JP2023000812A - Malignant pleural mesothelioma biomarker - Google Patents

Abstract

To provide a malignant pleural mesothelioma biomarker, and a use method therefor.SOLUTION: A specific protein group is detected in extracellular vesicles of bodily fluid or blood sample collected from a subject. With a biomarker, it is possible to, for example, examine malignant pleural mesothelioma, prevent or treat malignant pleural mesothelioma, screen active ingredients for a preventive or therapeutic agent for malignant pleural mesothelioma, and evaluate inducibility or exacerbation of malignant pleural mesothelioma.SELECTED DRAWING: None

Description

The present invention relates to malignant pleural mesothelioma biomarkers and the like.

It is known that the incidence of malignant pleural mesothelioma increases with exposure to asbestos, and that it often occurs 30 to 40 years after exposure. According to epidemiological studies, it is said that 10,000 malignant pleural mesothelioma patients die annually worldwide, and the prevalence of malignant pleural mesothelioma is expected to increase in the future.

A standard treatment for malignant pleural mesothelioma has not yet been established. If it can be detected at an early stage, radical surgery may be possible, so the development of early diagnosis technology is desired. In addition, malignant pleural mesothelioma cannot be diagnosed by bronchoscopy, but can be finally diagnosed by thoracoscopic VATS. Therefore, a simpler inspection method is desired.

Non-Patent Document 1 attempts to search for a biomarker for malignant pleural mesothelioma, but has not yet identified a useful biomarker.

Regan EA, et al., AJRCMB 2019 Aug;61 (2): 143.

An object of the present invention is to provide a biomarker for malignant pleural mesothelioma and a method for using the same.

As a result of intensive research, the present inventor found that a specific protein group in extracellular vesicles of body fluids or blood samples collected from a subject is useful as a biomarker for malignant pleural mesothelioma. . 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 for examining malignant pleural mesothelioma, comprising:
(1) Protein group (A) and protein group (B) in extracellular vesicles of body fluid or blood sample collected from a subject:
(A) Hermansky-Pudlak syndrome 4 protein, Biglycan, Decorin, Immunoglobulin kappa variable 1-27, Protein FAM3C, Erlin-1, Trafficking protein particle complex subunit 9, Proprotein convertase subtilisin/kexin type 9, Endosomal/lysosomal potassium channel TMEM175, Complement component C9, Complement component C8 alpha chain, Nidogen-2, EGF-containing fibulin-like extracellular matrix protein 1, Periostin, Emerin, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A, Complement factor H-related protein 5, Kunitz-type protease inhibitor 2, Signal-regulatory protein beta-1, Transmembrane emp24 domain-containing protein 5, Plexin domain-containing protein 2, Lanosterol synthase, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1, Transmembrane emp24 domain-containing protein 4, Post- GPI attachment to proteins factor 6, C4b-binding protein alpha chain, Tumor necrosis factor receptor superfamily member 5, ADP-ribosylation factor-binding protein GGA3, Low affinity immunoglobulin from gamma Fc region receptor II-c, Malectin, Disintegrin and metalloproteinase domain-containing protein 9, Prothrombin, Alpha-synuclein, Galectin-8, ELMO domain-containing protein 2, AP-2 complex subunit alpha-1, and Apoptosis regulator BAX (B) Pro-interleukin-16, Putative hydroxypyruvate isomerase, G protein-coupled receptor kinase 5, FERM, ARHGEF and pleckstrin domain-containing protein 1, 26S proteasome non-ATPase regulatory subunit 6, Dynein light chain 2 , cytoplasmic, Protein PRRC1, Myotubularin-related protein 2, T-cell surface glycoprotein CD4, Guanylate-binding protein 1, Spectrin beta chain, non-erythrocytic 2, Glutathione hydrolase 5 proenzyme, EGF-like repeat and discoidin I-like domain- containing protein 3, Bleomycin hydrolase, Acyl-CoA-binding protein, Band 4.1-like protein 2, Ankyrin-1, Glucosamine-6-phosphate isomerase 1, Rho family-interacting cell polarization regulator 1, Interleukin-18, Proteasome subunit beta type -3, Dedicator of cytokinesis protein 2, Band 4.1-like protein 3, WD repeat-c containing protein 37, Keratin, type II cytoskeletal 1, Afadin, Ubiquitin thioesterase OTUB1, Integrin alpha-9, 1,4-alpha-glucan-branching enzyme, Protein 4.1, Methylthioribose-1-phosphate isomerase, SAM and SH3 domain-containing protein 3, ADP-ribosylation factor-like protein 3, Pleckstrin homology domain-containing family O member 2, calcium/calmodulin-dependent protein kinase type 1, Deoxyribonuclease-1-like 1, Guanine nucleotide-binding protein subunit alpha-13, Angiogenin, At least one selected from the group consisting of Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, and Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform A testing method comprising the step of detecting the protein of

Section 2. When the detected protein contains at least one protein (protein A') selected from protein group (A),
(2a) determining that the subject has malignant pleural mesothelioma when the amount or concentration of protein A' detected in step (1) is equal to or greater than the cutoff value;
The inspection method according to Item 1, comprising:

Item 3. When the detected protein contains at least one protein (protein B') selected from protein group (B),
(2b) determining that the subject has malignant pleural mesothelioma when the amount or concentration of protein B' detected in step (1) is equal to or less than the cutoff value;
The inspection method according to Item 1 or 2, comprising:

Section 4. When the detected protein contains at least one protein (protein A') selected from the protein group (A) and at least one protein (protein B') selected from the protein group (B), further ,
(2c) The amount or concentration of protein A' detected in step (1) is equal to or greater than the cutoff value, and/or the amount or concentration of protein B' detected in step (1) is the cutoff value. determining that the subject has malignant pleural mesothelioma when:
The inspection method according to any one of Items 1 to 3, comprising:

Item 5. The protein group (A) is
(A1) Hermansky-Pudlak syndrome 4 protein, Biglycan, Decorin, Immunoglobulin kappa variable 1-27, Protein FAM3C, Erlin-1, Trafficking protein particle complex subunit 9, Proprotein convertase subtilisin/kexin type 9, Endosomal/lysosomal potassium channel TMEM175, Protein group consisting of Complement component C9 and Complement component C8 alpha chain, or (A2) Hermansky-Pudlak syndrome 4 protein, Prothrombin, Post-GPI attachment to proteins factor 6, Plexin domain-containing protein 2, Emerin, Immunoglobulin kappa variable 1 -27, Nidogen-2, and the protein group consisting of Endosomal/lysosomal potassium channel TMEM175, the inspection method according to any one of Items 1 to 4.

Item 6. The protein group (B) is
(B1) Pro-interleukin-16, Putative hydroxypyruvate isomerase, G protein-coupled receptor kinase 5, FERM, ARHGEF and pleckstrin domain-containing protein 1, 26S proteasome non-ATPase regulatory subunit 6, Dynein light chain 2, cytoplasmic, Protein PRRC1 , Myotubularin-related protein 2, T-cell surface glycoprotein CD4, Guanylate-binding protein 1, Spectrin beta chain, non-erythrocytic 2, Glutathione hydrolase 5 proenzyme, EGF-like repeat and discoidin I-like domain-containing protein 3, and A group of proteins consisting of Bleomycin hydrolase, or (B2) Protein PRRC1, Dedicator of cytokinesis protein 2, G protein-coupled receptor kinase 5, Integrin alpha-9, Calcium/calmodulin-dependent protein kinase type 1, Methylthioribose-1-phosphate isomerase, Ubiquitin thioesterase OTUB1, Afadin, Acyl-CoA-binding protein, Pro-interleukin-16, T-cell surface glycoprotein CD4, Interleukin-18, Glutathione hydrolase 5 proenzyme, ADP-ribosylation factor-like protein 3, and 26S proteasome non-ATPase Item 6. The inspection method according to any one of Items 1 to 5, wherein the protein group consists of regulatory subunit 6.

Item 7. Item 7. The test method according to any one of Items 1 to 6, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum.

Item 8. Item 8. The test method according to any one of Items 1 to 7, wherein the subject is a human.

