WO2020162441A1 - Granulomatous disease biomarker - Google Patents

Abstract

The present invention addresses the problem of providing a sarcoidosis biomarker and a method for using the biomarker. This problem is solved by a specific protein group in the extracellular vesicles in body fluid or a blood sample collected from a subject.

Description

Granulomatous disease biomarkers

The present invention relates to granuloma disease biomarkers and the like.

Sarcoidosis is a type of granulomatous disease, and it is an incurable disease of unknown cause in which non-caseating granulomas are formed throughout the body. Although there are cases in which granulomas are reversible and spontaneously healed, there are also cases with poor prognosis in which lesions appear in multiple organs including the lungs, margins, and heart, causing irreversible fibrosis and serious organ damage. To do. A single environmental factor of antigenic exposure, such as a bacterial infection, cannot be explained and multiple genetic factors are involved.

Despite the presence of ACE and sIL-2 receptors as serum biomarkers for sarcoidosis, their sensitivity and specificity are not sufficient, and the development of new biomarkers is urgently needed.

Although serum is considered to be an ideal sample that can be repeatedly collected non-invasively, it contains abundant proteins (albumin etc.) that are considered as contaminants from the viewpoint of biomarkers. Therefore, even if a biomarker is searched using serum as a sample, satisfactory results cannot be obtained. A recent report on a biomarker capable of differentiating between COPD and asthma (Non-patent Document 1) shows that serum containing a large amount of contaminants was used in a mass spectrometry technique by separation using a gel by a two-dimensional electrophoresis method. The only biomarkers released were serum contaminants containing prothrombin.

An object of the present invention is to provide a biomarker for granulomatous disease and a method for using the biomarker.

During the course of research, the present inventor focused on searching for biomarkers of granulomatous disease using a purified extracellular vesicle fraction as a sample. As a result of further intensive research based on this point of view, a specific protein group in extracellular vesicles or blood samples of body fluids collected from a subject is useful as a biomarker for granulomatous disease. Found. As a result of further research based on this finding, the present invention has been completed. That is, the present invention includes the following aspects.

Item 1. A method of testing for granulomatous disease, the method comprising:
(1) Protein group (AX) in extracellular vesicles or blood sample of body fluid collected from a subject:
(AX) Lipopolysaccharide-binding protein and a test method comprising a step of detecting at least one protein selected from the group consisting of a protein group consisting of Monocyte differentiation antigen CD14.

Item 2. A method of testing for granulomatous disease, the method comprising:
(1) Protein group (A) and protein group (B) in extracellular vesicles or blood samples of body fluid collected from a subject:
(A) Chitinase-3-like protein 1, Protein-tyrosine sulfotransferase 2, Myoferlin, Pancreas transcription factor 1 subunit alpha, RuvB-like 2, Protein FAM110D, Cystatin-A, Creatine kinase M-type, Rho GTPase-activating protein 4 , Glutathione hydrolase 5 proenzyme, Putative neutrophil cytosol factor 1C, Neutrophil cytosol factor 1, Putative neutrophil cytosol factor 1B, Sn1-specific diacylglycerol lipase beta, Protein FAM26E, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 2, Anoctamin CCR4-NOT transcription complex subunit 1, Interferon-induced helicase C domain-containing protein 1, T-cell surface glycoprotein CD3 zeta chain, Caspase-3, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2, Interferon regulatory factor 2-binding Protein 1, Endothelial lipase, Lipopolysaccharide-binding protein, Transthyretin, and Monocyte differentiation antigen CD14, and (B) Lysosome-associated membrane glycoprotein 2, Protein Jade-3, Tetraspanin-4, Oxysterol-binding protein-related protein 8, P2Y purinoceptor 12, Dematin, Tr ansforming acidic coiled-coil-containing protein 2, Ubiquitin carboxyl-terminal hydrolase MINDY-1, Septin-4, Ephrin-B1, Septin-5, Plexin-B3, Tetraspanin-32, H(+)/Cl(-) exchange transporter 5, Neutrophil collagenase, Septin-8, Septin-6, Metabotropic glutamate receptor 6, Metabotropic glutamate receptor 7, Metabotropic glutamate receptor 4, Metabotropic glutamate receptor 8, Gamma-enolase, and CD63 antigen An assay method comprising the step of detecting at least one protein that is

Item 3. When the detected protein contains at least one protein (protein A′) selected from the protein group (A),
(2a) when the amount or concentration of the protein A′ detected in the step (1) is a cutoff value or more, a step of determining that the subject has a granulomatous disease,
The inspection method according to Item 2, further comprising:

Item 4. When the detected protein contains at least one protein (protein B′) selected from the protein group (B),
(2b) when the amount or concentration of the protein B′ detected in the step (1) is less than or equal to a cutoff value, the step of determining that the subject has granulomatous disease,
The inspection method according to Item 2 or 3, including:

Item 5. When the detected protein contains at least one protein (protein A′) selected from protein group (A) and at least one protein (protein B′) selected from protein group (B), ,
(2c) The amount or concentration of protein A′ detected in the step (1) is a cutoff value or more, and/or the amount or concentration of protein B′ detected in the step (1) is a cutoff value. Step of determining that the subject is suffering from granulomatous disease, if
Item 5. The inspection method according to any one of Items 2 to 4, including:

Item 6. The protein group (A) is
(A1) Chitinase-3-like protein 1, Protein-tyrosine sulfotransferase 2, Myoferlin, Pancreas transcription factor 1 subunit alpha, RuvB-like 2, Protein FAM110D, Cystatin-A, Creatine kinase M-type, Rho GTPase-activating protein 4 , Glutathione hydrolase 5 proenzyme, Putative neutrophil cytosol factor 1C, Neutrophil cytosol factor 1, Putative neutrophil cytosol factor 1B, Sn1-specific diacylglycerol lipase beta, Protein FAM26E, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase-5, and Anoctamin. A group of,
(A2) CCR4-NOT transcription complex subunit 1, Interferon-induced helicase C domain-containing protein 1, and T-cell surface glycoprotein CD3 zeta chain,
(A3) Myoferlin, Glutathione hydrolase 5 proenzyme, Sn1-specific diacylglycerol lipase beta, Interferon-induced helicase C domain-containing protein 1, Caspase-3, and a group consisting of Lipopolysaccharide-binding protein, and (A4) Myoferlin, Protein FAM110D, CCR4-NOT transcription complex subunit 1, Keratin, type I cytoskeletal 39, Caspase-3, Endothelial lipase, Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase, and lipopolysaccharide-binding protein Item 6. The test method according to any one of Items 2 to 5, which is at least one protein group selected from the following.

Item 7. The protein group (B) is
(B1) Lysosome-associated membrane glycoprotein 2, and Protein Jade-3 group,
(B2) Tetraspanin-4, Oxysterol-binding protein-related protein 8, P2Y purinoceptor 12, and a group consisting of Dematin,
(B3) Protein Jade-3, P2Y purinoceptor 12, Dematin, Ubiquitin carboxyl-terminal hydrolase MINDY-1, Septin-4, Septin-5, Plexin-B3, Septin-8, Septin-6, Metabotropic glutamate receptor 6, Metabotropic glutamate group consisting of receptor 7, Metabotropic glutamate receptor 4, and Metabotropic glutamate receptor 8, and (B4) H(+)/Cl(-) exchange transporter 3, Metabotropic glutamate receptor 4, Metabotropic glutamate receptor 6, Metabotropic glutamate receptor 7, Metabotropic Item 7. The test method according to any one of Items 2 to 6, which is at least one protein group selected from the group consisting of glutamate receptor 8 and Mitogen-activated protein kinase 1.

Item 8. Item 8. The test method according to any one of Items 1 to 7, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum.

Item 9. The test method according to any one of Items 1 to 8, wherein the subject is a human.

Item 10. Protein group (A) and protein group (B):
(A) Chitinase-3-like protein 1, Protein-tyrosine sulfotransferase 2, Myoferlin, Pancreas transcription factor 1 subunit alpha, RuvB-like 2, Protein FAM110D, Cystatin-A, Creatine kinase M-type, Rho GTPase-activating protein 4 , Glutathione hydrolase 5 proenzyme, Putative neutrophil cytosol factor 1C, Neutrophil cytosol factor 1, Putative neutrophil cytosol factor 1B, Sn1-specific diacylglycerol lipase beta, Protein FAM26E, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 2, Anoctamin. CCR4-NOT transcription complex subunit 1, Interferon-induced helicase C domain-containing protein 1, T-cell surface glycoprotein CD3 zeta chain, Caspase-3, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2, Interferon regulatory factor 2-binding Protein 1, Endothelial lipase, Lipopolysaccharide-binding protein, Transthyretin, and Monocyte differentiation antigen CD14, and (B) Lysosome-associated membrane glycoprotein 2, Protein Jade-3, Tetraspanin-4, Oxysterol-binding protein-related protein 8, P2Y purinoceptor 12, Dematin, Tr ansforming acidic coiled-coil-containing protein 2, Ubiquitin carboxyl-terminal hydrolase MINDY-1, Septin-4, Ephrin-B1, Septin-5, Plexin-B3, Tetraspanin-32, H(+)/Cl(-) exchange transporter 5, Neutrophil collagenase, Septin-8, Septin-6, Metabotropic glutamate receptor 6, Metabotropic glutamate receptor 7, Metabotropic glutamate receptor 4, Metabotropic glutamate receptor 8, Gamma-enolase, and CD63 antigen An agent for detecting a granulomatous disease, which comprises a detection agent for at least one protein according to claim 1.

