WO2012086346A1

WO2012086346A1 - Diagnostic method for chronic inflammatory diseases using antibody against inhibitory co-stimulatory molecules as marker

Info

Publication number: WO2012086346A1
Application number: PCT/JP2011/076507
Authority: WO
Inventors: 康広三宅; 和秀山本; 松本　和幸
Original assignee: 国立大学法人岡山大学
Priority date: 2010-12-22
Filing date: 2011-11-17
Publication date: 2012-06-28
Also published as: JP5996438B2; JPWO2012086346A1

Abstract

Provided is a diagnostic method for chronic inflammatory diseases, the main condition of which is lymphocyte infiltration. Specifically provided is a diagnostic method which is capable of distinguishing chronic inflammatory diseases, the main condition of which is lymphocyte infiltration, from acute inflammation and inflammatory diseases, the main condition of which is not lymphocyte infiltration, said distinguishment having been impossible by conventional inflammation markers. The diagnostic method is enabled by measuring an antibody against inhibitory co-stimulatory molecules (such as PD-1, BTLA or CTLA-4) expressed in T cells and/or B cells. This diagnostic method enables distinguishment of chronic inflammatory diseases, the main condition of which is lymphocyte infiltration, from acute inflammation and inflammatory diseases, the main condition of which is not lymphocyte infiltration. A remarkable difference can be recognized among diseases that have similar clinical conditions by measuring the antibody, so that distinguishment among such diseases becomes possible.

Description

Method for testing chronic inflammatory disease using antibody to inhibitory costimulatory molecule as marker

The present invention relates to a test method for chronic inflammatory diseases. Specifically, it relates to a test method for chronic inflammatory diseases using an antibody against an inhibitory costimulatory molecule as a marker, more specifically, an anti-PD-1 (Programmed Cell Death protein 1) antibody, an anti-BTLA (B and T lymphocyte). attenuator) antibody and anti-CTLA-4 (Cytotoxic T lymphocyte antigen 4) antibody as a marker.

This application claims the priority of Japanese Patent Application No. 2010-286456, which is incorporated herein by reference.

In clinical practice, C-reactive protein (CRP) and amyloid A protein (SAA) are widely used as serum markers for assessing the presence and degree of inflammation. These are the degree of inflammation in both chronic inflammation and acute inflammation. An increase is observed depending on the situation. For this reason, chronic inflammation cannot be differentiated from acute inflammation depending on the degree of increase in CRP or SAA. In many of the diseases based on acute inflammation, such as acute hepatitis A, hepatitis B, vomiting diarrhea caused by norovirus and rotavirus infection, etc. To cure. However, in diseases based on chronic inflammation, the appearance of organ dysfunction or carcinogenesis occurs due to prolonged inflammation. Differentiating between acute and chronic inflammatory diseases is important for the selection of treatment and prognosis prediction for patients, but there are biomarkers for differentiating between acute and chronic inflammatory diseases. Absent.

Chronic inflammatory diseases include rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, mixed connective tissue disease, polymyositis, dermatomyositis, Hashimoto's disease, autoimmune hepatitis (AIH), primary biliary Autoimmune diseases represented by cirrhosis (PBC), primary sclerosing cholangitis (PSC), Crohn's disease (CD), ulcerative colitis (UC), represented by chronic hepatitis B and chronic hepatitis C Includes chronic viral infections. In some autoimmune diseases, various disease-specific markers (autoantibodies) have been identified. However, since many patients with autoimmune diseases often develop with nonspecific symptoms such as general malaise, fever, and joint pain, it is cost-effective to measure all specific autoantibodies for each disease. There is a problem with this point. In addition, the measurement of antinuclear antibodies is widely used as a screening test for autoimmune diseases, but it has been reported that about 26% are positive even in healthy individuals (Non-patent Document 1), and there is a problem in specificity. is there. Some chronic viral infections have established screening tests such as HBs antigen in chronic hepatitis B, HCV antibody in chronic hepatitis C, and blood EB virus DNA quantification in chronic active EB virus infection. is there. On the other hand, biomarkers as screening screening methods for autoimmune diseases have not been established, and it is necessary to develop biomarkers that can be tested more efficiently.

Among autoimmune diseases, autoimmune hepatitis (AIH) has not been identified as a disease-specific biomarker and is diagnosed according to various diagnostic criteria (Non-patent Documents 2 and 3). ). Autoimmune hepatitis is common in women after middle age, but many patients are taking any drug or health food at the time of onset. For this reason, an increasing number of cases require differentiation from liver damage caused by health foods and drugs (generally drug-induced liver damage). However, there is no biomarker useful for distinguishing between the two. Therefore, it is important to develop a biomarker useful for distinguishing between autoimmune hepatitis and drug-induced liver injury.