Item 9. Protein Group (A), and Protein Group (B):
(A) Hermansky-Pudlak syndrome 4 protein, Biglycan, Decorin, Immunoglobulin kappa variable 1-27, Protein FAM3C, Erlin-1, Trafficking protein particle complex subunit 9, Proprotein convertase subtilisin/kexin type 9, Endosomal/lysosomal potassium channel TMEM175, Complement component C9, Complement component C8 alpha chain, Nidogen-2, EGF-containing fibulin-like extracellular matrix protein 1, Periostin, Emerin, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A, Complement factor H-related protein 5, Kunitz-type protease inhibitor 2, Signal-regulatory protein beta-1, Transmembrane emp24 domain-containing protein 5, Plexin domain-containing protein 2, Lanosterol synthase, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1, Transmembrane emp24 domain-containing protein 4, Post- GPI attachment to proteins factor 6, C4b-binding protein alpha chain, Tumor necrosis factor receptor superfamily member 5, ADP-ribosylation factor-binding protein GGA3, Low affinity immunoglobulin from gamma Fc region receptor II-c, Malectin, Disintegrin and metalloproteinase domain-containing protein 9, Prothrombin, Alpha-synuclein, Galectin-8, ELMO domain-containing protein 2, AP-2 complex subunit alpha-1, and Apoptosis regulator BAX (B) Pro-interleukin-16, Putative hydroxypyruvate isomerase, G protein-coupled receptor kinase 5, FERM, ARHGEF and pleckstrin domain-containing protein 1, 26S proteasome non-ATPase regulatory subunit 6, Dynein light chain 2 , cytoplasmic, Protein PRRC1, Myotubularin-related protein 2, T-cell surface glycoprotein CD4, Guanylate-binding protein 1, Spectrin beta chain, non-erythrocytic 2, Glutathione hydrolase 5 proenzyme, EGF-like repeat and discoidin I-like domain- containing protein 3, Bleomycin hydrolase, Acyl-CoA-binding protein, Band 4.1-like protein 2, Ankyrin-1, Glucosamine-6-phosphate isomerase 1, Rho family-interacting cell polarization regulator 1, Interleukin-18, Proteasome subunit beta type -3, Dedicator of cytokinesis protein 2, Band 4.1-like protein 3, WD repeat-c containing protein 37, Keratin, type II cytoskeletal 1, Afadin, Ubiquitin thioesterase OTUB1, Integrin alpha-9, 1,4-alpha-glucan-branching enzyme, Protein 4.1, Methylthioribose-1-phosphate isomerase, SAM and SH3 domain-containing protein 3, ADP-ribosylation factor-like protein 3, Pleckstrin homology domain-containing family O member 2, calcium/calmodulin-dependent protein kinase type 1, Deoxyribonuclease-1-like 1, Guanine nucleotide-binding protein subunit alpha-13, Angiogenin, At least one selected from the group consisting of Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, and Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform A diagnostic agent for malignant pleural mesothelioma, comprising a protein-detecting agent for

Item 10. Protein group (A):
(A) Hermansky-Pudlak syndrome 4 protein, Biglycan, Decorin, Immunoglobulin kappa variable 1-27, Protein FAM3C, Erlin-1, Trafficking protein particle complex subunit 9, Proprotein convertase subtilisin/kexin type 9, Endosomal/lysosomal potassium channel TMEM175, Complement component C9, Complement component C8 alpha chain, Nidogen-2, EGF-containing fibulin-like extracellular matrix protein 1, Periostin, Emerin, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A, Complement factor H-related protein 5, Kunitz-type protease inhibitor 2, Signal-regulatory protein beta-1, Transmembrane emp24 domain-containing protein 5, Plexin domain-containing protein 2, Lanosterol synthase, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1, Transmembrane emp24 domain-containing protein 4, Post- GPI attachment to proteins factor 6, C4b-binding protein alpha chain, Tumor necrosis factor receptor superfamily member 5, ADP-ribosylation factor-binding protein GGA3, Low affinity immunoglobulin from gamma Fc region receptor II-c, Malectin, Disintegrin and metalloproteinase domain-containing protein 9, Prothrombin, Alpha-synuclein, Galectin-8, ELMO domain-containing protein 2, AP-2 complex subunit alpha-1, and Apoptosis regulator BAX an inhibitor of at least one protein selected from the group of proteins consisting of and protein group (B):
(B) Pro-interleukin-16, Putative hydroxypyruvate isomerase, G protein-coupled receptor kinase 5, FERM, ARHGEF and pleckstrin domain-containing protein 1, 26S proteasome non-ATPase regulatory subunit 6, Dynein light chain 2, cytoplasmic, Protein PRRC1 , Myotubularin-related protein 2, T-cell surface glycoprotein CD4, Guanylate-binding protein 1, Spectrin beta chain, non-erythrocytic 2, Glutathione hydrolase 5 proenzyme, EGF-like repeat and discoidin I-like domain-containing protein 3, Bleomycin hydrolase, Acyl-CoA-binding protein, Band 4.1-like protein 2, Ankyrin-1, Glucosamine-6-phosphate isomerase 1, Rho family-interacting cell polarization regulator 1, Interleukin-18, Proteasome subunit beta type-3, Dedicator of cytokinesis protein 2, Band 4.1-like protein 3, WD repeat-containing protein 37, Keratin, type II cytoskeletal 1, Afadin, Ubiquitin thioesterase OTUB1, Integrin alpha-9, 1,4-alpha-glucan-branching enzyme, Protein 4.1, Methylthioribose-1-phosphate isomerase, SAM and SH3 domain-containing protein 3, ADP-ribosyl cation factor-like protein 3, Pleckstrin homology domain-containing family O member 2, calcium/calmodulin-dependent protein kinase type 1, deoxyribonuclease-1-like 1, guanine nucleotide-binding protein subunit alpha-13, angiogenin, serine/threonine- at least one protein enhancer selected from the group of proteins consisting of protein phosphatase 2A catalytic subunit beta isoform, Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, and Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform A prophylactic or therapeutic agent for malignant pleural mesothelioma containing at least one drug selected from the group.

Item 11. Protein group (A) and protein group (B) in extracellular vesicles of bodily fluids or blood samples taken from animals treated with the test substance:
(A) Hermansky-Pudlak syndrome 4 protein, Biglycan, Decorin, Immunoglobulin kappa variable 1-27, Protein FAM3C, Erlin-1, Trafficking protein particle complex subunit 9, Proprotein convertase subtilisin/kexin type 9, Endosomal/lysosomal potassium channel TMEM175, Complement component C9, Complement component C8 alpha chain, Nidogen-2, EGF-containing fibulin-like extracellular matrix protein 1, Periostin, Emerin, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A, Complement factor H-related protein 5, Kunitz-type protease inhibitor 2, Signal-regulatory protein beta-1, Transmembrane emp24 domain-containing protein 5, Plexin domain-containing protein 2, Lanosterol synthase, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1, Transmembrane emp24 domain-containing protein 4, Post- GPI attachment to proteins factor 6, C4b-binding protein alpha chain, Tumor necrosis factor receptor superfamily member 5, ADP-ribosylation factor-binding protein GGA3, Low affinity immunoglobulin from gamma Fc region receptor II-c, Malectin, Disintegrin and metalloproteinase domain-containing protein 9, Prothrombin, Alpha-synuclein, Galectin-8, ELMO domain-containing protein 2, AP-2 complex subunit alpha-1, and Apoptosis regulator BAX (B) Pro-interleukin-16, Putative hydroxypyruvate isomerase, G protein-coupled receptor kinase 5, FERM, ARHGEF and pleckstrin domain-containing protein 1, 26S proteasome non-ATPase regulatory subunit 6, Dynein light chain 2 , cytoplasmic, Protein PRRC1, Myotubularin-related protein 2, T-cell surface glycoprotein CD4, Guanylate-binding protein 1, Spectrin beta chain, non-erythrocytic 2, Glutathione hydrolase 5 proenzyme, EGF-like repeat and discoidin I-like domain- containing protein 3, Bleomycin hydrolase, Acyl-CoA-binding protein, Band 4.1-like protein 2, Ankyrin-1, Glucosamine-6-phosphate isomerase 1, Rho family-interacting cell polarization regulator 1, Interleukin-18, Proteasome subunit beta type -3, Dedicator of cytokinesis protein 2, Band 4.1-like protein 3, WD repeat-c containing protein 37, Keratin, type II cytoskeletal 1, Afadin, Ubiquitin thioesterase OTUB1, Integrin alpha-9, 1,4-alpha-glucan-branching enzyme, Protein 4.1, Methylthioribose-1-phosphate isomerase, SAM and SH3 domain-containing protein 3, ADP-ribosylation factor-like protein 3, Pleckstrin homology domain-containing family O member 2, calcium/calmodulin-dependent protein kinase type 1, Deoxyribonuclease-1-like 1, Guanine nucleotide-binding protein subunit alpha-13, Angiogenin, At least one selected from the group consisting of Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, and Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform A screening method for an active ingredient of a preventive or therapeutic agent for malignant pleural mesothelioma, wherein the amount or concentration of the protein of is used as an index.