Item 11. Protein group (A):
(A) Chitinase-3-like protein 1, Protein-tyrosine sulfotransferase 2, Myoferlin, Pancreas transcription factor 1 subunit alpha, RuvB-like 2, Protein FAM110D, Cystatin-A, Creatine kinase M-type, Rho GTPase-activating protein 4 , Glutathione hydrolase 5 proenzyme, Putative neutrophil cytosol factor 1C, Neutrophil cytosol factor 1, Putative neutrophil cytosol factor 1B, Sn1-specific diacylglycerol lipase beta, Protein FAM26E, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 2, Anoctamin CCR4-NOT transcription complex subunit 1, Interferon-induced helicase C domain-containing protein 1, T-cell surface glycoprotein CD3 zeta chain, Caspase-3, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2, Interferon regulatory factor 2-binding At least one protein inhibitor selected from the protein group consisting of protein 1, Endothelial lipase, Lipopolysaccharide-binding protein, Transthyretin, and Monocyte differentiation antigen CD14, and protein group (B):
(B) Lysosome-associated membrane glycoprotein 2, Protein Jade-3, Tetraspanin-4, Oxysterol-binding protein-related protein 8, P2Y purinoceptor 12, Dematin, Transforming acidic coiled-coil-containing protein 2, Ubiquitin carboxyl-terminal hydrolase MINDY -1, Septin-4, Ephrin-B1, Septin-5, Plexin-B3, Tetraspanin-32, H(+)/Cl(-) exchange transporter 5, Neutrophil collagenase, Septin-8, Septin-6, Metabotropic glutamate receptor 6, Metabotropic glutamate receptor 7, Metabotropic glutamate receptor 4, Metabotropic glutamate receptor 8, Gamma-enolase, and at least one selected from the group consisting of at least one protein enhancer selected from the protein group consisting of CD63 antigen A prophylactic or therapeutic agent for granulomatous disease, which comprises the drug.

Item 12. Protein group (A) and protein group (B) in extracellular vesicles or blood samples of body fluids collected from animals treated with the test substance:
(A) Chitinase-3-like protein 1, Protein-tyrosine sulfotransferase 2, Myoferlin, Pancreas transcription factor 1 subunit alpha, RuvB-like 2, Protein FAM110D, Cystatin-A, Creatine kinase M-type, Rho GTPase-activating protein 4 , Glutathione hydrolase 5 proenzyme, Putative neutrophil cytosol factor 1C, Neutrophil cytosol factor 1, Putative neutrophil cytosol factor 1B, Sn1-specific diacylglycerol lipase beta, Protein FAM26E, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 2, Anoctamin CCR4-NOT transcription complex subunit 1, Interferon-induced helicase C domain-containing protein 1, T-cell surface glycoprotein CD3 zeta chain, Caspase-3, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2, Interferon regulatory factor 2-binding Protein 1, Endothelial lipase, Lipopolysaccharide-binding protein, Transthyretin, and Monocyte differentiation antigen CD14, and (B) Lysosome-associated membrane glycoprotein 2, Protein Jade-3, Tetraspanin-4, Oxysterol-binding protein-related protein 8, P2Y purinoceptor 12, Dematin, Tr ansforming acidic coiled-coil-containing protein 2, Ubiquitin carboxyl-terminal hydrolase MINDY-1, Septin-4, Ephrin-B1, Septin-5, Plexin-B3, Tetraspanin-32, H(+)/Cl(-) exchange transporter 5, Neutrophil collagenase, Septin-8, Septin-6, Metabotropic glutamate receptor 6, Metabotropic glutamate receptor 7, Metabotropic glutamate receptor 4, Metabotropic glutamate receptor 8, Gamma-enolase, and CD63 antigen A method for screening an active ingredient of a prophylactic or therapeutic agent for granulomatous disease, which comprises using the amount or concentration of at least one protein as an index.

Item 13. When the value of the index for the protein group (A) is lower than the amount or concentration of the corresponding protein in extracellular vesicles or blood samples of body fluid collected from animals not treated with the test substance, the test The step of selecting a substance as an active ingredient of a prophylactic or therapeutic agent for granulomatous disease, and the value of the index relating to protein group (B) is the extracellular small amount of the body fluid collected from an animal not treated with the test substance. At least one step selected from the group consisting of selecting the test substance as an active ingredient of a prophylactic or therapeutic agent for granulomatous disease when the amount or concentration of the corresponding protein in the vesicle or blood sample is higher. Item 13. A screening method according to Item 12, which comprises:

Item 14. Protein group (A) and protein group (B) in extracellular vesicles or blood samples of body fluids collected from animals treated with the test substance:
(A) Chitinase-3-like protein 1, Protein-tyrosine sulfotransferase 2, Myoferlin, Pancreas transcription factor 1 subunit alpha, RuvB-like 2, Protein FAM110D, Cystatin-A, Creatine kinase M-type, Rho GTPase-activating protein 4 , Glutathione hydrolase 5 proenzyme, Putative neutrophil cytosol factor 1C, Neutrophil cytosol factor 1, Putative neutrophil cytosol factor 1B, Sn1-specific diacylglycerol lipase beta, Protein FAM26E, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 2, Anoctamin CCR4-NOT transcription complex subunit 1, Interferon-induced helicase C domain-containing protein 1, T-cell surface glycoprotein CD3 zeta chain, Caspase-3, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2, Interferon regulatory factor 2-binding Protein 1, Endothelial lipase, Lipopolysaccharide-binding protein, Transthyretin, and Monocyte differentiation antigen CD14, and (B) Lysosome-associated membrane glycoprotein 2, Protein Jade-3, Tetraspanin-4, Oxysterol-binding protein-related protein 8, P2Y purinoceptor 12, Dematin, Tr ansforming acidic coiled-coil-containing protein 2, Ubiquitin carboxyl-terminal hydrolase MINDY-1, Septin-4, Ephrin-B1, Septin-5, Plexin-B3, Tetraspanin-32, H(+)/Cl(-) exchange transporter 5, Neutrophil collagenase, Septin-8, Septin-6, Metabotropic glutamate receptor 6, Metabotropic glutamate receptor 7, Metabotropic glutamate receptor 4, Metabotropic glutamate receptor 8, Gamma-enolase, and CD63 antigen A method for evaluating the induction or exacerbation of granulomatous disease, which comprises using the amount or concentration of at least one protein as an index.

Item 15. When the value of the index relating to the protein group (A) is higher than the amount or concentration of the corresponding protein in the extracellular vesicle or blood sample of the body fluid collected from the animal not treated with the test substance, the test The step of determining that the substance has a granulomatous disease-inducing or malignant state, and the value of the above-mentioned index relating to the protein group (B) is the extracellular small amount of the body fluid collected from the animal not treated with the test substance. At least one step selected from the group consisting of the step of determining that the test substance has a granulomatous disease-inducing or malignant condition when it is lower than the amount or concentration of the corresponding protein in the vesicle or blood sample. Item 15. The evaluation method according to Item 14, which comprises:

According to the present invention, a biomarker for granulomatous disease can be provided. By using the biomarker, examination of granulomatous disease, prevention or treatment of granulomatous disease, prevention or treatment of granulomatous disease, screening of active ingredient of drug Granuloma disease-induced or malignant Evaluation etc. may be possible.

The particle diameter of the extracellular vesicle fraction is shown (Test Example 1). Control shows the results of the extracellular vesicle fraction obtained from the healthy subject, and Sarcoidosis shows the results of the extracellular vesicle fraction obtained from the sarcoidosis subject. The number of particles in the extracellular vesicle fraction is shown (Test Example 1). Control shows the results of the extracellular vesicle fraction obtained from the healthy subject, and Sarcoidosis shows the results of the extracellular vesicle fraction obtained from the sarcoidosis subject. The electron microscopic observation image of an extracellular vesicle fraction is shown (Test Example 1). Control shows the results of the extracellular vesicle fraction obtained from the healthy subject, and Sarcoidosis shows the results of the extracellular vesicle fraction obtained from the sarcoidosis subject. The image displayed in the lower left of each image is a magnified image of one extracellular vesicle. The result of Western blotting of the extracellular vesicle fraction is shown (Test Example 1). Control shows the results of the extracellular vesicle fraction obtained from the healthy subject, and Sarcoidosis shows the results of the extracellular vesicle fraction obtained from the sarcoidosis subject. The left side of the band picture shows the antigen of the primary antibody used. The quantification result (SRM analysis result) of Test Example 4 for Monocyte differentiation antigen CD14 (CD14) is shown. The vertical axis represents the quantitative value. The quantitative results (SRM analysis results) of Test Example 4 for Lipopolysaccharide-binding protein (LBP) are shown. The vertical axis represents the quantitative value. The result of Western blotting of the extracellular vesicle fraction is shown (Test Example 5). Each lane shows the results for a separate sample of each analyte. The result of the immunoelectron microscopy of the extracellular vesicle fraction is shown (Test Example 6). The result of immunostaining of lung tissue is shown (Test Example 7). The ROC curve and AUC value when using Monocyte differentiation antigen CD14 (CD14) and Lipopolysaccharide-binding protein (LBP) as a sarcoidosis biomarker are shown (Test Example 8). Shows the ROC curve when using ACE and CD14 in combination as a sarcoidosis biomarker, and the ROC curve when using ACE, sIL-2, CD14 and LBP in combination as a sarcoidosis biomarker, and an AUC curve ( Test example 9).

In the present specification, the expressions “containing” and “including” include the concepts of “containing”, “including”, “consisting essentially of” and “consisting solely of”.

1. Method for Examining Granulomatous Disease The present invention, in one aspect thereof, is a method for examining a granulomatous disease, which comprises (1) extracellular vesicles or a blood sample of a body fluid collected from a subject, a protein group. (A) and a test method (herein referred to as "test method of the present invention") including a step (step 1) of detecting at least one protein selected from the group consisting of protein group (B) There is also a thing.) This will be described below.

1-1. Process (1)
The type of “granulomatous disease” to be tested is not particularly limited. Many diseases that form granulomas in the lungs include infectious diseases such as tuberculosis/nontuberculous mycobacteriosis and mycosis, as well as sarcoidosis and Wegener granulomatosis, rheumatoid nodules, and hypersensitivity pneumonitis. Non-infectious (or unknown cause) diseases are also included.

Particularly preferred granulomatous disease is sarcoidosis.

All classes, grades, and stages of granulomatous disease in various classification criteria related to the progression of granulomatous disease can be tested. In addition, all types of sarcoidosis in various classifications regarding lesion sites of sarcoidosis (eg, pulmonary sarcoidosis, ocular sarcoidosis, nerve sarcoidosis, cardiac sarcoidosis, cutaneous sarcoidosis, muscle sarcoidosis, etc.) can be tested.

The subject is a target organism of the test method of the present invention, and its species is not particularly limited. Examples of the biological species of the subject include various mammalian animals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, and preferably humans.

The condition of the subject is not particularly limited. As the subject, for example, a sample that is not known to have a granulomatous disease, a sample that has already been determined to have a granulomatous disease by another method, and does not have a granulomatous disease And a sample under treatment for granulomatous disease, a sample after treatment for granulomatous disease, and the like.

