hammerhead structure are made complementary to the desired cleavage site of mRNA.
  • ribozyme nucleic acid has an advantage that it does not attack genomic DNA because it uses only RNA as a substrate.
  • the target sequence is single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase.
  • ribozymes when used in the form of expression vectors containing the DNA that encodes them, they should be hybrid ribozymes in which tRNA-modified sequences are further linked in order to promote the transfer of transcripts to the cytoplasm. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
  • 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 agent of the present invention is not particularly limited, and can take a form normally used in each application depending on the application of the agent of the present invention.
  • the form when the use is a medicine, health enhancer, nutritional supplement (eg supplement), etc., for example, tablets (inner disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drops, etc.) ), Pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jelly preparations suitable for oral intake Forms (oral dosage forms), nasal drops, inhalants, rectal suppositories, inserts, enemas, jellies, injections, patches, lotions, creams, etc. Oral dosage form).
  • tablets inner disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drops, etc.
  • Pills granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jelly preparations suitable for oral intake Forms (oral dosage forms), nasal drops,
  • a liquid, gel or solid food such as juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, infant formula , Cake mix, powdered or liquid dairy products, bread, cookies and the like.
  • an oral composition for example, liquid (solution, emulsion, suspension, etc.), semi-solid (gel, cream, paste, etc.), solid (tablet, particulate agent, capsule, Film, kneaded material, molten solid, waxy solid, elastic solid, etc.), more specifically, dentifrice (toothpaste, liquid toothpaste, liquid toothpaste, powder toothpaste etc.), mouthwash,
  • a coating agent for example, chewing gum, tablet candy, candy, gummi, film, troche, etc.
  • the agent of the present invention may further contain other components as necessary.
  • Other components are not particularly limited as long as they are components that can be blended in, for example, pharmaceuticals, food compositions, oral compositions, health enhancers, nutritional supplements (such as supplements), etc. , Carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, and the like.
  • the content of the inhibitor of the target protein of the agent of the present invention depends on the type of inhibitor, application, use mode, application target, application target state, and the like, and is not limited. For example, 0.0001 to 100 % By weight, preferably 0.001 to 50% by weight.
  • the amount of application (eg, 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 a medicinal effect, and is generally 0.1 to 1000 per day as the weight of the active ingredient. mg / kg body weight.
  • the above dose is preferably administered once a day or divided into 2 to 3 times a day, and can be appropriately increased or decreased depending on age, disease state, and symptoms.
  • Chronic prophylaxis of obstructive pulmonary disease or therapeutic agent screening method the invention of the active ingredient, in one aspect thereof, in the extracellular vesicles fluid taken from animals treated with test substance, protein group (A)
  • a method for screening an active ingredient of a prophylactic or therapeutic agent for chronic obstructive pulmonary disease using the amount or concentration of at least one protein selected from the above as an index (hereinafter referred to as “the active ingredient screening method of the present invention”) May also be indicated). This will be described below.
  • Body fluid, extracellular vesicles, protein group (A), chronic obstructive pulmonary disease, measurement of the amount or concentration of the target protein, etc. are the same as defined in “1. Method for examining chronic obstructive pulmonary disease” above. .
  • Species of animal species are not particularly limited. Examples of animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, and preferably mice.
  • test substance can be widely used regardless of whether it is a naturally occurring compound or a man-made compound.
  • composition which mixed not only the refined compound but various compounds, and the extract of animals and plants can also be used.
  • the compounds include not only low molecular compounds but also high molecular compounds such as proteins, nucleic acids, and polysaccharides.
  • the active ingredient screening method of the present invention is such that the value of the index is the amount or concentration of the corresponding protein in the extracellular vesicles of a body fluid collected from an animal not treated with the test substance (control value).
  • the test substance is selected as an active ingredient of a prophylactic or therapeutic agent for chronic obstructive pulmonary disease (or a candidate substance for an active ingredient of prophylactic or therapeutic agent for chronic obstructive pulmonary disease) Including.