Item 12. If the value of the indicator for protein group (A) is lower than the amount or concentration of the corresponding protein in extracellular vesicles of bodily fluids or blood samples collected from animals not treated with the test substance, the test substance a step of selecting the substance as an active ingredient of a prophylactic or therapeutic agent for malignant pleural mesothelioma; At least one selected from the group consisting of the step of selecting the test substance as an active ingredient of a preventive or therapeutic agent for malignant pleural mesothelioma when the amount or concentration of the corresponding protein in the vesicles or blood sample is higher than that of the corresponding protein. Item 12. The screening method according to Item 11, comprising the step of

Item 13. Protein group (A) and protein group (B) in extracellular vesicles of bodily fluids or blood samples taken from animals treated with the test substance:
(A) Hermansky-Pudlak syndrome 4 protein, Biglycan, Decorin, Immunoglobulin kappa variable 1-27, Protein FAM3C, Erlin-1, Trafficking protein particle complex subunit 9, Proprotein convertase subtilisin/kexin type 9, Endosomal/lysosomal potassium channel TMEM175, Complement component C9, Complement component C8 alpha chain, Nidogen-2, EGF-containing fibulin-like extracellular matrix protein 1, Periostin, Emerin, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A, Complement factor H-related protein 5, Kunitz-type protease inhibitor 2, Signal-regulatory protein beta-1, Transmembrane emp24 domain-containing protein 5, Plexin domain-containing protein 2, Lanosterol synthase, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1, Transmembrane emp24 domain-containing protein 4, Post- GPI attachment to proteins factor 6, C4b-binding protein alpha chain, Tumor necrosis factor receptor superfamily member 5, ADP-ribosylation factor-binding protein GGA3, Low affinity immunoglobulin from gamma Fc region receptor II-c, Malectin, Disintegrin and metalloproteinase domain-containing protein 9, Prothrombin, Alpha-synuclein, Galectin-8, ELMO domain-containing protein 2, AP-2 complex subunit alpha-1, and Apoptosis regulator BAX (B) Pro-interleukin-16, Putative hydroxypyruvate isomerase, G protein-coupled receptor kinase 5, FERM, ARHGEF and pleckstrin domain-containing protein 1, 26S proteasome non-ATPase regulatory subunit 6, Dynein light chain 2 , cytoplasmic, Protein PRRC1, Myotubularin-related protein 2, T-cell surface glycoprotein CD4, Guanylate-binding protein 1, Spectrin beta chain, non-erythrocytic 2, Glutathione hydrolase 5 proenzyme, EGF-like repeat and discoidin I-like domain- containing protein 3, Bleomycin hydrolase, Acyl-CoA-binding protein, Band 4.1-like protein 2, Ankyrin-1, Glucosamine-6-phosphate isomerase 1, Rho family-interacting cell polarization regulator 1, Interleukin-18, Proteasome subunit beta type -3, Dedicator of cytokinesis protein 2, Band 4.1-like protein 3, WD repeat-c containing protein 37, Keratin, type II cytoskeletal 1, Afadin, Ubiquitin thioesterase OTUB1, Integrin alpha-9, 1,4-alpha-glucan-branching enzyme, Protein 4.1, Methylthioribose-1-phosphate isomerase, SAM and SH3 domain-containing protein 3, ADP-ribosylation factor-like protein 3, Pleckstrin homology domain-containing family O member 2, calcium/calmodulin-dependent protein kinase type 1, Deoxyribonuclease-1-like 1, Guanine nucleotide-binding protein subunit alpha-13, Angiogenin, At least one selected from the group consisting of Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, and Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform A method for evaluating the inducibility or exacerbation of malignant pleural mesothelioma, using the amount or concentration of protein as an index.

Item 14. If the value of the indicator for protein group (A) is higher than the amount or concentration of the corresponding protein in extracellular vesicles of body fluids or blood samples collected from animals not treated with the test substance, the test substance Determining a substance as inducing or exacerbating malignant pleural mesothelioma, and the value of said indicator for protein group (B) is determined in extracellular body fluids collected from animals not treated with the test substance At least one selected from the group consisting of the step of determining that the test substance has the potential to induce or exacerbate malignant pleural mesothelioma when the amount or concentration is lower than the amount or concentration of the corresponding protein in the vesicles or blood sample. 14. The evaluation method according to Item 13, comprising the step of

According to the present invention, a biomarker for malignant pleural mesothelioma can be provided. By using the biomarker, examination of malignant pleural mesothelioma, prevention or treatment of malignant pleural mesothelioma, screening of active ingredients for prevention or treatment of malignant pleural mesothelioma, induction of malignant pleural mesothelioma It may be possible to evaluate sexuality or exacerbation, etc.

As used herein, the expressions "contain" and "include" include the concepts "contain", "include", "consist essentially of" and "consist only of".

1. Method for Examining Malignant Pleural Mesothelioma In one aspect of the present invention, there is provided a method for examining malignant pleural mesothelioma, comprising: An inspection method (herein, "inspection method of the present invention") comprising a step (step 1) of detecting at least one protein selected from the group consisting of protein group (A) and protein group (B) It is also shown as.) related. This will be explained below.

1-1. Process (1)
The type of "malignant pleural mesothelioma" to be examined is not particularly limited. All classes, grades, and stages of malignant pleural mesothelioma according to various classification criteria for progression of malignant pleural mesothelioma can be tested. In addition, all types of malignant pleural mesothelioma (for example, epithelial type, sarcoma type, biphasic type, etc.) in various classifications related to lesion sites of malignant pleural mesothelioma can be examined.

A subject is a target organism of the test 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.

The subject's condition is not particularly limited. Subjects include, for example, subjects with unknown malignant pleural mesothelioma, subjects already determined to have malignant pleural mesothelioma by another method, and subjects with malignant pleural mesothelioma. Specimens that have already been determined by another method to have not been treated, specimens during treatment for malignant pleural mesothelioma, specimens after treatment for malignant pleural mesothelioma, and the like.

The detection sample in step (1) is preferably extracellular vesicles of body fluids.

Body fluids are not particularly limited. 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, and the like, preferably whole blood and serum. , plasma and cerebrospinal fluid, more preferably whole blood, serum and plasma. One type of body fluid may be used alone, or two or more types may be used in combination.

A bodily fluid can be collected from a subject by methods known to those skilled in the art. For example, 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.

In the present specification, blood itself such as whole blood, serum, plasma, etc., or a blood-derived sample is referred to as a "blood sample."

Extracellular vesicles are not particularly limited as long as they are membrane vesicles 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 according to 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.

The detection target in step (1) is at least one protein selected from the group consisting of protein group (A) and protein group (B) (in this specification, these are collectively referred to as "target protein" There is also.)

Protein group (A) includes (A) Hermansky-Pudlak syndrome 4 protein, Biglycan, Decorin, Immunoglobulin kappa variable 1-27, Protein FAM3C, Erlin-1, Trafficking protein particle complex subunit 9, Proprotein convertase subtilisin/kexin type 9, Endosomal/lysosomal potassium channel TMEM175, Complement component C9, Complement component C8 alpha chain, Nidogen-2, EGF-containing fibulin-like extracellular matrix protein 1, Periostin, Emerin, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A, Complement factor H- related protein 5, Kunitz-type protease inhibitor 2, Signal-regulatory protein beta-1, Transmembrane emp24 domain-containing protein 5, Plexin domain-containing protein 2, Lanosterol synthase, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1, Transmembrane emp24 domain -containing protein 4, Post-GPI attachment to proteins factor 6, C4b-binding protein alpha chain, Tumor necrosis factor receptor superfamily member 5, ADP-ribosylation factor-binding protein GGA3, Low affinity imm unoglobulin gamma Fc region receptor II-c, Malectin, Disintegrin and metalloproteinase domain-containing protein 9, Prothrombin, Alpha-synuclein, Galectin-8, ELMO domain-containing protein 2, AP-2 complex subunit alpha-1, and Apoptosis regulator BAX It is a protein group consisting of

Protein group (A) is a protein group whose amount in malignant pleural mesothelioma specimens is higher than that in normal specimens.

Among the protein group (A), the protein group (A1) is preferable from the viewpoint that the quantitative ratio to the normal specimen is higher:
(A1) Hermansky-Pudlak syndrome 4 protein, Biglycan, Decorin, Immunoglobulin kappa variable 1-27, Protein FAM3C, Erlin-1, Trafficking protein particle complex subunit 9, Proprotein convertase subtilisin/kexin type 9, Endosomal/lysosomal potassium channel TMEM175, A protein group consisting of complement component C9 and complement component C8 alpha chain, more preferably protein group (A11):
(A11) A protein group consisting of Hermansky-Pudlak syndrome 4 protein, Biglycan, Decorin, and Immunoglobulin kappa variable 1-27.