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

The body fluid is not particularly limited. Examples of the body fluid include whole blood, serum, plasma, spinal fluid, saliva, joint fluid, urine, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal discharge, etc., preferably whole blood, serum , Plasma, and cerebrospinal fluid, and more preferably whole blood, serum, and plasma. As the body fluid, one type may be used alone, or two or more types may be used in combination.

Body fluid can be collected from a subject by a method known to those skilled in the art. For example, whole blood can be collected by collecting blood using a syringe or the like. Serum is a portion obtained by removing blood cells and a specific blood coagulation factor from whole blood, and can be obtained, for example, as a supernatant after coagulating whole blood. Plasma is a portion obtained by removing blood cells from whole blood, and can be obtained, for example, as a supernatant when subjected to centrifugation under conditions in which whole blood is not coagulated.

In the present specification, blood itself such as whole blood, serum, plasma or a sample derived from blood is referred to as “blood sample”.

Extracellular vesicles are not particularly limited as long as they are membrane vesicles secreted and released from cells. Extracellular vesicles are usually defined as membrane vesicles that carry intracellular and local cell-to-cell information transfer by transporting intracellular proteins and genetic information (mRNA, microRNA, etc.) to the outside of the cell. It Examples of extracellular vesicles include exosomes, microvesicles, apoptotic bodies, ectosomes, microparticles, secretory microvesicles, and the like.

Extracellular vesicles can be purified, separated, concentrated, etc. from body fluids according to known methods or according to known methods. Examples of methods for purifying, separating, concentrating extracellular vesicles include ultracentrifugation (eg pellet down method, sucrose cushion method, density gradient centrifugation etc.), method using immunoaffinity carrier, gel filtration method, field Flow fractionation method, FACS method and the like can be mentioned. In addition, purification, separation, concentration, etc. of extracellular vesicles can be performed using a commercially available kit. These methods may be used alone 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 the present specification, these are collectively referred to as “target protein”). There is also).

Protein group (A) is (A) Chitinase-3-like protein1, Protein-tyrosinesulfotransferase2, Myoferlin, Pancreas transcription factor 1 subunit, alpha, RuvB-like2, ProteinFAM110D, Cystatin-A, Creatine kinase M-type , Rho GTPase-activating protein 4, Glutathione hydrolase 5 proenzyme, Putative neutrophil cytosol factor 1C, Neutrophil cytosol factor 1, Putative neutrophil cytosol factor 1B,Sn1-specific diacylglycerol lipase beta,Eteinhosto phosphatase2, Anoctamin-5, CCR4-NOT transcription complex subunit1, Interferon-induced helicaseC domain-containing protein 1, T-cell surface glycoprotein CD3 zeta chain, Caspase-3, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase2 , Interferon regulatory factor2-binding protein1, Endothelial lipase, Lipopolysaccharide-binding protein, Transthyretin, and Monocyte differentiation antigen CD14.

The protein group (A) is a protein group in which the amount in the granulomatous disease specimen is higher than that in the normal specimen.

Among the protein group (A), the protein group (AX) is particularly preferable from the viewpoint of diagnostic ability:
(AX) Lipopolysaccharide-binding protein, and a group of proteins consisting of Monocyte differentiation antigen CD14.

Among the protein group (A), from the viewpoint that the amount ratio to the normal sample is higher, the protein group (A1) and the protein group (A2) are preferably:
(A1) Chitinase-3-like protein 1, Protein-tyrosine sulfotransferase 2, Myoferlin, Pancreas transcription factor 1 subunit alpha, RuvB-like 2, Protein FAM110D, Cystatin-A, Creatine kinase M-type, Rho GTPase-activating protein 4 , Glutathione hydrolase 5 proenzyme, Putative neutrophil cytosol factor 1C, Neutrophil cytosol factor 1, Putative neutrophil cytosol factor 1B, Sn1-specific diacylglycerol lipase beta, Protein FAM26E, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase-5, and Anoctamin. And a group consisting of (A2) CCR4-NOT transcription complex subunit 1, Interferon-induced helicase C domain-containing protein 1, and T-cell surface glycoprotein CD3 zeta chain.

Of the protein group (A), the protein group (A3):
(A3) Myoferlin, Glutathione hydrolase 5 proenzyme, Sn1-specific diacylglycerol lipase beta, Interferon-induced helicase C domain-containing protein 1, Caspase-3, and lipopolysaccharide-binding protein.

Among the protein group (A), the protein group (A4) is preferably from the viewpoint of more reflecting the therapeutic effect on the granulomatous disease.
(A4) Myoferlin, Protein FAM110D, CCR4-NOT transcription complex subunit 1, Keratin, type I cytoskeletal 39, Caspase-3, Endothelial lipase, Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase, and Lipopolysaccharide- An example is a group consisting of binding proteins.

Protein group (B) is (B) Lysosome-associated membrane glycoprotein2, ProteinJade-3, Tetraspanin-4, Oxysterol-binding protein-related protein8, P2Y purinoceptor 12, Dematin, Transforming acidic coiled-coil-containing protein 2 , Ubiquitin carboxyl-terminal hydrolase MINDY-1, Septin-4, Ephrin-B1, Septin-5, Plexin-B3, Tetraspanin-32, H(+)/Cl(-)  exchange transporter 5, Neutrophil collagenase, Septin-8, It is a protein group consisting of Septin-6, Metabotropic glutamate receptor 6, Metabotropic glutamate receptor 7, Metabotropic glutamate receptor 4, Metabotropic glutamate receptor 8, Gamma-enolase, and CD63 antigen.

The protein group (B) is a protein group whose amount in the granulomatous disease sample is lower than that in the normal sample.

Among the protein group (B), preferably the protein group (B1) and the protein group (B2):
(B1) Lysosome-associated membrane glycoprotein 2, and Protein Jade-3 group, and (B2) Tetraspanin-4, Oxysterol-binding protein-related protein 8, P2Y purinoceptor 12, and Dematin group,
Are listed.

Among the protein group (B), the protein group (B3):
(B3) Protein Jade-3, P2Y purinoceptor 12, Dematin, Ubiquitin carboxyl-terminal hydrolase MINDY-1, Septin-4, Septin-5, Plexin-B3, Septin-8, Septin-6, Metabotropic glutamate receptor 6, Metabotropic glutamate a group consisting of receptor 7, Metabotropic glutamate receptor 4, and Metabotropic glutamate receptor 8,
Are listed.

Among the protein group (B), from the viewpoint of more reflecting the therapeutic effect on the granulomatous disease, the protein group (B4) is preferably:
(B4) H(+)/Cl(-) exchange transporter 3, Metabotropic glutamate receptor 4, Metabotropic glutamate receptor 6, Metabotropic glutamate receptor 7, Metabotropic glutamate receptor 8, and Mitogen-activated protein kinase 1.

In the case of humans, the proteins of the protein groups (A) to (B) are proteins specified by UniProtKB accession numbers shown in Tables 1 to 4 in the examples described later. In the case of other species, it is the ortholog of the protein identified by the UniProtKB accession number shown in Tables 1-4.

The number of target proteins in step (1) may be only one type, or may be a combination of two or more types. By combining more (for example, 2, 5, 10, 20, 30, 40, 45 or more) detection targets, it is possible to perform more accurate tests for granulomatous disease, etc. become.

-Detection is usually performed by measuring the amount or concentration of the target protein. The “concentration” is not limited to the absolute concentration, but may be a relative concentration, a weight per unit volume, or raw data measured to know the 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 a mass spectrometry method for detecting a peptide constituting a target protein, an immunological measurement method using an antibody that specifically recognizes the target protein, and the like. The amino acid sequence information of the target protein can be obtained by searching a database of EBI (http://www.ebi.ac.uk/IPI/IPIhelp.html) based on the UniProtKB accession number.

As the immunological measurement method, immunohistochemical staining method, ELISA method, EIA method, RIA method, Western blotting method and the like can be preferably exemplified.

Mass spectrometry is a method in which a peptide sample is made into gaseous ions by using an ion source (ionization), and the peptide sample ionized by moving in a vacuum in the analysis section by using electromagnetic force or by a flight time difference is subjected to mass charge. It refers to a measurement method that uses a mass spectrometer that can be separated and detected according to the ratio, and methods for ionizing using an ion source include EI method, CI method, FD method, FAB method, MALDI method, ESI method. Etc. can be appropriately selected, and methods for separating ionized peptide samples in the analysis unit include magnetic field deflection type, quadrupole type, ion trap type, time of flight (TOF) type, Fourier transform A separation method such as an ion cyclotron resonance type can be appropriately selected. In addition, tandem mass spectrometry (MS/MS) or triple quadrupole mass spectrometry combining two or more mass spectrometry methods can be used. Further, when the sample is a sample containing a phosphorylated peptide, the sample can be concentrated using iron ion-immobilized affinity chromatography (Fe-IMAC) before introducing the sample into the mass spectrometer. In addition, by using liquid chromatography (LC) or HPLC, the peptides constituting the target protein can be separated and purified and used as a sample. Moreover, the detection unit and the data processing method can be appropriately selected. In addition, when detecting and quantifying a peptide constituting a target protein by mass spectrometry by mass spectrometry, a peptide that is composed of the same amino acid sequence as the peptide and labeled with a stable isotope of known concentration is used as an internal standard. can do. As such a stable isotope-labeled peptide, if one or more of the amino acids in the peptide constituting the target protein to be detected is a stable isotope-labeled peptide containing at least one of 15N, 13C, 18O, and 2H, the amino acid is The type, position, number, etc. can be appropriately selected, and such a stable isotope-labeled peptide is prepared by the F-moc method (Amblard., et al. Methods Mol Biol.298) using an amino acid labeled with a stable isotope. :3-24 (2005)) and the like, but can be chemically synthesized by iTRAQ (registered trademark) reagent, ICAT (registered trademark) reagent, ICPL (registered trademark) reagent, NBS (registered trademark) reagent, etc. It can also be prepared using the labeling reagent of.

According to the test method of the present invention including the step (1), it is possible to provide the amount and/or the concentration of the target protein, which is a detection index for granulomatous disease, thereby assisting the detection of granulomatous disease and the like. can do.

The test results of the test method of the present invention including the step (1) include the elucidation of the pathological condition of the granulomatous disease, the prognosis of the granulomatous disease, the stratification of the subject, the selection of the treatment method (individualized medicine, treatment response). Sex), intractability in granulomatous disease, evaluation of remodeling, differentiation of histological type and phenotype of granulomatous disease, and the like.