  • 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.
  • the present invention is selected from the protein group (A) in an extracellular vesicle of a body fluid collected from an animal treated with a test substance.
  • a method for evaluating the induction or malignancy of chronic obstructive pulmonary disease using the amount or concentration of at least one kind of protein as an index (in this specification, sometimes referred to as “the toxicity evaluation method of the present invention”). ) This will be described below.
  • Chronic obstructive pulmonary disease For body fluids, extracellular vesicles, protein group (A), chronic obstructive pulmonary disease, measurement of the amount or concentration of the target protein, animal species, test substances, etc., see “1. Chronic obstructive pulmonary disease” above. The definition is the same as in the “examination method” and “6. screening method for active ingredients of preventive or therapeutic agents for chronic obstructive lung disease”.
  • the value of the above-mentioned index is the amount or concentration of the corresponding protein in the extracellular vesicles of body fluid collected from an animal that has not been treated with the test substance (control value).
  • the test substance is determined to have induction or malignancy of chronic obstructive pulmonary disease.
  • Corresponding protein means the same protein as the target protein used as an index.
  • “High” means, for example, that the index value is 2 times, 5 times, 10 times, 20 times, 50 times, 100 times the control value.
  • Test Example 1 Preparation of extracellular vesicle fraction
  • the cyst fraction was prepared.
  • the extracellular vesicle fraction was prepared by stepwise ultracentrifugation. Specifically, it was performed as follows.
  • the serum obtained in Test Examples 1 and 2 and the serum of normal mice (11-week-old C57BL6 / J male mice) were diluted with PBS and centrifuged (2000 g, 4 ° C., 30 minutes). The supernatant was passed through a 0.22 ⁇ m filter and ultracentrifuged (100,000 g, 4 ° C., 90 minutes).
  • the pellet was washed with PBS and then ultracentrifuged under the same conditions. This washing-ultracentrifugation step was repeated twice in total.
  • the finally obtained pellet was suspended in PBS to obtain an extracellular vesicle fraction.
  • the number and particle diameter of the extracellular vesicles were measured for the obtained extracellular vesicle fraction.
  • the measurement was carried out using a 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, tracking (tracking) each individual scattered light reflected on the screen, and using each moving speed (diffusion coefficient) in the liquid.
  • the particle diameter (hydrodynamic diameter) can be calculated. The results are shown in FIG.
  • extracellular vesicles were observed by immunoelectron microscopy. Specifically, it was performed 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 fix the extracellular vesicles to the form bar of the grid. After washing 3 times with PBS, blocking reaction (1% BSA / PBS, 10 minutes) followed by 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).
  • exosomes are present in the obtained fraction, and the number and particle diameters of the fractions are substantially different between healthy subjects and chronic obstructive pulmonary disease subjects. I found that there was no.
  • TMT label-LC-MS / MS proteomics analysis was performed according to a previously reported document (Journal of Proteome Research., 2017, 16 (2), pp 1077-1086.). The outline is as follows. The peptide sample obtained by trypsin digesting the protein in the extracellular vesicle fraction obtained in Test Example 1 was labeled with TMT 10-plex reagent. The labeled sample was fractionated with an SCX column and subjected to LC-MS / MS analysis using a mass spectrometer (LTQ-Orbitrap XL, manufactured by Thermo Fisher Scientific).
  • the obtained raw data is analyzed using an analysis program (Proteome Discoverer ver.1.3 (Thermo Fisher Scientific) equipped with Mascot v2.3.1 (Matrix Science) for UniProt / SwissProt) to quantify various proteins. I got the data.
  • COPD group Protein in the extracellular vesicle fraction of COPD patients (COPD group) was significantly higher than the amount in the extracellular vesicle fraction of healthy subjects (control group). It was determined that it had a certain degree of effectiveness as a marker and was selected. Table 1 shows the amount ratio of the selected protein and the control group.