Among the protein group (A), the protein group (A2) is preferable from the viewpoint of a higher significant difference from the normal specimen:
(A2) Consists of Hermansky-Pudlak syndrome 4 protein, Prothrombin, Post-GPI attachment to proteins factor 6, Plexin domain-containing protein 2, Emerin, Immunoglobulin kappa variable 1-27, Nidogen-2, and Endosomal/lysosomal potassium channel TMEM175 A group of proteins, more preferably a group of proteins (A21):
(A21) A protein group consisting of Hermansky-Pudlak syndrome 4 protein and Prothrombin.

Protein group (B) consists of (B) Pro-interleukin-16, Putative hydroxypyruvate isomerase, G protein-coupled receptor kinase 5, FERM, ARHGEF and pleckstrin domain-containing protein 1, 26S proteasome non-ATPase regulatory subunit 6, Dynein light chain 2, cytoplasmic, Protein PRRC1, Myotubularin-related protein 2, T-cell surface glycoprotein CD4, Guanylate-binding protein 1, Spectrin beta chain, non-erythrocytic 2, Glutathione hydrolase 5 proenzyme, EGF-like repeat and discoidin I-like domain-containing protein 3, Bleomycin hydrolase, Acyl-CoA-binding protein, Band 4.1-like protein 2, Ankyrin-1, Glucosamine-6-phosphate isomerase 1, Rho family-interacting cell polarization regulator 1, Interleukin-18, Proteasome subunit beta type-3, Dedicator of cytokinesis protein 2, Band 4.1-like protein 3, WD repeat-containing protein 37, Keratin, type II cytoskeletal 1, Afadin, Ubiquitin thioesterase OTUB1, Integrin alpha-9, 1,4-alpha-glucan -branching enzyme, Protein 4.1, Methylthioribose-1-phosphate isomerase, SAM and SH3 domain-containing protein 3, ADP-ribosylation factor-like protein 3, Pleckstrin homology domain-containing family O member 2, Calcium/calmodulin-dependent protein kinase type 1, Deoxyribonuclease-1-like 1, Guanine nucleotide-binding protein subunit alpha-13, Angiogenin, Serine/ It is a protein group consisting of threonine-protein phosphatase 2A catalytic subunit beta isoform, Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, and Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform.

Protein group (B) is a protein group whose amount in malignant pleural mesothelioma specimens is lower than that in normal specimens.

Among the protein group (B), the protein group (B1) is preferable from the viewpoint that the quantitative ratio to the normal sample is lower:
(B1) Pro-interleukin-16, Putative hydroxypyruvate isomerase, G protein-coupled receptor kinase 5, FERM, ARHGEF and pleckstrin domain-containing protein 1, 26S proteasome non-ATPase regulatory subunit 6, Dynein light chain 2, cytoplasmic, Protein PRRC1 , Myotubularin-related protein 2, T-cell surface glycoprotein CD4, Guanylate-binding protein 1, Spectrin beta chain, non-erythrocytic 2, Glutathione hydrolase 5 proenzyme, EGF-like repeat and discoidin I-like domain-containing protein 3, and A group of proteins consisting of Bleomycin hydrolase, more preferably a group of proteins (B11):
(B11) A group of proteins consisting of Pro-interleukin-16, Putative hydroxypyruvate isomerase, G protein-coupled receptor kinase 5, and FERM, ARHGEF and pleckstrin domain-containing protein 1.

Among the protein group (B), the protein group (B2) is preferable from the viewpoint of a higher significant difference from the normal specimen:
(B2) Protein PRRC1, Dedicator of cytokinesis protein 2, G protein-coupled receptor kinase 5, Integrin alpha-9, Calcium/calmodulin-dependent protein kinase type 1, Methylthioribose-1-phosphate isomerase, Ubiquitin thioesterase OTUB1, Afadin, Acyl- A group of proteins consisting of CoA-binding protein, Pro-interleukin-16, T-cell surface glycoprotein CD4, Interleukin-18, Glutathione hydrolase 5 proenzyme, ADP-ribosylation factor-like protein 3, and 26S proteasome non-ATPase regulatory subunit 6 more preferably the protein group (B21):
(B21) Protein group consisting of protein PRRC1, dedicator of cytokinesis protein 2, G protein-coupled receptor kinase 5, integrin alpha-9, and calcium/calmodulin-dependent protein kinase type 1.

The proteins of the protein groups (A) to (B) are proteins specified by UniProtKB accession numbers shown in Tables 1 to 4 in Examples below, in the case of humans. For other species, it is an orthologue of the protein identified by the UniProtKB accession numbers shown in Tables 1-4.

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), it is possible to more accurately perform tests for malignant pleural mesothelioma. be possible.

Detection is usually performed by measuring the amount or concentration of the protein of interest. "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 is a method capable of specifically detecting 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 and the like can be preferably exemplified as immunological assay methods.

In mass spectrometry, 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. In addition, tandem mass spectrometry (MS/MS) combining two or more mass spectrometry methods and triple quadrupole mass spectrometry can be used. Alternatively, 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. In addition, peptides constituting the target protein can be separated and purified and used as a sample by liquid chromatography (LC) or HPLC. Also, the detection unit and data processing method can be selected as appropriate. When mass spectrometry is used to detect and quantify a peptide that constitutes a target protein, 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. As such 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 can be produced by the F-moc method (Amblard., et al. Methods Mol Biol. 298) using stable isotope-labeled amino acids. : 3-24 (2005)), iTRAQ (registered trademark) reagent, ICAT (registered trademark) reagent, ICPL (registered trademark) reagent, NBS (registered trademark) reagent, etc. can also be produced using a labeling reagent of

According to the testing method of the present invention including step (1), the amount and/or concentration of the target protein, which is a detection indicator for malignant pleural mesothelioma, can be provided, thereby detecting malignant pleural mesothelioma, etc. can assist.

The test results obtained by the test method of the present invention including step (1) are used to elucidate the pathology of malignant pleural mesothelioma, predict the prognosis of malignant pleural mesothelioma, stratify subjects, and select treatment methods (personalized medicine, response to treatment), intractability in malignant pleural mesothelioma, evaluation of remodeling, differentiation of histological types and phenotypes of malignant pleural mesothelioma, and the like.

1-2. Process (2)
As one aspect of the inspection method of the present invention,
When the detected protein contains at least one protein (protein A') selected from protein group (A),
(2a) determining that the subject has malignant pleural mesothelioma when the amount or concentration of protein A' detected in step (1) is equal to or greater than the cutoff value;
It is preferred to include According to the testing method of the present invention including step 2a, it is possible to determine malignant pleural mesothelioma.

As one aspect of the inspection method of the present invention,
When the detected protein contains at least one protein (protein B') selected from protein group (B),
(2b) determining that the subject has malignant pleural mesothelioma when the amount or concentration of protein B' detected in step (1) is equal to or less than the cutoff value;
is preferably included. According to the examination method of the present invention including step 2b, it is possible to determine malignant pleural mesothelioma.

As one aspect of the inspection method of the present invention,
When the detected protein contains at least one protein (protein A') selected from the protein group (A) and at least one protein (protein B') selected from the protein group (B), further ,
(2c) The amount or concentration of protein A' detected in step (1) is equal to or greater than the cutoff value, and/or the amount or concentration of protein B' detected in step (1) is the cutoff value. determining that the subject has malignant pleural mesothelioma when:
is preferably included. According to the examination method of the present invention including step 2c, it is possible to determine malignant pleural mesothelioma.

The cut-off value can be appropriately set by a person skilled in the art from the viewpoint of sensitivity, specificity, positive predictive value, negative predictive value, etc. It can be a determined value each time or a predetermined value based on the amount and/or concentration of the protein of interest in the extracellular vesicles of the bodily fluid or the blood sample. The cut-off value is, for example, the amount and / or concentration of the target protein in extracellular vesicles of body fluids or blood samples collected from subjects not suffering from malignant pleural mesothelioma (when there are multiple subjects, average, median, etc.), for example, 0.7 to 1.5 times. In addition, it is also possible to set by performing statistical analysis (more specifically, a method using Youden index is exemplified) based on receiver operating characteristic (ROC) curve analysis, etc. can.

In a preferred embodiment of step (2), when the subject is a specimen during or after treatment for malignant pleural mesothelioma, the cutoff value is, for example, the amount of the target protein in a past sample of the same specimen and / Or a concentration-based value can be used to determine the therapeutic effect.