The biomarker of the present invention can also be used as a companion biomarker. Since September 2016, adalimumab (genetical recombination) (Humira (registered trademark)), a fully human anti-human TNF-α monoclonal antibody preparation, has been used for non-infectious uveitis including middle and posterior uveitis. Insurance was applied to the treatment. Therefore, the biomarker of the present invention can be utilized as, for example, a companion biomarker for adalimumab.

1-2. Process (2)
The inspection method of the present invention, as one aspect,
When the detected protein contains at least one protein (protein A′) selected from the protein group (A),
(2a) when the amount or concentration of the protein A′ detected in the step (1) is a cutoff value or more, a step of determining that the subject has a granulomatous disease,
It is preferable to include. According to the inspection method of the present invention including the step 2a, it becomes possible to determine a granulomatous disease.

The inspection method of the present invention, as one aspect,
When the detected protein contains at least one protein (protein B′) selected from the protein group (B),
(2b) when the amount or concentration of the protein B′ detected in the step (1) is less than or equal to a cutoff value, the step of determining that the subject has granulomatous disease,
It is preferable to include. According to the inspection method of the present invention including the step 2b, it becomes possible to determine a granulomatous disease.

The inspection method of the present invention, as one aspect,
When the detected protein contains at least one protein (protein A′) selected from protein group (A) and at least one protein (protein B′) selected from protein group (B), ,
(2c) The amount or concentration of protein A′ detected in the step (1) is a cutoff value or more, and/or the amount or concentration of protein B′ detected in the step (1) is a cutoff value. Step of determining that the subject is suffering from granulomatous disease, if
It is preferable to include. According to the inspection method of the present invention including the step 2c, it becomes possible to determine a granulomatous disease.

The cut-off value can be appropriately set by those skilled in the art from the viewpoints of sensitivity, specificity, positive predictive value, negative predictive value, etc., for example, it was collected from a subject not suffering from granulomatous disease. Based on the amount and/or the concentration of the target protein in the extracellular vesicles of the body fluid or the blood sample, the value can be set to a value determined in advance or a value determined in advance. The cutoff value is, for example, the amount and/or concentration of the protein of interest in extracellular vesicles or blood samples of body fluid collected from a subject not suffering from granulomatous disease (in the case of multiple subjects, the average Value, median value, etc.), for example, 0.7 to 1.5 times.

In a preferred embodiment of step (2), when the subject is a sample during or after the treatment of granulomatous disease, the cut-off value is, for example, the amount of the protein of interest in the past sample for the same sample and/or Alternatively, the therapeutic effect can be determined by setting the value based on the concentration.

2. If it is determined that the subject is suffering from a granulomatous disease by the test method of the present invention including the step of diagnosing granulomatous disease with higher accuracy (2), the test method of the present invention further comprises: By combining the steps of applying a diagnosis of a granulomatous disease by a doctor, the granulomatous disease can be diagnosed with higher accuracy. Further, since the inspection method of the present invention can detect a granulomatous disease more accurately, by combining the above-described steps with the inspection method of the present invention, the patient suffers from "granulomatous disease more efficiently and more accurately. Can be diagnosed.

3. When it is determined that the subject is suffering from a granulomatous disease by the test method of the present invention including the treatment step (2) for granulomatous disease, further to the test method of the present invention, or the above "2. As described in “Diagnosis of Granulomatous Disease with Higher Accuracy”, in the case of being diagnosed as suffering from granulomatous disease, a combination of the inspection method of the present invention and the step of applying a diagnosis by a doctor On the other hand, further, (3) treating the disease of the subject by performing a step of treating the disease determined or diagnosed as having the granulomatous disease, to the subject. It will be possible. Further, since the inspection method of the present invention can detect a granulomatous disease more accurately, a step for the inspection method of the present invention or a combination of the inspection method of the present invention and the step of applying a diagnosis by a doctor By combining the three, a subject suffering from a granulomatous disease can be treated more efficiently and more reliably.

The method for treating the granulomatous disease is not particularly limited, but typically includes drug treatment. As a medicine to be used for drug administration, in addition to systemic administration of steroids, eyedrops of steroids, inhalation of steroids, administration of antibacterial agents and the like are also performed as local therapy. When the disease state cannot be controlled even by systemic steroid administration, immunosuppressive drugs such as methotrexate and azathioprine can be used. .. The drug can be used alone, in combination of two, or in combination of three or more.

By using the test method of the present invention, it is also possible to select a suitable drug to be used for medication treatment from the drugs, for example, the drugs exemplified above.

Four. A test agent for a granulomatous disease, a test kit The present invention, in one embodiment thereof, comprises a detection agent for at least one protein selected from the group consisting of a protein group (A) and a protein group (B), The present invention relates to a test agent for tumorous diseases (herein sometimes referred to as "test agent of the present invention"). This will be described below.

The protein group (A), protein group (B), granulomatous disease, etc. are the same as defined in "1. Granulomatous disease test method" above.

The detection agent is not particularly limited as long as it can specifically detect the target protein. Examples of the detection agent include an antibody against the target protein.

-The detection agent may be modified as long as its function is not significantly impaired. Examples of the modification include addition and introduction of a labeling substance such as a fluorescent dye, a luminescent substance, a dye, an enzyme, a protein, a radioisotope, a chemiluminescent substance, colloidal gold and biotin.

The detection agent can be immobilized on any solid phase before use. Therefore, 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 a probe is immobilized, or another example, an ELISA plate on which an antibody is immobilized).

The solid phase used for immobilization is not particularly limited as long as it can immobilize an antibody and the like, and examples thereof include a glass plate, nylon membrane, microbeads, silicon chip, capillary or other substrate. it can. Immobilization of the detection agent on 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) recognizes" means that the target protein can be specifically detected by, for example, Western blotting or ELISA, but is not limited thereto and one skilled in the art can 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, and some of the above-mentioned antibodies having antigen-binding properties such as Fab fragments and fragments produced by Fab expression libraries. The antibody of the present invention also includes an antibody having an antigen-binding property with respect to a polypeptide having at least continuous 8 amino acids, preferably 15 amino acids, and more preferably 20 amino acids in the amino acid sequence of the target protein.

The method for producing these antibodies is already well known, and the antibody of the present invention can also be produced according to these conventional methods (Current protocols in Molecular Biology, Chapter 11.12 to 11.13 (2000)). Specifically, when the antibody of the present invention is a polyclonal antibody, a target protein expressed and purified in Escherichia coli or the like according to a conventional method is used, or an oligopeptide having a partial amino acid sequence of the target protein is synthesized according to a conventional method. Then, it is possible to immunize a non-human animal such as a rabbit and obtain it from the serum of 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 in E. coli or the like and purified according to a conventional method, or an oligopeptide having a partial amino acid sequence of the target protein, and the resulting spleen cells are It can be obtained from hybridoma cells prepared by cell fusion with myeloma cells (Current protocols in Molecular Biology edit. Ausubelet et.al. (1987) Publish. John Wiley and Sons. Section 11.4 to 11.11).

The target protein used as an immunogen for the production of antibodies is based on known gene sequence information, DNA cloning, construction of each plasmid, transfection into a host, culture of transformants, and recovery of the protein from the culture. It can be obtained by the operation of. These operations follow methods known to those skilled in the art, or methods described in the literature (Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)), etc. Can be done by

Specifically, a recombinant DNA (expression vector) capable of expressing a gene encoding a protein of interest in a desired host cell is prepared, introduced into a host cell for transformation, and the transformant is cultured. By recovering the target protein from the obtained culture, a protein as an immunogen for producing the antibody of the present invention can be obtained. The partial peptide of the target protein can also be produced by a general chemical synthesis method (peptide synthesis) according to known gene sequence information.

The antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of the target protein. The oligo(poly)peptide used for producing such an antibody does not need to have a functional biological activity, but it is desirable that it has immunogenic properties similar to those of the target protein. An oligo(poly)peptide that preferably has this immunogenic property and consists of at least 8 consecutive amino acids, preferably 15 amino acids, and more preferably 20 amino acids in the amino acid sequence of the target protein can be exemplified.

The production of antibodies against such oligo(poly)peptides can also be carried out by enhancing the immunological reaction with 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 treatments such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Active substances, human adjuvants such as BCG (bacillus Calmette-Guerin) and Corynebacterium parvum are included.

The test agent of the present invention may be in the form of a composition. The composition may contain other components as necessary. Other components include, for example, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, and fragrances. , 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 above-mentioned detection agent or the above-mentioned composition containing the same, an agent that can be used for detection of a target protein in extracellular vesicles of a body fluid of a subject or a blood sample. Specific examples of such substances include various reagents (eg, secondary antibody, buffer, etc.), instruments (eg, instruments for purification, separation, concentration of extracellular vesicles (eg, columns)) and the like.

Five. Preventive or therapeutic agent for granulomatous disease The present invention is, in one aspect thereof, an inhibitor of at least one protein selected from the protein group (A), and at least one protein selected from the protein group (B). The present invention relates to a prophylactic or therapeutic agent for granulomatous disease, which contains at least one drug selected from the group consisting of protein enhancers (herein, sometimes referred to as "the drug of the present invention"). This will be described below.

The protein group (A), protein group (B), granulomatous disease, etc. are the same as defined in "1. Granulomatous disease test method" above.

Examples of the inhibitor include an antibody against the target protein. As the antibody, the same antibodies as those described above in “4. Test agents and test kits for granulomatous disease” can be used.

Another example of the inhibitor is an expression inhibitor of the 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-gene editing agent of target protein by CRISPR/Cas system.

The expression suppression means the expression amount of the target protein, its mRNA, etc., for example, 1/2, 1/3, 1/5, 1/10, 1/20, 1/30, 1/50, 1/100. , 1/200, 1/300, 1/500, 1/1000, 1/10000 or less, which means that the expression level of these is set to 0.

Examples of the enhancer include an enhancer of expression of the target protein.

The target protein expression enhancer is not particularly limited as long as it can enhance the expression level of the target protein, its mRNA, its precursor, etc., and examples thereof include an expression vector of the target protein.

The expression enhancement means that the expression level of the target protein, its mRNA or the like is increased to, for example, 2, 3, 5, 10, 20, 30, 50, 100, 200, 300, 500, 1000, 10,000 times. means.