  • Test Example 3 Detection of Chronic Obstructive Pulmonary Disease Biomarker in Extracellular Vesicle Fraction by Western Blot Western blot using an antibody against the chronic obstructive pulmonary disease biomarker (Test Example 2) Blotted.
  • the primary antibodies used were anti-Fibulin-1 antibody (Abcam, ab211536), anti-EFEMP-1 antibody (GeneTex, GTX111657), anti-CD99 antibody (Abcam, ab108297), anti-CD18 antibody (Abcam, ab185723), anti-OIT 3 antibody (MyBioSource, MBS2028378), anti-VAMP3 (Cellubrevin) antibody (Abcam, ab181749), and anti-CD81 (TAPA-1) antibody (Abcam, ab155760).
  • antibodies against exosome markers anti-Flotillin-1 antibody (BD Bioscience, 610821) and anti-CD63 antibody (Proteintech, 12632-1-AP) were also used as controls. The results are shown in FIG.
  • the chronic obstructive pulmonary disease biomarker can be detected even by Western blotting, and the expression level difference can be confirmed between the healthy subject and the chronic obstructive pulmonary disease subject. .
  • Test Example 4 Detection of chronic obstructive pulmonary disease biomarkers in lung tissue by immunostaining Tissue array (BMX, Lung disease and normal tissue array, 16 cases / 48 cores; Lung disease spectrum tissue array, including TNM and pathology grade, 24 cases / 48 cores ) was immunostained with an antibody against a chronic obstructive pulmonary disease biomarker (Test Example 2).
  • the primary antibodies used were anti-Fibulin-1 antibody (Abcam, ab211536), anti-EFEMP-1 antibody (GeneTex, GTX111657), anti-CD99 antibody (Abcam, ab108297), anti-CD18 antibody (Abcam, ab185723), and anti-CD81 ( TAPA-1) antibody (Abcam, ab155760). The results are shown in FIG.
  • the examined chronic obstructive pulmonary disease biomarker is also expressed in the lung tissue and specifically highly expressed in the lung tissue of the chronic obstructive pulmonary disease subject. I understood. No increase in expression was observed in lung tissues of healthy subjects and pulmonary fibrosis subjects.
  • Test Example 5 Evaluation of Chronic Obstructive Pulmonary Disease Biomarker AUC, cutoff value, sensitivity, and specificity were calculated for the chronic obstructive pulmonary disease biomarker (Test Example 2). Specifically, it is as follows. JMP v. 8.0 (SAS Institute, Cary, NC, USA) was used. Sensitivity is plotted on the vertical axis and (1-specificity) is plotted on the horizontal axis. As for the ROC curve of multi-marker, logistic regression analysis was performed on Fibulin-1 and SSC5D, the prediction probability was calculated, and the ROC curve was similarly drawn from the prediction probability. The results are shown in Table 2.
  • Test Example 6 Analysis of relationship between chronic obstructive pulmonary disease biomarker and respiratory function
  • Vital capacity When I inhaled all the air and exhaled it, I examined how much air was exhaled.
  • % Vital capacity (VC) The ratio of the measured vital capacity to the predicted vital capacity (reference value) calculated from age and sex was examined.
  • Forced vital capacity (FVC) The amount of air exhaled and breathed out was examined.
  • 1 second amount The amount of air exhaled during the first second of the forced vital capacity was examined.
  • 1 second rate The ratio of the 1 second amount to the forced vital capacity was examined.

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Abstract

L'invention fournit un biomarqueur de la bronchopneumopathie chronique obstructive, et un procédé de mise en œuvre de celui-ci. Plus précisément, l'invention concerne un groupe de protéines (A) se trouvant dans une vésicule extracellulaire de fluide corporelle prélevée chez un sujet.
PCT/JP2018/009529 2017-03-14 2018-03-12 Biomarqueur de la bronchopneumopathie chronique obstructive WO2018168779A1 (fr)

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