2. When a subject is determined to have malignant pleural mesothelioma by the testing method of the present invention including the diagnostic step (2) for diagnosing malignant pleural mesothelioma with higher accuracy, the testing method of the present invention is performed. Furthermore, by combining the step of applying diagnosis of malignant pleural mesothelioma by a doctor, malignant pleural mesothelioma can be diagnosed with higher accuracy. In addition, since the testing method of the present invention can more accurately detect malignant pleural mesothelioma, combining the above-described steps with the testing method of the present invention enables more efficient and more accurate identification of patients suffering from malignant pleural mesothelioma. It can be diagnosed as

3. When the subject is determined to be suffering from malignant pleural mesothelioma by the testing method of the present invention including the treatment step (2) for malignant pleural mesothelioma, the testing method of the present invention additionally or above Diagnosis of malignant pleural mesothelioma with higher accuracy”, the step of applying the examination method of the present invention and the diagnosis by a doctor when diagnosed as suffering from malignant pleural mesothelioma. and (3) treating a subject determined or diagnosed as suffering from malignant pleural mesothelioma by performing a step of treating the disease in the subject can be treated. In addition, since the examination method of the present invention can more accurately detect malignant pleural mesothelioma, the examination method of the present invention, or the combination of the examination method of the present invention and the step of applying a diagnosis by a doctor. By combining step 3, a subject suffering from malignant pleural mesothelioma can be treated more efficiently and reliably.

Methods for treating malignant pleural mesothelioma are not particularly limited, but typically include drug therapy. Various anticancer agents and the like are exemplified as pharmaceuticals used for medication therapy. Anticancer agents include, for example, pemetrexed, cisplatin, carboplatin, gemcitabine, vinorelbine and the like. One, two, or a combination of three or more medicaments can be used.

Moreover, by using the testing method of the present invention, it is also possible to select an appropriate drug to be used for medication treatment from among the drugs, for example, the drugs exemplified above.

Methods for treating malignant pleural mesothelioma include, in addition to the above, surgical therapy (operation), radiation therapy, and the like.

Four. Test agent and test kit for malignant pleural mesothelioma In one aspect of the present invention, an agent for detecting at least one protein selected from the group consisting of protein group (A) and protein group (B) is included. The present invention relates to a diagnostic agent for malignant pleural mesothelioma (in the present specification, it may be referred to as the "diagnostic agent of the present invention"). This will be explained below.

Protein group (A), protein group (B), malignant pleural mesothelioma, etc. are defined in the same manner as in the above "1. Method for testing malignant pleural mesothelioma".

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. For this reason, 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. As another example, 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. Here, "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.

Methods for producing these antibodies are already well known, and 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)). Specifically, when 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. On the other hand, in the case of a monoclonal antibody, 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. Sections 11.4 to 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 transformants, 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.

Specifically, a recombinant DNA (expression vector) 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. 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.

Antibodies 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. Such 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.

Five. Preventive or therapeutic agent for malignant pleural mesothelioma In one aspect of the present invention, an inhibitor of at least one protein selected from protein group (A) and at least one protein selected from protein group (B) A prophylactic or therapeutic agent for malignant pleural mesothelioma containing at least one drug selected from the group consisting of protein enhancers of (in the present specification, sometimes referred to as "the drug of the present invention"). Regarding. This will be explained below.

Protein group (A), protein group (B), malignant pleural mesothelioma, etc. are defined in the same manner as in the above "1. Method for testing malignant pleural mesothelioma".

Inhibitors include, for example, antibodies to the protein of interest. As for the antibody, the same antibody as described in the above "4. Test agent and test kit for malignant pleural mesothelioma" can be used.

Another example of an inhibitor is an expression inhibitor for a target protein.

The expression inhibitor 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. For example, gene-specific small interfering RNA (siRNA) of the target protein, target Gene-specific microRNA (miRNA) of protein, gene-specific antisense nucleic acid of target protein, expression vectors thereof; gene-specific ribozyme of target protein; gene editing agent of target protein by CRISPR/Cas system.

In addition, 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.

The enhancer includes, for example, an expression enhancer for the target protein.

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, and the like, and examples thereof include expression vectors for the target protein.

In addition, expression enhancement means that the expression level of the target protein, its mRNA, etc. is enhanced, for example, 2, 3, 5, 10, 20, 30, 50, 100, 200, 300, 500, 1000, 10000 times. means.

The gene siRNA for 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. In one embodiment, the siRNA is preferably, for example, 18 bases or longer, 19 bases or longer, 20 bases or longer, or 21 bases or longer. In addition, 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. For example, a length with a lower limit of 18 bases and an upper limit of 25 bases, 24 bases, 23 bases, or 22 bases; A length with a lower limit of 20 bases and an upper limit of 25, 24, 23, or 22 bases; a lower limit of 21 bases with an upper limit of 25, 24, 23, or 22 bases. Combinations of lengths that are bases are envisioned.

The siRNA may be shRNA (small hairpin RNA). A portion of shRNA can be designed to form a stem-loop structure. For example, for shRNA, 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. And the length of sequence a is 19-25 bases, preferably 19-21 bases.

A gene-specific siRNA for a protein of interest may have additional bases at the 5' or 3' ends. 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', ttttt-3 ', uuuuu-3', and the like, but are not limited to these.

The siRNA may have an overhang sequence (overhang) at the 3' end, and specific examples include 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)” can be mentioned. 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. For the target sequence whose specificity has been confirmed, 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. In addition, for shRNA, 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.

A gene-specific miRNA for a protein of interest is optional as long as it inhibits translation of the gene encoding the protein of interest. For example, 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. In one embodiment, the gene-specific miRNA of the protein of interest is preferably pre-miRNA or mature miRNA, more preferably mature miRNA. Such gene-specific miRNAs for 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 selectively degrade the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase 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. More specifically, 5' terminal hairpin loop, 5' terminal untranslated region, translation initiation codon, protein coding region, ORF translation termination codon, 3' terminal untranslated region, 3' terminal palindromic region of gene of target protein Alternatively, 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).

The nucleotide molecules that make up the above-described gene-specific siRNA of the protein of interest, gene-specific miRNA of the protein of interest, and gene-specific antisense nucleic acid of the protein of interest have stability (chemical and/or enzymatic) and specific activity ( Various chemical modifications may be included in order to improve affinity with RNA). For example, in order to prevent degradation by hydrolases such as nucleases, 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. In addition, the hydroxyl group at the 2' position of the sugar (ribose) of each nucleotide is replaced by -OR (R=CH ₃ (2'-O-Me), CH ₂ CH ₂ OCH ₃ (2'-O-MOE), CH _{2 CH2NHC (} NH) _NH2 , _{CH2CONHCH3 ,} or _CH2CH2CN ). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or cationic functional group to the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. may be applied. In addition, 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 automatic DNA/RNA synthesizer. In addition, antisense nucleic acids containing the various modifications described above can also be chemically synthesized by known techniques.

Gene-specific siRNA of target protein, gene-specific miRNA of target protein, expression vector of gene-specific antisense nucleic acid of target protein, expression vector of target protein, gene-specific siRNA of target protein, gene of target protein There is no particular limitation as long as the specific miRNA, the gene-specific antisense nucleic acid of the target protein, the mRNA of the target protein, etc. are incorporated in an expressible state. Typically, 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. Promoters are not particularly limited, for example, RNA polymerase II (polII) promoters such as CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter; mouse and human U6-snRNA promoters; Examples 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. Moreover, the types of drug-resistant genes and the types of vectors can be exemplified by those described above.

Another example of a gene expression inhibitor for a target protein includes a gene-specific ribozyme for the target protein. The term "ribozyme" in the narrow sense means RNA having enzymatic activity that cleaves nucleic acid, but in the present application 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. When the mRNA of the gene of the target protein has a double-stranded structure by itself, 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. [Proc. Natl. Acad. Sci. USA, 98(10): 5572-5577 (2001)]. Furthermore, when a ribozyme is used in the form of an expression vector containing the DNA encoding it, it should be a hybrid ribozyme in which a tRNA-modified sequence is further ligated in order to promote translocation of the transcript into 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 drug of the present invention is not particularly limited, and can take a form commonly used in 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-enhancing 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, jellies, injections, patches, lotions, and creams (non oral dosage forms).

As for the form, when the application is a food composition, 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.

As for the form, when the application is an oral composition, 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, moisturizers, 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 a day or in 2 to 3 divided doses, and can be adjusted appropriately according to age, condition and symptoms.

6. Method for Screening Active Ingredients for Preventive or Curative Agents for Malignant Pleural Mesothelioma An active ingredient (or a candidate substance thereof) for a prophylactic or therapeutic agent for malignant pleural mesothelioma, wherein the amount or concentration of at least one protein selected from the group consisting of (A) and protein group (B) is used as an index. (In the present specification, it may also be referred to as the "active ingredient screening method of the present invention."). This will be explained below.

For body fluids, extracellular vesicles, blood samples, protein group (A), protein group (B), malignant pleural mesothelioma, measurement of the amount or concentration of the target protein, etc., see "1. Malignant pleural mesothelioma It is the same as the definition in "Inspection method".