The gene siRNA of the target protein is not particularly limited as long as it is a double-stranded RNA molecule that specifically suppresses the expression of the gene encoding the target protein. In one embodiment, the siRNA preferably has a length of, for example, 18 bases or more, 19 bases or more, 20 bases or more, or 21 bases or more. Moreover, 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. It is assumed that the upper limit value and the lower limit value of the siRNA length described here are arbitrarily combined. For example, the lower limit is 18 bases, the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases length; the lower limit is 19 bases, the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases Certain length; lower limit is 20 bases, upper limit is 25 bases, 24 bases, 23 bases, or 22 bases Length; lower limit is 21 bases, upper limit is 25 bases, 24 bases, 23 bases, or 22 Combinations of lengths that are bases are envisioned.

SiRNA may be shRNA (small hairpin RNA). shRNA can be designed such that a part thereof forms a stem-loop structure. For example, in shRNA, if the sequence of a certain region is sequence a and the complementary strand to sequence a is sequence b, then these sequences are present in one RNA strand in the order of sequence a, spacer, sequence b. And can be designed to be 45-60 bases in length. Sequence a is a sequence of a partial region of the base sequence encoding the target protein to be targeted, and the target region is not particularly limited, and any region can be used as a candidate. The length of the sequence a is 19 to 25 bases, preferably 19 to 21 bases.

The gene-specific siRNA of the target protein may have an additional base at the 5'or 3'end. The length of the additional base is usually about 2 to 4 bases. The additional base may be DNA or RNA, but the use of DNA may improve the stability of nucleic acid in some cases. Such additional base sequences include, for example, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3'. Examples include, but are not limited to, sequences such as', uuuuu-3'.

SiRNA may have a protruding sequence (overhang) at the 3'end, and specific examples thereof include dTdT (dT represents deoxythymidine). In addition, it may be a blunt end with no terminal addition. The siRNA may have a different number of bases in the sense strand and the antisense strand, and for example, the “asymmetrical interfering RNA (where the antisense strand has a protruding sequence (overhang) at the 3′ end and 5′ end) ( aiRNA)". A typical aiRNA has an antisense strand of 21 bases, a sense strand of 15 bases, and an overhang structure of 3 bases at each end of the antisense strand.

The position of the target sequence of the gene-specific siRNA of the protein of interest is not particularly limited, but in one embodiment, it is targeted from a region other than the 5'-UTR and the start codon to about 50 bases and the 3'-UTR. It is desirable to choose the sequence. Regarding the candidate group of the selected target sequence, BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ It is preferable to check the specificity of the selected target sequence by using a homology search software such as ). Regarding the target sequence whose specificity was confirmed, a sense strand having a 3'terminal overhang of TT or UU at 19-21 bases after AA (or NA), a sequence complementary to the 19-21 bases and TT or Double-stranded RNA consisting of an antisense strand having a 3'-terminal overhang of UU may be designed as siRNA. In addition, for shRNA that is a precursor of siRNA, any linker sequence capable of forming a loop structure (for example, about 5-25 bases) is appropriately selected, and the sense strand and the antisense strand are linked via the linker sequence. It can be designed by connecting.

The sequences of siRNA and/or shRNA can be searched using search software provided for free 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 (registered trademark) Expression Vector (http://www.ambion.com/ (jp/techlib/misc/psilencer_converter.html) GeneSeer (http://codex.cshl.edu/scripts/newsearchhairpin.cgi) provided by RNAi Codex.

siRNA is a DNA/RNA automatic synthesizer that synthesizes the sense and antisense strands of the target sequence on mRNA, and denatures 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. In addition, it can also be prepared by synthesizing shRNA as a precursor of siRNA and cleaving it with an RNA cleaving protein dicer.

The gene-specific miRNA of the target protein is optional as long as it inhibits the translation of the gene encoding the target protein. For example, miRNAs may pair with the 3'untranslated region (UTR) of the target and inhibit its translation rather than cleaving the target mRNA like siRNA. The miRNA may be any of pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA), and mature miRNA. The length of miRNA is not particularly limited, the length of pri-miRNA is usually several hundred to several thousand bases, the length of pre-miRNA is usually 50 to 80 bases, and the length of mature miRNA is usually 18 ~30 bases. 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 miRNA of the target protein may be synthesized by a known method, or may be purchased from a company that provides synthetic RNA.

The gene-specific antisense nucleic acid of the target protein is a nucleic acid containing a base sequence complementary or substantially complementary to the base sequence of mRNA of the gene encoding the target protein, or a part thereof, and is specific to the mRNA. It is a nucleic acid having a function of suppressing target protein synthesis by forming a stable and stable double chain and binding. The antisense nucleic acid may be DNA, RNA, or a DNA/RNA chimera. When the antisense nucleic acid is DNA, the RNA:DNA hybrid formed by the target RNA and the antisense DNA is recognized by the endogenous ribonuclease H (RNaseH) and causes selective degradation of the target RNA. Therefore, in the case of antisense DNA that directs 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 the target protein. The intron sequence can be determined by comparing the genomic sequence with the cDNA base sequence of the gene of the target protein using a homology search program such as BLAST or FASTA.

The length of the target region of the gene-specific antisense nucleic acid of the target protein is not limited as long as the antisense nucleic acid hybridizes with the result that the translation into the target protein is inhibited. The gene-specific antisense nucleic acid of the target protein may be the entire sequence or a partial sequence of mRNA encoding the target protein. Considering the problems of easiness of synthesis, antigenicity, intracellular transfer, etc., an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferable, but not limited thereto. More specifically, 5'end hairpin loop of the gene of the target protein, 5'end untranslated region, translation initiation codon, protein coding region, ORF translation stop codon, 3'end untranslated region, 3'end palindromic region Alternatively, a 3′-end hairpin loop or the like may 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 of the gene of the target protein or the initial transcription product to inhibit translation into a protein but also binds to these genes that are double-stranded DNA. It may be one that can form a triplex and inhibit transcription into RNA (antigene).

The nucleotide molecules constituting the gene-specific siRNA of the target protein, the gene-specific miRNA of the target protein, and the gene-specific antisense nucleic acid of the target protein described above have stability (chemical and/or counterenzyme) and specific activity ( Various chemical modifications may be included in order to improve (affinity with RNA). For example, in order to prevent degradation by a hydrolase such as nuclease, a phosphate residue (phosphate) of each nucleotide constituting an antisense nucleic acid is converted into, for example, phosphorothioate (PS), methylphosphonate, or phosphorodithioate. It may be replaced with a chemically modified phosphate residue such as phosphorodithioate. In addition, the hydroxyl group at the 2'position of the sugar (ribose) of each nucleotide is replaced with -OR (R=CH ₃ (2'-O-Me), CH ₂ CH ₂ OCH ₃ (2'-O-MOE), CH ₂ CH ₂ NHC(NH)NH ₂ , CH ₂ CONHCH ₃ , or CH ₂ CH ₂ CN etc.). Further, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. May be given. In addition, a part of the nucleotide molecule constituting siRNA or miRNA may be replaced with natural DNA.

Gene-specific siRNA of the target protein, gene-specific miRNA of the target protein, and gene-specific antisense nucleic acid of the target protein are targets of mRNA or early transcription products based on the cDNA sequence or genomic DNA sequence of the gene of the target protein. It can be prepared by determining the sequence and using a commercially available DNA/RNA automatic synthesizer to synthesize a sequence complementary thereto. In addition, any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a known method.

For 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, etc., gene-specific siRNA of target protein, gene of target protein The specific miRNA, the gene-specific antisense nucleic acid of the target protein, the mRNA of the target protein, etc. are not particularly limited as long as they are incorporated in an expressible state. Typically, the expression vector comprises a promoter sequence and a gene-specific siRNA of the protein of interest, a gene-specific miRNA of the protein of interest, a gene-specific antisense nucleic acid of the protein of interest, or a coding sequence of the protein of interest (if necessary). And a polynucleotide containing a transcription termination signal sequence), and optionally other sequences. The promoter is not particularly limited, and examples thereof include RNAV polymerase II (polII) promoters such as CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, and CAG promoter; mouse and human U6-snRNA promoters, Examples include RNA polymerase III (pol III) type promoters such as human H1-RNase P RNA promoter and human valine-tRNA promoter. The other sequence is not particularly limited, and various known sequences that can be contained in the expression vector can be adopted. Examples of such sequences include an origin of replication, a drug resistance gene, and the like. Moreover, the types of drug resistance genes and types of vectors can be exemplified as described above.

Another example of the gene expression inhibitor of the target protein is a gene-specific ribozyme of the target protein. The term "ribozyme" means, in a narrow sense, RNA having an enzymatic activity of cleaving nucleic acid, but in the present application, it also includes DNA as long as it has sequence-specific nucleic acid cleaving activity. The most versatile ribozyme nucleic acid is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammerhead type and hairpin type are known. The hammerhead type exerts enzyme activity with about 40 bases, and several bases at both ends adjacent to the part having the hammerhead structure (about 10 bases in total) are made into sequences complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme nucleic acid has an 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, by using a hybrid ribozyme in which an RNA motif derived from a viral nucleic acid that can specifically bind to RNA helicase is used, the target sequence is single-stranded. [Proc.Natl. Acad. Sci. USA, 98(10): 5572-5577 (2001)]. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the translocation of the transcript to the cytoplasm, it should be a hybrid ribozyme further linked with a tRNA-modified sequence. You can also do it [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 may be a form usually used in each application depending on the use of the drug of the present invention.

Examples of the form include a tablet (a disintegrating tablet in the oral cavity, a chewable tablet, an effervescent tablet, a lozenge, a jelly-like drop agent, etc.) when the application is a medicine, a health promoting agent, a nutritional supplement (supplement, etc.) ), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jelly preparations suitable for oral ingestion Formulations suitable for parenteral ingestion (oral formulation), nasal drops, inhalants, rectal suppositories, intercalates, enemas, jellies, injections, patches, lotions, creams, etc. Oral formulation form).

As the form, when the use is a food composition, liquid, gel-like or solid food, for example, juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, baby milk powder , Cake mix, powdered or liquid dairy products, bread, cookies and the like.

As a form, when the application is an oral composition, for example, liquid (solution, emulsion, suspension, etc.), semisolid (gel, cream, paste, etc.), solid (tablet, particulate, capsule, Film agents, kneaded products, molten solids, waxy solids, elastic solids, etc.), more specifically, dentifrice (toothpaste, liquid toothpaste, liquid toothpaste, powdered toothpaste, etc.), mouthwash, Examples thereof include coating agents, patches, refreshing agents in the mouth, and foods (for example, chewing gum, tablet confectionery, candy, gummies, films, troches, etc.).

The drug of the present invention may further contain other components, if necessary. Other components are not particularly limited as long as they are components that can be blended into, for example, a medicine, a food composition, a composition for oral cavity, a health promoting agent, a nutritional supplement (supplement, etc.) and the like. , Carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents and the like.

The total content of the inhibitor and enhancer of the target protein of the agent of the present invention depends on the type of the inhibitor and enhancer, the use, the mode of use, the application target, the state of the application target, and the like. Although not included, it can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

The application (eg, administration, ingestion, inoculation, etc.) amount of the composition of the present invention is not particularly limited as long as it is an effective amount that exerts a medicinal effect. Usually, the weight of the active ingredient is generally 0.1 to 1000 per day. mg/kg body weight. It is preferable to administer the above-mentioned dose once a day or in 2 to 3 divided doses, and the dose may be appropriately adjusted depending on the age, disease state, and symptom.

6. Method for screening active ingredient of prophylactic or therapeutic agent for granulomatous disease The present invention, in one aspect thereof, comprises a group of proteins in extracellular vesicles or blood samples of body fluid collected from an animal treated with a test substance ( Screening for an active ingredient (or a candidate substance thereof) of a prophylactic or therapeutic agent for granulomatous disease, which uses as an index the amount or concentration of at least one protein selected from the group consisting of A) and the protein group (B). The present invention relates to a method (herein sometimes referred to as “the method for screening an active ingredient of the present invention”). This will be described below.

For body fluids, extracellular vesicles, blood samples, protein group (A), protein group (B), granulomatous disease, measurement of the amount or concentration of the target protein, etc., see “1. Granuloma Disease Testing Method” above. Is the same as the definition in ".

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

As the test substance, a naturally occurring compound or an artificially made compound can be widely used. Further, not only the purified compound, but also a composition in which various compounds are mixed and an extract of animals and plants can be used. The compound is not limited to low molecular weight compounds, and includes high molecular weight compounds such as proteins, nucleic acids, and polysaccharides.

More specifically, the method for screening the active ingredient of the present invention, the value of the index relating to the protein group (A) is the extracellular vesicle or blood sample of the body fluid collected from the animal not treated with the test substance. When lower than the amount or concentration of the corresponding protein, the step of selecting the test substance as an active ingredient of a prophylactic or therapeutic agent for granulomatous disease, and the value of the index for protein group (B) is the test substance As an active ingredient of a prophylactic or therapeutic agent for granulomatous disease when the amount or concentration of the corresponding protein in extracellular vesicles or blood sample of body fluid collected from an animal not treated with At least one step selected from the group consisting of selecting steps is included.

Corresponding protein means the same protein as the target protein used as an index.

“Low” means that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value, for example.

“High” means that the index value is, for example, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times the control value.

7. In an aspect of the present invention, there is provided a method for evaluating a granulomatous disease inducing or malignant malignancy , a protein group (A) in an extracellular vesicle or a blood sample of a body fluid collected from an animal treated with a test substance. , And a method for assessing the inducibility or exacerbation of granulomatous disease using the amount or concentration of at least one protein selected from the group consisting of protein group (B) as an index (in the present specification, "the present invention "Toxicity evaluation method". This will be described below.

For body fluids, extracellular vesicles, protein group (A), protein group (B), granulomatous disease, measurement of the amount or concentration of target protein, animal species of animals, test substances, etc., see “1. The definition is the same as the definition in “Test method for tumorous disease” and “6. Method for screening active ingredient of preventive or therapeutic agent for granulomatous disease”.

More specifically, the toxicity evaluation method of the present invention, the value of the index for the protein group (A) corresponds to the extracellular vesicles or blood samples of body fluid collected from animals not treated with the test substance. When the amount or concentration of the protein is higher than the step of determining that the test substance has a granulomatous disease-inducing or malignant state, and the value of the index for the protein group (B) is the test substance. The test substance is determined to be inducing or exacerbating a granulomatous disease when it is lower than the amount or concentration of the corresponding protein in the extracellular vesicles or blood sample of the body fluid collected from the untreated animal. At least one step selected from the group consisting of:

Corresponding protein means the same protein as the target protein used as an index.

“High” means that the index value is, for example, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times the control value.

“Low” means that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value, for example.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

Test example 1. Preparation of extracellular vesicle fraction 1
Extracellular vesicle fractions were prepared from the serum of each human subject (7 persons) diagnosed as having sarcoidosis and the serum of each healthy human subject (5 persons). The extracellular vesicle fraction was prepared using the extracellular vesicle purification column (EV-Second, manufactured by GL Sciences Inc.) with the same volume of serum for each sample.

For sarcoidosis subjects, extracellular vesicle fractions were prepared in the same manner as above, after steroid treatment (predonin 15-30 mg/body was administered as the initial dose).

Next, the number of extracellular vesicle particles and the particle size of the obtained extracellular vesicle fraction were measured. Specifically, the measurement was performed using a nanosite (Nippon Quantum Design Co., Ltd., Nanoparticle Tracking Analysis (NTA) Version 2.3 Build 0025). This is an analysis based on the difference in Brownian motion velocity for each particle size. The movement of each scattered light reflected on the screen is tracked (tracked), and the movement velocity (diffusion coefficient) of each scattered light The particle diameter (fluid dynamic diameter) in can be calculated. The results are shown in FIGS. 1 and 2.

Furthermore, extracellular vesicles were observed by immunoelectron microscopy. Specifically, it carried out as follows. The fixed extracellular vesicle solution was placed on the grid in an amount of 5 to 8 μL and allowed to stand for 15 minutes to fix the extracellular vesicles to the form bar of the grid. After washing 3 times with PBS, blocking reaction (1% BSA/PBS, 10 minutes) is performed, followed by primary antibody reaction (Invitrogen AHS0902 Mouse (monoclonal) Anti-Human Leukemia and Platelet Associated Antigen CD9 Clone: MM2/57, Diluted 100 times, 2 hours and a half, at room temperature). After washing 6 times with PBS, a secondary antibody reaction (CRLEMGMHL10 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, it was treated with 1% GA/PBS for 15 minutes and further washed 3 times with PBS. It was washed with water, treated with 0.5% UA (uranyl acetate), dried and observed with an electron microscope. The obtained results are shown in FIG.

Furthermore, the extracellular vesicle fraction was Western blotted using an antibody against the exosome marker (anti-CD9 antibody (Invitrogen, clone=MM2/57)). The results are shown in Fig. 4.

As shown in FIGS. 1 to 4, exosomes were present in the obtained fractions, and it was found that there is almost no difference in the number of particles and the particle size between the healthy subject and the sarcoidosis subject. It was

Test example 2. Proteomics analysis (non-label, LC-MS/MS)
The protein in the extracellular vesicle fraction obtained in Test Example 1 was quantified by LC-MS/MS analysis (non-label method). Specifically, it carried out as follows.

(Sample preparation)
The extracellular vesicle fraction was dried under reduced pressure, dissolved in 8 M urea, reduced with 5 mM TCEP at 37° C. for 30 minutes, and alkylated with 25 mM iodoacetamide at room temperature for 45 minutes. Then, the sample was diluted 8 times with 50 mM ammonium bicarbonate, put into a 96-well filter plate, and shaken with 5 μL of immobilized trypsin beads (manufactured by Thermo Fisher Scientific) at 37° C. for 6 hours for digestion. The trypsin digest was desalted using Oasis HLB 96-well μElution Plate (Waters Corporation, USA) and subjected to LC-MS/MS analysis.

(LC/MS/MS analysis)
The dried peptide sample was resuspended in a 2% acetonitrile solution containing 0.1% trifluoroacetic acid and an Orbitrap Fusion Lumos mass spectrometer (Thermo) equipped with an Ultimate 3000 RSLC nano-flow HPLC system (DIONEX, USA). It was analyzed using Fisher Scientific). 75 μm × 150 mm C18 tip-column (manufactured by Nikyo Technos) using solvent A (0.1% formic acid) and solvent B (0.1% formic acid in acetonitrile) at a flow rate of 250 nL/min, 95 Samples were separated on a multi-step linear gradient of 2-30% Solvent B in 30 minutes and then 30-95% Solvent B in 10 minutes.

The eluted peptides were ionized at a spray voltage of 2000 V and MS data were acquired by the data dependent fragment method. The measurement scan (survey scan) was performed with m/z 350 to 1500, mass resolution of 50,000, and AGC target value of 1.0×10 ⁶ ion count. One MS scan (Full scan) and MS/MS scan for precursor ions selected from the mass spectrum are analyzed in a cycle of up to 2 seconds. MS/MS is a linear ion trap with an AGC target value of 5000 ion counts. The CID cleavage mode was used.

Using the Proteome Discoverer 2.2 software (ThermoFischer Scientific), Mascot and SEQUEST database searches were performed to identify and quantify proteins. Mass spectrometry data was searched against the UniProt mouse protein database. The search parameters are: EnzymeName=Trypsin, Precursor MassTolerance = 10ppm, Fragment Mass Tolerance = 0.8 Da, Dynamic Modification = Oxidation (Met), StaticModification = Carbamidomethyl (Cys). Less than 1% of FDR (false discovery rate) in Decoy Database Search was used as a significant identification criterion for peptides. Peptide and protein quantification and standardization were performed using standard functions of Proteome Discoverer 2.2 software.

(Statistical analysis and bioinformatics analysis)
Sarcoidosis test subjects and healthy test subjects were compared, and by t-test, 366 proteins having expression levels significantly (P<0.05) apart between the two groups were extracted. Furthermore, by bioinformatics analysis, 50 proteins considered to be highly associated with sarcoidosis were selected as effective sarcoidosis biomarkers.

In addition, among the 50 selected biomarkers, 14 proteins with expression levels significantly (t-test, P<0.05) apart between the two groups of the sarcoidosis subject and the post-sarcoidosis treatment subject were selected. Extracted.

(result)
Among the selected sarcoidosis biomarkers, the proteins whose expression level in the sarcoidosis subject was higher than that in the healthy subject are shown in Table 1. In Table 1, "fold change" is a value obtained by dividing the average expression level of sarcoidosis subjects by the average expression level of healthy subjects (average expression level of sarcoidosis subjects/average expression amount of healthy subjects). Indicates. ∞ indicates that it was not detected in the healthy subject but was detected in the sarcoidosis subject.