The animal species is not particularly limited. Examples of animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.

A wide range of test substances can be used regardless of whether they are naturally occurring compounds or artificially produced compounds. In addition, not only 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.

More specifically, the active ingredient screening method of the present invention is characterized in that the value of the indicator for protein group (A) is obtained in extracellular vesicles of body fluids collected from animals not treated with the test substance or in blood samples A step of selecting the test substance as an active ingredient of a prophylactic or therapeutic agent for malignant pleural mesothelioma when the amount or concentration is lower than that of the corresponding protein, and the index value for the protein group (B) is When the amount or concentration of the corresponding protein in extracellular vesicles of body fluids or blood samples collected from animals not treated with the substance is higher than the amount or concentration of the corresponding protein, the test substance is effective as an agent for the prevention or treatment of malignant pleural mesothelioma. It comprises at least one step selected from the group consisting of steps selected as components.

The corresponding protein means the same protein as the target protein used as an indicator.

"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-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold higher than the control value.

7. In one aspect of the present invention, a method for evaluating the inducibility or exacerbation of malignant pleural mesothelioma is characterized by the protein group (A ), and a method for evaluating the induction or exacerbation of malignant pleural mesothelioma (herein, " It may be indicated as "the toxicity evaluation method of the present invention"). This will be explained below.

Body fluids, extracellular vesicles, protein group (A), protein group (B), malignant pleural mesothelioma, measurement of the amount or concentration of the target protein, animal species, test substance, etc. are described in "1. Method for examining malignant pleural mesothelioma" and "6. Screening method for active ingredient of preventive or therapeutic agent for malignant pleural mesothelioma".

More specifically, the toxicity evaluation method of the present invention is characterized in that the values of the indicators related to the protein group (A) correspond to extracellular vesicles of body fluids collected from animals not treated with the test substance or blood samples. a step of determining that the test substance has malignant pleural mesothelioma-inducing or exacerbating properties when the amount or concentration is higher than that of the protein, and the value of the indicator for the protein group (B) is equal to the test substance If the amount or concentration of the corresponding protein in extracellular vesicles or blood samples of body fluids collected from animals not treated with At least one step selected from the group consisting of the steps of determining

The corresponding protein means the same protein as the target protein used as an indicator.

"High" means, for example, that the index value is 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold higher than 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.

EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.

Test example 1. Preparation of extracellular vesicle fraction Extracellular vesicle fractions were prepared from the sera of 6 human subjects diagnosed with malignant pleural mesothelioma and the sera of 4 healthy human subjects. prepared. The extracellular vesicle fraction was prepared using an extracellular vesicle preparation kit (MagCaptur™ Exosome Isolation Kit PS, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) with the same volume of serum for each sample. rice field.

Subsequently, the obtained extracellular vesicle fraction was measured for the number of extracellular vesicle particles and the particle size. 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.

In addition, extracellular vesicles were observed by immunoelectron microscopy. Specifically, it was carried out as follows. 5 to 8 μL 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, blocking reaction (1% BSA/PBS, 10 minutes) was performed, 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). After washing with PBS six times, secondary antibody reaction (CRL EMGMHL10 Anti-IgG (H+L), Mouse, Goat-Poly, Gold 10 nm, EM, 200-fold dilution, 1 hour, room temperature) was performed. After washing 6 times with PBS, the cells were treated with 1% GA/PBS for 15 minutes, and washed 3 times with PBS. It was washed with water, treated with 0.5% UA (uranyl acetate), dried and observed under an electron microscope.

Furthermore, extracellular vesicle fractions were subjected to Western blotting using antibodies against exosome markers (anti-CD9 antibody and anti-CD63 antibody).

As a result of the above analysis, it was confirmed that exosomes are present in the obtained fraction.

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.

1M Tris-HCl pH 8.5 containing 10% sodium deoxycholate was added to 100 μl of exosome eluate and sonicated for 10 minutes at "High" using BIORUPTOR UCW-201 (SonicBio Corporation). Samples were treated with 10 mM dithiothreitol for 30 minutes at 50° C. and then alkylated with 30 mM iodoacetamide for 30 minutes at room temperature in the dark. Alkylated samples were incubated with 60 mM cysteine at room temperature for 10 minutes, then diluted to 200 μL with 50 mM ammonium bicarbonate and digested with 500 ng Trypsin/Lys-C Mix overnight at 37°C. Digested peptides were precipitated, acidified with 30 μL 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.

Pretreatment for shotgun proteome analysis was performed as previously reported (PMID: 31779068). Peptides were injected directly into a 75 μm × 20 cm PicoFrit emitter (New Objective) packed with C18 core-shell particles (CAPCELL CORE MP 2.7 μm, 160 Å 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. Peptides eluted from the column were analyzed by overlapping window DIA-MS (PMID: 30671891, PMID: 31779068) using a Q Exactive HF-X (Thermo Fisher Scientific). 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 isolation width of MS2 was set to 4 m/z and the overlapping window pattern of 500-780 m/z used window placement optimized in Skyline (PMID: 20147306).

MS files were searched against a 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). Search parameters for the 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 the EncyclopeDIA algorithm of Scaffold DIA (PMID: 30510204). For each peptide, the four highest quality fragment ions were selected and quantified. Protein quantification was estimated from summed peptide quantification.

Malignant pleural mesothelioma subjects and healthy subjects were compared, and 78 proteins with significantly (P<0.05) different expression levels between the two groups were extracted by t-test.

(result)
Among the extracted malignant pleural mesothelioma biomarkers, Tables 1 and 2 show proteins whose expression levels were higher in malignant pleural mesothelioma subjects than in healthy subjects. In Tables 1 and 2, "Fold change" is the value obtained by dividing the average expression level of malignant pleural mesothelioma subjects by the average expression level of healthy subjects (average expression level of malignant pleural mesothelioma subjects value/average expression level of healthy subjects). Table 1 is a table in which proteins are arranged in descending order of fold change, and Table 2 is a table in which proteins are arranged in ascending order of p value.

Among the extracted malignant pleural mesothelioma biomarkers, Tables 3 and 4 show proteins whose expression levels in malignant pleural mesothelioma subjects were lower than those in healthy subjects. In Tables 3 and 4, "Fold change" is the value obtained by dividing the average expression level of malignant pleural mesothelioma subjects by the average expression level of healthy subjects (average expression level of malignant pleural mesothelioma subjects value/average expression level of healthy subjects). Table 3 is a table in which proteins are arranged in ascending order of fold change, and Table 4 is a table in which proteins are arranged in ascending order of p value.

Claims (14)