Among the sarcoidosis biomarkers in Table 1, the proteins reduced by the treatment (proteins whose expression level in the subject after sarcoidosis treatment was lower than that in the sarcoidosis subject are shown in Table 2.

Among the selected sarcoidosis biomarkers, those in which the expression level of the sarcoidosis subject was lower than that of the healthy subject are shown in Table 3. In Table 3, "fold change" is the value obtained by dividing the average expression level of sarcoidosis subjects by the average expression level of healthy subjects (average expression level of sarcoidosis subjects/average expression amount of healthy subjects). Indicates. 0 indicates that it was not detected in the sarcoidosis subject, but was detected in the healthy subject.

Among the sarcoidosis biomarkers in Table 3, proteins increased by treatment (proteins whose expression level in subjects after sarcoidosis treatment was higher than those in sarcoidosis subjects are shown in Table 4.

Test example 3. Preparation of extracellular vesicle fraction 2
As the subjects, human subjects (46 subjects) diagnosed as having sarcoidosis and healthy human subjects (10 subjects) were adopted. The characteristics of the sample are shown in Table 5.

Serum was collected from the subject and an extracellular vesicle fraction was prepared in the same manner as in Test Example 1.

Test example 4. Proteomics analysis 2 (SRM/MRM)
The protein selected in Test Example 2 was subjected to SRM proteomics analysis according to a previously reported document (Molecular & Cellular Proteomics 13: 10.1074/mcp.M113.037093, 1471-1484, 2014.). The outline is as follows. Based on the amino acid sequence information of the protein, one or two peptides (trypsin digestion fragments) that are specifically detected by the SRM method are selected, and a stable isotope-labeled peptide (SI peptide having the same amino acid sequence as the peptide for each is selected. ) Was used as the internal standard peptide. The protein of the extracellular vesicle fraction obtained in Test Example 3 was trypsin-digested, mixed with an internal standard peptide, and subjected to relative quantitative analysis by the SRM method using a mass spectrometer (TSQ Vantage, Thermo Fisher Scientific). did.

Based on the obtained quantitative data, two proteins highly expressed in sarcoidosis (Monocyte differentiation antigen CD14 (CD14) and Lipopolysaccharide-binding protein (LBP)) were selected as more reliable biomarkers. The quantification results of these two biomarkers are shown in FIGS. 5 and 6.

Analysis of CD14 expression in extracellular vesicles collected from patients with tuberculosis, which is a granulomatous disease similar to sarcoidosis, revealed that CD14 expression levels were updated in the extracellular vesicles of patients with tuberculosis. I understood.

Test example 5. Detection of Sarcoidosis Biomarker in Extracellular Vesicle Fraction by Western Blot Regarding the protein of the extracellular vesicle fraction obtained in Test Example 3, the sarcoidosis biomarkers (Monocyte differentiation antigen CD14 (CD14) and Lipopolysaccharide-binding protein ( Western blot using an antibody against LBP)). The primary antibodies used were anti-CD14 antibody (CST, product code: #56082) and anti-LBP antibody (abcam, product code: ab231182). Furthermore, as a control, an antibody against an exosome marker (anti-CD9 antibody (manufactured by Invitrogen, product code: AHS0902)) was also used. The results are shown in Fig. 7.

As shown in FIG. 7, it was found that the sarcoidosis biomarker can be detected by Western blot and the difference in the expression level between the healthy subject and the sarcoidosis subject can be confirmed.

Test example 6. Detection of Sarcoidosis Biomarker in Extracellular Vesicle Fraction by Immunoelectron Microscopy Regarding the extracellular vesicle fraction obtained in Test Example 3, sarcoidosis biomarkers (Monocyte differentiation antigen CD14 (CD14), and Lipopolysaccharide-binding protein ( Immunoelectron microscopy was performed using an antibody against LBP)). The primary antibody used is the same as in Test Example 5. Specifically, the same procedure as in Test Example 1 was performed. A representative example of the obtained results is shown in FIG.

As shown in Fig. 8, it was found that CD14 and LBP exist on the membrane of extracellular vesicles.

Test example 7. Detection of sarcoidosis biomarker in lung tissue by immunostaining Lung tissue collected from monkey subjects with sarcoidosis was tested using antibodies against sarcoidosis biomarkers (Monocyte differentiation antigen CD14 (CD14) and Lipopolysaccharide-binding protein (LBP)). Immunostained. The primary antibody used is the same as in Test Example 5. The results are shown in Fig. 9.

As shown in FIG. 9, it was found that the investigated sarcoidosis biomarkers had enhanced expression in the lung tissue of sarcoidosis subjects. From this, it was suggested that this biomarker is closely related to the pathological condition.

Test Example 8. Evaluation of the diagnostic ability of sarcoidosis biomarkers 1
Based on the quantification results of Test Example 4, an ROC curve was prepared when Monocyte differentiation antigen CD14 (CD14) and Lipopolysaccharide-binding protein (LBP) were used alone as sarcoidosis biomarkers. Specifically, using the statistical software JMP, an ROC curve was created with the vertical axis representing the sensitivity (positive rate) and the horizontal axis representing the value obtained by subtracting specificity from 1 (1-specificity) (false positive rate). .. The results are shown in Fig. 10. Furthermore, the AUC when both CD14 and LBP were used was calculated by logistic regression analysis and found to be 0.89.

AUC using existing serum biomarkers (ACE) for sarcoidosis has been reported to be 0.69. On the other hand, as shown in FIG. 10, the AUC when using CD14 and LBP alone as biomarkers was 0.8 or more, and by combining these, an AUC close to 0.9 was obtained. From this, it was found that these biomarkers have a better diagnostic ability than existing biomarkers.

Test example 9. Evaluation of diagnostic ability of sarcoidosis biomarker 2
Based on the quantification result of Test Example 4 and the quantification result of the existing sarcoidosis biomarker (ACE and sIL-2) in the serum of the sample adopted in Test Example 3, when ACE and CD14 were used in combination as a sarcoidosis biomarker The ROC curve of ACE, sIL-2, CD14 and LBP used in combination as a sarcoidosis biomarker were prepared in the same manner as in Test Example 8. The results are shown in Fig. 11.

From the results of Test Example 8 and FIG. 11, it was found that the diagnostic ability can be further improved by combining the existing biomarkers.

Claims (15)