A method for examining malignant pleural mesothelioma, comprising:
(1) Protein group (A) and protein group (B) in extracellular vesicles of body fluid or blood sample collected from a subject:
(A) Hermansky-Pudlak syndrome 4 protein, Biglycan, Decorin, Immunoglobulin kappa variable 1-27, Protein FAM3C, Erlin-1, Trafficking protein particle complex subunit 9, Proprotein convertase subtilisin/kexin type 9, Endosomal/lysosomal potassium channel TMEM175, Complement component C9, Complement component C8 alpha chain, Nidogen-2, EGF-containing fibulin-like extracellular matrix protein 1, Periostin, Emerin, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A, Complement factor H-related protein 5, Kunitz-type protease inhibitor 2, Signal-regulatory protein beta-1, Transmembrane emp24 domain-containing protein 5, Plexin domain-containing protein 2, Lanosterol synthase, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1, Transmembrane emp24 domain-containing protein 4, Post- GPI attachment to proteins factor 6, C4b-binding protein alpha chain, Tumor necrosis factor receptor superfamily member 5, ADP-ribosylation factor-binding protein GGA3, Low affinity immunoglobulin from gamma Fc region receptor II-c, Malectin, Disintegrin and metalloproteinase domain-containing protein 9, Prothrombin, Alpha-synuclein, Galectin-8, ELMO domain-containing protein 2, AP-2 complex subunit alpha-1, and Apoptosis regulator BAX (B) Pro-interleukin-16, Putative hydroxypyruvate isomerase, G protein-coupled receptor kinase 5, FERM, ARHGEF and pleckstrin domain-containing protein 1, 26S proteasome non-ATPase regulatory subunit 6, Dynein light chain 2 , cytoplasmic, Protein PRRC1, Myotubularin-related protein 2, T-cell surface glycoprotein CD4, Guanylate-binding protein 1, Spectrin beta chain, non-erythrocytic 2, Glutathione hydrolase 5 proenzyme, EGF-like repeat and discoidin I-like domain- containing protein 3, Bleomycin hydrolase, Acyl-CoA-binding protein, Band 4.1-like protein 2, Ankyrin-1, Glucosamine-6-phosphate isomerase 1, Rho family-interacting cell polarization regulator 1, Interleukin-18, Proteasome subunit beta type -3, Dedicator of cytokinesis protein 2, Band 4.1-like protein 3, WD repeat-c containing protein 37, Keratin, type II cytoskeletal 1, Afadin, Ubiquitin thioesterase OTUB1, Integrin alpha-9, 1,4-alpha-glucan-branching enzyme, Protein 4.1, Methylthioribose-1-phosphate isomerase, SAM and SH3 domain-containing protein 3, ADP-ribosylation factor-like protein 3, Pleckstrin homology domain-containing family O member 2, calcium/calmodulin-dependent protein kinase type 1, Deoxyribonuclease-1-like 1, Guanine nucleotide-binding protein subunit alpha-13, Angiogenin, At least one selected from the group consisting of Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, and Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform A testing method comprising the step of detecting the protein of When the detected protein contains at least one protein (protein A') selected from protein group (A),
(2a) determining that the subject has malignant pleural mesothelioma when the amount or concentration of protein A' detected in step (1) is equal to or greater than the cutoff value;
The inspection method according to claim 1, comprising: When the detected protein contains at least one protein (protein B') selected from protein group (B),
(2b) determining that the subject has malignant pleural mesothelioma when the amount or concentration of protein B' detected in step (1) is equal to or less than the cutoff value;
The inspection method according to claim 1 or 2, comprising: When the detected protein contains at least one protein (protein A') selected from the protein group (A) and at least one protein (protein B') selected from the protein group (B), further ,
(2c) The amount or concentration of protein A' detected in step (1) is equal to or greater than the cutoff value, and/or the amount or concentration of protein B' detected in step (1) is the cutoff value. determining that the subject has malignant pleural mesothelioma when:
The inspection method according to any one of claims 1 to 3, comprising. The protein group (A) is
(A1) Hermansky-Pudlak syndrome 4 protein, Biglycan, Decorin, Immunoglobulin kappa variable 1-27, Protein FAM3C, Erlin-1, Trafficking protein particle complex subunit 9, Proprotein convertase subtilisin/kexin type 9, Endosomal/lysosomal potassium channel TMEM175, Protein group consisting of Complement component C9 and Complement component C8 alpha chain, or (A2) Hermansky-Pudlak syndrome 4 protein, Prothrombin, Post-GPI attachment to proteins factor 6, Plexin domain-containing protein 2, Emerin, Immunoglobulin kappa variable 1 -27, Nidogen-2, and the protein group consisting of Endosomal/lysosomal potassium channel TMEM175, the test method according to any one of claims 1 to 4. The protein group (B) is
(B1) Pro-interleukin-16, Putative hydroxypyruvate isomerase, G protein-coupled receptor kinase 5, FERM, ARHGEF and pleckstrin domain-containing protein 1, 26S proteasome non-ATPase regulatory subunit 6, Dynein light chain 2, cytoplasmic, Protein PRRC1 , Myotubularin-related protein 2, T-cell surface glycoprotein CD4, Guanylate-binding protein 1, Spectrin beta chain, non-erythrocytic 2, Glutathione hydrolase 5 proenzyme, EGF-like repeat and discoidin I-like domain-containing protein 3, and A group of proteins consisting of Bleomycin hydrolase, or (B2) Protein PRRC1, Dedicator of cytokinesis protein 2, G protein-coupled receptor kinase 5, Integrin alpha-9, Calcium/calmodulin-dependent protein kinase type 1, Methylthioribose-1-phosphate isomerase, Ubiquitin thioesterase OTUB1, Afadin, Acyl-CoA-binding protein, Pro-interleukin-16, T-cell surface glycoprotein CD4, Interleukin-18, Glutathione hydrolase 5 proenzyme, ADP-ribosylation factor-like protein 3, and 26S proteasome non-ATPase The inspection method according to any one of claims 1 to 5, wherein the protein group consists of regulatory subunit 6. The test method according to any one of claims 1 to 6, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum. The test method according to any one of claims 1 to 7, wherein the subject is a human. Protein Group (A), and Protein Group (B):
(A) Hermansky-Pudlak syndrome 4 protein, Biglycan, Decorin, Immunoglobulin kappa variable 1-27, Protein FAM3C, Erlin-1, Trafficking protein particle complex subunit 9, Proprotein convertase subtilisin/kexin type 9, Endosomal/lysosomal potassium channel TMEM175, Complement component C9, Complement component C8 alpha chain, Nidogen-2, EGF-containing fibulin-like extracellular matrix protein 1, Periostin, Emerin, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A, Complement factor H-related protein 5, Kunitz-type protease inhibitor 2, Signal-regulatory protein beta-1, Transmembrane emp24 domain-containing protein 5, Plexin domain-containing protein 2, Lanosterol synthase, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1, Transmembrane emp24 domain-containing protein 4, Post- GPI attachment to proteins factor 6, C4b-binding protein alpha chain, Tumor necrosis factor receptor superfamily member 5, ADP-ribosylation factor-binding protein GGA3, Low affinity immunoglobulin from gamma Fc region receptor II-c, Malectin, Disintegrin and metalloproteinase domain-containing protein 9, Prothrombin, Alpha-synuclein, Galectin-8, ELMO domain-containing protein 2, AP-2 complex subunit alpha-1, and Apoptosis regulator BAX (B) Pro-interleukin-16, Putative hydroxypyruvate isomerase, G protein-coupled receptor kinase 5, FERM, ARHGEF and pleckstrin domain-containing protein 1, 26S proteasome non-ATPase regulatory subunit 6, Dynein light chain 2 , cytoplasmic, Protein PRRC1, Myotubularin-related protein 2, T-cell surface glycoprotein CD4, Guanylate-binding protein 1, Spectrin beta chain, non-erythrocytic 2, Glutathione hydrolase 5 proenzyme, EGF-like repeat and discoidin I-like domain- containing protein 3, Bleomycin hydrolase, Acyl-CoA-binding protein, Band 4.1-like protein 2, Ankyrin-1, Glucosamine-6-phosphate isomerase 1, Rho family-interacting cell polarization regulator 1, Interleukin-18, Proteasome subunit beta type -3, Dedicator of cytokinesis protein 2, Band 4.1-like protein 3, WD repeat-c containing protein 37, Keratin, type II cytoskeletal 1, Afadin, Ubiquitin thioesterase OTUB1, Integrin alpha-9, 1,4-alpha-glucan-branching enzyme, Protein 4.1, Methylthioribose-1-phosphate isomerase, SAM and SH3 domain-containing protein 3, ADP-ribosylation factor-like protein 3, Pleckstrin homology domain-containing family O member 2, calcium/calmodulin-dependent protein kinase type 1, Deoxyribonuclease-1-like 1, Guanine nucleotide-binding protein subunit alpha-13, Angiogenin, At least one selected from the group consisting of Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, and Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform A diagnostic agent for malignant pleural mesothelioma, comprising a protein-detecting agent for Protein group (A):
(A) Hermansky-Pudlak syndrome 4 protein, Biglycan, Decorin, Immunoglobulin kappa variable 1-27, Protein FAM3C, Erlin-1, Trafficking protein particle complex subunit 9, Proprotein convertase subtilisin/kexin type 9, Endosomal/lysosomal potassium channel TMEM175, Complement component C9, Complement component C8 alpha chain, Nidogen-2, EGF-containing fibulin-like extracellular matrix protein 1, Periostin, Emerin, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A, Complement factor H-related protein 5, Kunitz-type protease inhibitor 2, Signal-regulatory protein beta-1, Transmembrane emp24 domain-containing protein 5, Plexin domain-containing protein 2, Lanosterol synthase, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1, Transmembrane emp24 domain-containing protein 4, Post- GPI attachment to proteins factor 6, C4b-binding protein alpha chain, Tumor necrosis factor receptor superfamily member 5, ADP-ribosylation factor-binding protein GGA3, Low affinity immunoglobulin from gamma Fc region receptor II-c, Malectin, Disintegrin and metalloproteinase domain-containing protein 9, Prothrombin, Alpha-synuclein, Galectin-8, ELMO domain-containing protein 2, AP-2 complex subunit alpha-1, and Apoptosis regulator BAX an inhibitor of at least one protein selected from the group of proteins consisting of and protein group (B):