1. A method of testing for granulomatous disease, the method comprising:
   (1) Protein group (AX) in extracellular vesicles or blood sample of body fluid collected from a subject:
   (AX) Lipopolysaccharide-binding protein and a test method comprising a step of detecting at least one protein selected from the group consisting of a protein group consisting of Monocyte differentiation antigen CD14.
2. A method of testing for granulomatous disease, the method comprising:
   (1) Protein group (A) and protein group (B) in extracellular vesicles or blood sample of body fluid collected from a subject:
   (A) Chitinase-3-like protein 1, Protein-tyrosine sulfotransferase 2, Myoferlin, Pancreas transcription factor 1 subunit alpha, RuvB-like 2, Protein FAM110D, Cystatin-A, Creatine kinase M-type, Rho GTPase-activating protein 4 , Glutathione hydrolase 5 proenzyme, Putative neutrophil cytosol factor 1C, Neutrophil cytosol factor 1, Putative neutrophil cytosol factor 1B, Sn1-specific diacylglycerol lipase beta, Protein FAM26E, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 2, Anoctamin. CCR4-NOT transcription complex subunit 1, Interferon-induced helicase C domain-containing protein 1, T-cell surface glycoprotein CD3 zeta chain, Caspase-3, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2, Interferon regulatory factor 2-binding Protein 1, Endothelial lipase, Lipopolysaccharide-binding protein, Transthyretin, and Monocyte differentiation antigen CD14, and (B) Lysosome-associated membrane glycoprotein 2, Protein Jade-3, Tetraspanin-4, Oxysterol-binding protein-related protein 8, P2Y purinoceptor 12, Dematin, Tr ansforming acidic coiled-coil-containing protein 2, Ubiquitin carboxyl-terminal hydrolase MINDY-1, Septin-4, Ephrin-B1, Septin-5, Plexin-B3, Tetraspanin-32, H(+)/Cl(-) exchange transporter 5, Neutrophil collagenase, Septin-8, Septin-6, Metabotropic glutamate receptor 6, Metabotropic glutamate receptor 7, Metabotropic glutamate receptor 4, Metabotropic glutamate receptor 8, Gamma-enolase, and CD63 antigen An assay method comprising the step of detecting at least one protein that is
3. When the detected protein contains at least one protein (protein A′) selected from the protein group (A),
   (2a) when the amount or concentration of the protein A′ detected in the step (1) is a cutoff value or more, a step of determining that the subject has a granulomatous disease,
   The inspection method according to claim 2, further comprising:
4. When the detected protein contains at least one protein (protein B′) selected from the protein group (B),
   (2b) when the amount or concentration of the protein B′ detected in the step (1) is less than or equal to a cutoff value, the step of determining that the subject has granulomatous disease,
   The inspection method according to claim 2, further comprising:
5. When the detected protein contains at least one protein selected from protein group (A) (protein A′) and at least one protein selected from protein group (B) (protein B′), ,
   (2c) The amount or concentration of protein A′ detected in the step (1) is a cutoff value or more, and/or the amount or concentration of protein B′ detected in the step (1) is a cutoff value. Step of determining that the subject is suffering from granulomatous disease, if
   The inspection method according to any one of claims 2 to 4, further comprising:
6. The protein group (A) is
   (A1) Chitinase-3-like protein 1, Protein-tyrosine sulfotransferase 2, Myoferlin, Pancreas transcription factor 1 subunit alpha, RuvB-like 2, Protein FAM110D, Cystatin-A, Creatine kinase M-type, Rho GTPase-activating protein 4 , Glutathione hydrolase 5 proenzyme, Putative neutrophil cytosol factor 1C, Neutrophil cytosol factor 1, Putative neutrophil cytosol factor 1B, Sn1-specific diacylglycerol lipase beta, Protein FAM26E, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase-5, and Anoctamin. A group of,
   (A2) CCR4-NOT transcription complex subunit 1, Interferon-induced helicase C domain-containing protein 1, and T-cell surface glycoprotein CD3 zeta chain,
   (A3) Myoferlin, Glutathione hydrolase 5 proenzyme, Sn1-specific diacylglycerol lipase beta, Interferon-induced helicase C domain-containing protein 1, Caspase-3, and a group consisting of Lipopolysaccharide-binding protein, and (A4) Myoferlin, Protein FAM110D, CCR4-NOT transcription complex subunit 1, Keratin, type I cytoskeletal 39, Caspase-3, Endothelial lipase, Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase, and lipopolysaccharide-binding protein The test method according to any one of claims 2 to 5, which is at least one protein group selected from the following.
7. The protein group (B) is
   (B1) Lysosome-associated membrane glycoprotein 2, and Protein Jade-3 group,
   (B2) Tetraspanin-4, Oxysterol-binding protein-related protein 8, P2Y purinoceptor 12, and a group consisting of Dematin,
   (B3) Protein Jade-3, P2Y purinoceptor 12, Dematin, Ubiquitin carboxyl-terminal hydrolase MINDY-1, Septin-4, Septin-5, Plexin-B3, Septin-8, Septin-6, Metabotropic glutamate receptor 6, Metabotropic glutamate group consisting of receptor 7, Metabotropic glutamate receptor 4, and Metabotropic glutamate receptor 8, and (B4) H(+)/Cl(-) exchange transporter 3, Metabotropic glutamate receptor 4, Metabotropic glutamate receptor 6, Metabotropic glutamate receptor 7, Metabotropic The test method according to any one of claims 2 to 6, which is at least one protein group selected from the group consisting of glutamate receptor 8 and mitogen-activated protein kinase 1.
8. The test method according to any one of claims 1 to 7, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum.
9. 9. The inspection method according to claim 1, wherein the subject is a human.
10. Protein group (A) and protein group (B):
    (A) Chitinase-3-like protein 1, Protein-tyrosine sulfotransferase 2, Myoferlin, Pancreas transcription factor 1 subunit alpha, RuvB-like 2, Protein FAM110D, Cystatin-A, Creatine kinase M-type, Rho GTPase-activating protein 4 , Glutathione hydrolase 5 proenzyme, Putative neutrophil cytosol factor 1C, Neutrophil cytosol factor 1, Putative neutrophil cytosol factor 1B, Sn1-specific diacylglycerol lipase beta, Protein FAM26E, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 2, Anoctamin CCR4-NOT transcription complex subunit 1, Interferon-induced helicase C domain-containing protein 1, T-cell surface glycoprotein CD3 zeta chain, Caspase-3, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2, Interferon regulatory factor 2-binding Protein 1, Endothelial lipase, Lipopolysaccharide-binding protein, Transthyretin, and Monocyte differentiation antigen CD14, and (B) Lysosome-associated membrane glycoprotein 2, Protein Jade-3, Tetraspanin-4, Oxysterol-binding protein-related protein 8, P2Y purinoceptor 12, Dematin, Tr ansforming acidic coiled-coil-containing protein 2, Ubiquitin carboxyl-terminal hydrolase MINDY-1, Septin-4, Ephrin-B1, Septin-5, Plexin-B3, Tetraspanin-32, H(+)/Cl(-) exchange transporter 5, Neutrophil collagenase, Septin-8, Septin-6, Metabotropic glutamate receptor 6, Metabotropic glutamate receptor 7, Metabotropic glutamate receptor 4, Metabotropic glutamate receptor 8, Gamma-enolase, and CD63 antigen A diagnostic agent for granulomatous disease, which comprises a detection agent for at least one protein according to claim 1.
11. Protein group (A):
    (A) Chitinase-3-like protein 1, Protein-tyrosine sulfotransferase 2, Myoferlin, Pancreas transcription factor 1 subunit alpha, RuvB-like 2, Protein FAM110D, Cystatin-A, Creatine kinase M-type, Rho GTPase-activating protein 4 , Glutathione hydrolase 5 proenzyme, Putative neutrophil cytosol factor 1C, Neutrophil cytosol factor 1, Putative neutrophil cytosol factor 1B, Sn1-specific diacylglycerol lipase beta, Protein FAM26E, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 2, Anoctamin CCR4-NOT transcription complex subunit 1, Interferon-induced helicase C domain-containing protein 1, T-cell surface glycoprotein CD3 zeta chain, Caspase-3, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2, Interferon regulatory factor 2-binding At least one protein inhibitor selected from the protein group consisting of protein 1, Endothelial lipase, Lipopolysaccharide-binding protein, Transthyretin, and Monocyte differentiation antigen CD14, and protein group (B):
    (B) Lysosome-associated membrane glycoprotein 2, Protein Jade-3, Tetraspanin-4, Oxysterol-binding protein-related protein 8, P2Y purinoceptor 12, Dematin, Transforming acidic coiled-coil-containing protein 2, Ubiquitin carboxyl-terminal hydrolase MINDY -1, Septin-4, Ephrin-B1, Septin-5, Plexin-B3, Tetraspanin-32, H(+)/Cl(-) exchange transporter 5, Neutrophil collagenase, Septin-8, Septin-6, Metabotropic glutamate receptor 6, Metabotropic glutamate receptor 7, Metabotropic glutamate receptor 4, Metabotropic glutamate receptor 8, Gamma-enolase, and at least one selected from the group consisting of at least one protein enhancer selected from the protein group consisting of CD63 antigen A prophylactic or therapeutic agent for granulomatous disease, which comprises the drug.
12. Protein group (A) and protein group (B) in extracellular vesicles or blood samples of body fluids collected from animals treated with the test substance:
    (A) Chitinase-3-like protein 1, Protein-tyrosine sulfotransferase 2, Myoferlin, Pancreas transcription factor 1 subunit alpha, RuvB-like 2, Protein FAM110D, Cystatin-A, Creatine kinase M-type, Rho GTPase-activating protein 4 , Glutathione hydrolase 5 proenzyme, Putative neutrophil cytosol factor 1C, Neutrophil cytosol factor 1, Putative neutrophil cytosol factor 1B, Sn1-specific diacylglycerol lipase beta, Protein FAM26E, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 2, Anoctamin. CCR4-NOT transcription complex subunit 1, Interferon-induced helicase C domain-containing protein 1, T-cell surface glycoprotein CD3 zeta chain, Caspase-3, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2, Interferon regulatory factor 2-binding Protein 1, Endothelial lipase, Lipopolysaccharide-binding protein, Transthyretin, and Monocyte differentiation antigen CD14, and (B) Lysosome-associated membrane glycoprotein 2, Protein Jade-3, Tetraspanin-4, Oxysterol-binding protein-related protein 8, P2Y purinoceptor 12, Dematin, Tr ansforming acidic coiled-coil-containing protein 2, Ubiquitin carboxyl-terminal hydrolase MINDY-1, Septin-4, Ephrin-B1, Septin-5, Plexin-B3, Tetraspanin-32, H(+)/Cl(-) exchange transporter 5, Neutrophil collagenase, Septin-8, Septin-6, Metabotropic glutamate receptor 6, Metabotropic glutamate receptor 7, Metabotropic glutamate receptor 4, Metabotropic glutamate receptor 8, Gamma-enolase, and CD63 antigen A method for screening an active ingredient of a prophylactic or therapeutic agent for granulomatous disease, which comprises using the amount or concentration of at least one protein as an index.
13. When the value of the index for the protein group (A) is lower than the amount or concentration of the corresponding protein in extracellular vesicles or blood samples of body fluid collected from animals not treated with the test substance, the test The step of selecting a substance as an active ingredient of a prophylactic or therapeutic agent for granulomatous disease, and the value of the index relating to protein group (B) is the extracellular small amount of the body fluid collected from an animal not treated with the test substance. At least one step selected from the group consisting of selecting the test substance as an active ingredient of a prophylactic or therapeutic agent for granulomatous disease when the amount or concentration of the corresponding protein in the vesicle or blood sample is higher. The screening method according to claim 12, which comprises:
14. Protein group (A) and protein group (B) in extracellular vesicles or blood samples of body fluids collected from animals treated with the test substance:
    (A) Chitinase-3-like protein 1, Protein-tyrosine sulfotransferase 2, Myoferlin, Pancreas transcription factor 1 subunit alpha, RuvB-like 2, Protein FAM110D, Cystatin-A, Creatine kinase M-type, Rho GTPase-activating protein 4 , Glutathione hydrolase 5 proenzyme, Putative neutrophil cytosol factor 1C, Neutrophil cytosol factor 1, Putative neutrophil cytosol factor 1B, Sn1-specific diacylglycerol lipase beta, Protein FAM26E, Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 2, Anoctamin CCR4-NOT transcription complex subunit 1, Interferon-induced helicase C domain-containing protein 1, T-cell surface glycoprotein CD3 zeta chain, Caspase-3, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2, Interferon regulatory factor 2-binding Protein 1, Endothelial lipase, Lipopolysaccharide-binding protein, Transthyretin, and Monocyte differentiation antigen CD14, and (B) Lysosome-associated membrane glycoprotein 2, Protein Jade-3, Tetraspanin-4, Oxysterol-binding protein-related protein 8, P2Y purinoceptor 12, Dematin, Tr ansforming acidic coiled-coil-containing protein 2, Ubiquitin carboxyl-terminal hydrolase MINDY-1, Septin-4, Ephrin-B1, Septin-5, Plexin-B3, Tetraspanin-32, H(+)/Cl(-) exchange transporter 5, Neutrophil collagenase, Septin-8, Septin-6, Metabotropic glutamate receptor 6, Metabotropic glutamate receptor 7, Metabotropic glutamate receptor 4, Metabotropic glutamate receptor 8, Gamma-enolase, and CD63 antigen A method for evaluating the inducibility or exacerbation of granulomatous disease, which comprises using the amount or concentration of at least one protein as an index.
15. When the value of the index relating to the protein group (A) is higher than the amount or concentration of the corresponding protein in the extracellular vesicle or blood sample of the body fluid collected from the animal not treated with the test substance, the test The step of determining that the substance has a granulomatous disease-inducing or malignant state, and the value of the above-mentioned index relating to the protein group (B) is the extracellular small amount of the body fluid collected from the animal not treated with the test substance. At least one step selected from the group consisting of the step of determining that the test substance has a granulomatous disease-inducing or malignant condition when it is lower than the amount or concentration of the corresponding protein in the vesicle or blood sample. The evaluation method according to claim 14, comprising:

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