(B) Pro-interleukin-16, Putative hydroxypyruvate isomerase, G protein-coupled receptor kinase 5, FERM, ARHGEF and pleckstrin domain-containing protein 1, 26S proteasome non-ATPase regulatory subunit 6, Dynein light chain 2, cytoplasmic, Protein PRRC1 , Myotubularin-related protein 2, T-cell surface glycoprotein CD4, Guanylate-binding protein 1, Spectrin beta chain, non-erythrocytic 2, Glutathione hydrolase 5 proenzyme, EGF-like repeat and discoidin I-like domain-containing protein 3, Bleomycin hydrolase, Acyl-CoA-binding protein, Band 4.1-like protein 2, Ankyrin-1, Glucosamine-6-phosphate isomerase 1, Rho family-interacting cell polarization regulator 1, Interleukin-18, Proteasome subunit beta type-3, Dedicator of cytokinesis protein 2, Band 4.1-like protein 3, WD repeat-containing protein 37, Keratin, type II cytoskeletal 1, Afadin, Ubiquitin thioesterase OTUB1, Integrin alpha-9, 1,4-alpha-glucan-branching enzyme, Protein 4.1, Methylthioribose-1-phosphate isomerase, SAM and SH3 domain-containing protein 3, ADP-ribosyl cation factor-like protein 3, Pleckstrin homology domain-containing family O member 2, calcium/calmodulin-dependent protein kinase type 1, deoxyribonuclease-1-like 1, guanine nucleotide-binding protein subunit alpha-13, angiogenin, serine/threonine- at least one protein enhancer selected from the group of proteins consisting of protein phosphatase 2A catalytic subunit beta isoform, Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, and Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform A prophylactic or therapeutic agent for malignant pleural mesothelioma containing at least one drug selected from the group. Protein group (A) and protein group (B) in extracellular vesicles of bodily fluids or blood samples taken from animals treated with the test substance:
(A) Hermansky-Pudlak syndrome 4 protein, Biglycan, Decorin, Immunoglobulin kappa variable 1-27, Protein FAM3C, Erlin-1, Trafficking protein particle complex subunit 9, Proprotein convertase subtilisin/kexin type 9, Endosomal/lysosomal potassium channel TMEM175, Complement component C9, Complement component C8 alpha chain, Nidogen-2, EGF-containing fibulin-like extracellular matrix protein 1, Periostin, Emerin, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A, Complement factor H-related protein 5, Kunitz-type protease inhibitor 2, Signal-regulatory protein beta-1, Transmembrane emp24 domain-containing protein 5, Plexin domain-containing protein 2, Lanosterol synthase, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1, Transmembrane emp24 domain-containing protein 4, Post- GPI attachment to proteins factor 6, C4b-binding protein alpha chain, Tumor necrosis factor receptor superfamily member 5, ADP-ribosylation factor-binding protein GGA3, Low affinity immunoglobulin from gamma Fc region receptor II-c, Malectin, Disintegrin and metalloproteinase domain-containing protein 9, Prothrombin, Alpha-synuclein, Galectin-8, ELMO domain-containing protein 2, AP-2 complex subunit alpha-1, and Apoptosis regulator BAX (B) Pro-interleukin-16, Putative hydroxypyruvate isomerase, G protein-coupled receptor kinase 5, FERM, ARHGEF and pleckstrin domain-containing protein 1, 26S proteasome non-ATPase regulatory subunit 6, Dynein light chain 2 , cytoplasmic, Protein PRRC1, Myotubularin-related protein 2, T-cell surface glycoprotein CD4, Guanylate-binding protein 1, Spectrin beta chain, non-erythrocytic 2, Glutathione hydrolase 5 proenzyme, EGF-like repeat and discoidin I-like domain- containing protein 3, Bleomycin hydrolase, Acyl-CoA-binding protein, Band 4.1-like protein 2, Ankyrin-1, Glucosamine-6-phosphate isomerase 1, Rho family-interacting cell polarization regulator 1, Interleukin-18, Proteasome subunit beta type -3, Dedicator of cytokinesis protein 2, Band 4.1-like protein 3, WD repeat-c containing protein 37, Keratin, type II cytoskeletal 1, Afadin, Ubiquitin thioesterase OTUB1, Integrin alpha-9, 1,4-alpha-glucan-branching enzyme, Protein 4.1, Methylthioribose-1-phosphate isomerase, SAM and SH3 domain-containing protein 3, ADP-ribosylation factor-like protein 3, Pleckstrin homology domain-containing family O member 2, calcium/calmodulin-dependent protein kinase type 1, Deoxyribonuclease-1-like 1, Guanine nucleotide-binding protein subunit alpha-13, Angiogenin, At least one selected from the group consisting of Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, and Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform A screening method for an active ingredient of a preventive or therapeutic agent for malignant pleural mesothelioma, wherein the amount or concentration of the protein of is used as an index. If the value of the indicator for protein group (A) is lower than the amount or concentration of the corresponding protein in extracellular vesicles of bodily fluids or blood samples collected from animals not treated with the test substance, the test substance a step of selecting the substance as an active ingredient of a prophylactic or therapeutic agent for malignant pleural mesothelioma; At least one selected from the group consisting of the step of selecting the test substance as an active ingredient of a preventive or therapeutic agent for malignant pleural mesothelioma when the amount or concentration of the corresponding protein in the vesicles or blood sample is higher than that of the corresponding protein. The screening method according to claim 11, comprising the step of Protein group (A) and protein group (B) in extracellular vesicles of bodily fluids or blood samples taken from animals treated with the test substance:
(A) Hermansky-Pudlak syndrome 4 protein, Biglycan, Decorin, Immunoglobulin kappa variable 1-27, Protein FAM3C, Erlin-1, Trafficking protein particle complex subunit 9, Proprotein convertase subtilisin/kexin type 9, Endosomal/lysosomal potassium channel TMEM175, Complement component C9, Complement component C8 alpha chain, Nidogen-2, EGF-containing fibulin-like extracellular matrix protein 1, Periostin, Emerin, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A, Complement factor H-related protein 5, Kunitz-type protease inhibitor 2, Signal-regulatory protein beta-1, Transmembrane emp24 domain-containing protein 5, Plexin domain-containing protein 2, Lanosterol synthase, Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1, Transmembrane emp24 domain-containing protein 4, Post- GPI attachment to proteins factor 6, C4b-binding protein alpha chain, Tumor necrosis factor receptor superfamily member 5, ADP-ribosylation factor-binding protein GGA3, Low affinity immunoglobulin from gamma Fc region receptor II-c, Malectin, Disintegrin and metalloproteinase domain-containing protein 9, Prothrombin, Alpha-synuclein, Galectin-8, ELMO domain-containing protein 2, AP-2 complex subunit alpha-1, and Apoptosis regulator BAX (B) Pro-interleukin-16, Putative hydroxypyruvate isomerase, G protein-coupled receptor kinase 5, FERM, ARHGEF and pleckstrin domain-containing protein 1, 26S proteasome non-ATPase regulatory subunit 6, Dynein light chain 2 , cytoplasmic, Protein PRRC1, Myotubularin-related protein 2, T-cell surface glycoprotein CD4, Guanylate-binding protein 1, Spectrin beta chain, non-erythrocytic 2, Glutathione hydrolase 5 proenzyme, EGF-like repeat and discoidin I-like domain- containing protein 3, Bleomycin hydrolase, Acyl-CoA-binding protein, Band 4.1-like protein 2, Ankyrin-1, Glucosamine-6-phosphate isomerase 1, Rho family-interacting cell polarization regulator 1, Interleukin-18, Proteasome subunit beta type -3, Dedicator of cytokinesis protein 2, Band 4.1-like protein 3, WD repeat-c containing protein 37, Keratin, type II cytoskeletal 1, Afadin, Ubiquitin thioesterase OTUB1, Integrin alpha-9, 1,4-alpha-glucan-branching enzyme, Protein 4.1, Methylthioribose-1-phosphate isomerase, SAM and SH3 domain-containing protein 3, ADP-ribosylation factor-like protein 3, Pleckstrin homology domain-containing family O member 2, calcium/calmodulin-dependent protein kinase type 1, Deoxyribonuclease-1-like 1, Guanine nucleotide-binding protein subunit alpha-13, Angiogenin, At least one selected from the group consisting of Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform, and Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform A method for evaluating the inducibility or exacerbation of malignant pleural mesothelioma, using the amount or concentration of protein as an index. If the value of the indicator for protein group (A) is higher than the amount or concentration of the corresponding protein in extracellular vesicles of body fluids or blood samples collected from animals not treated with the test substance, the test substance Determining a substance as inducing or exacerbating malignant pleural mesothelioma, and the value of said indicator for protein group (B) is determined in extracellular body fluids collected from animals not treated with the test substance At least one selected from the group consisting of determining that the test substance has the potential to induce or exacerbate malignant pleural mesothelioma when the amount or concentration is lower than the amount or concentration of the corresponding protein in the vesicles or blood sample. The evaluation method according to claim 13, comprising the step of