In many chronic inflammatory diseases including autoimmune diseases, T cell activation plays a central role in the pathological state. For antigen-specific activation of T cells, the antigen presented by the major histocompatibility complex (MHC) on the antigen-presenting cell is recognized by the T cell receptor (TCR), which is expressed specifically by the T cell. CD28, which is a type of co-stimulatory molecule expressed on the surface of T cells, is required to react with B7 (CD80 and CD86), which are ligands on antigen-presenting cells (FIG. 1,). (See left figure). Even if T cells recognize antigens by TCR, if there is no co-stimulation signal from CD28, not only T cell activation will occur, but also antigen-specificity that will not respond to subsequent re-stimulation with the same antigen It becomes a state called clonal anergy. On the surface of activated T cells, expression of inhibitory costimulatory molecules such as CTLA-4 (Cytotoxic T lymphocyte antigen 4) and PD-1 is observed.

Recently, CTLA-4 molecules against the B7 antigen for the purpose of inactivating T cells activated in patients with rheumatoid arthritis have been disclosed (Patent Documents 1 and 2). In addition, when TLA selective co-stimulatory agent (generic name: abatacept) by gene recombination including CTLA-4 part and modified Fc region derived from human IgG1 is insufficient for existing treatment for rheumatoid arthritis It was approved as a medicine in Japan on the condition that it is limited to In addition, treatment with anti-CTLA-4 antibody or anti-PD-1 antibody administration has been examined for the purpose of cancer cell-specific T cell activation in malignant tumors (Patent Documents 3 to 5, Non-Patent Document 4). In mice knocked out of PD-1, it has been reported that antinuclear antibodies appear in the blood and spontaneously develop autoimmune diseases similar to autoimmune hepatitis, autoimmune gastritis, and lupus nephritis (patent) Document 6, Non-patent document 5).

However, there is no report on a test method using an antibody of a costimulatory molecule as a marker.

JP-A-8-47391 Japanese Unexamined Patent Publication No. 9-202800 JP 2006-340714 A JP 2009-155338 A JP 2009-155338 A JP 2007-023047

An object of the present invention is to provide a method for examining a chronic inflammatory disease. Specifically, a test method that can distinguish chronic inflammation and acute inflammation that could not be distinguished by conventional inflammation markers, more specifically, a test method for autoimmune diseases and a prognostic test method after transplantation are provided. The task is to do.

In order to solve the above problems, the inventors of the present application focused on the activation state of T cells related to many chronic inflammatory diseases including autoimmune diseases, and as a result of earnest examination, The present inventors have found for the first time that an antibody against an inhibitory costimulatory molecule that can be used as a marker for chronic inflammatory diseases has completed the present invention.

That is, this invention consists of the following.
1. A method for examining a chronic inflammatory disease, comprising measuring an antibody against an inhibitory costimulatory molecule in a collected biological specimen as a marker.
2. The inhibitory costimulatory molecule is PD-1 (Programmed Cell Death protein 1) expressed on the surface of T cells and / or B cells, characterized in that the anti-PD-1 antibody is measured as a marker. The test | inspection method of chronic inflammatory disease of description.
3. 2. Chronic inflammation according to item 1 above, wherein the inhibitory costimulatory molecule is BTLA (B and T lymphocyte attenuator) expressed on the surface of T cells and / or B cells, and the anti-BTLA antibody is measured as a marker. Of testing for sexually transmitted diseases
4). The inhibitory costimulatory molecule is CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4) expressed on the surface of T cells and / or B cells, and is measured using an anti-CTLA-4 antibody as a marker, 1 above The test | inspection method of the chronic inflammatory disease of description.
5. 2. The method for examining a chronic inflammatory disease according to item 1, wherein an anti-PD-1 antibody and an anti-BTLA antibody are each measured as a marker as an antibody against an inhibitory costimulatory molecule.
6). 6. The method for examining a chronic inflammatory disease according to item 4 or 5, wherein an anti-CTLA-4 antibody is further measured as an antibody against an inhibitory costimulatory molecule, respectively.
7. 7. The method for examining a chronic inflammatory disease according to any one of items 1 to 6, wherein the chronic inflammatory disease is an autoimmune disease, a viral chronic disease, or a chronic rejection reaction after transplantation of a biological component.
8). 8. The chronic inflammatory disease according to item 7 above, wherein the autoimmune disease is any one selected from autoimmune hepatitis, Crohn's disease, ulcerative colitis, primary sclerosing cholangitis and primary biliary cirrhosis. Inspection method.
9. 8. The method for examining a chronic inflammatory disease according to item 7, wherein the viral chronic disease is chronic hepatitis B or chronic hepatitis C.
10. 8. The method for examining a chronic inflammatory disease according to item 7 above, wherein the chronic rejection after transplantation of a biological component is chronic rejection after transplantation of an organ, tissue and / or cell.
11. 11. The method for examining a chronic inflammatory disease according to any one of the preceding

items

1, 2, and 5 to 10, wherein the chronic inflammatory disease is a chronic inflammatory disease having lymphocyte infiltration as a main pathological condition and comprising the following steps: :
1) a step of measuring the anti-PD-1 antibody titer present in the collected specimen;
2) A step of determining that the measured anti-PD-1 antibody titer is a chronic inflammatory disease centered on lymphocyte infiltration when the measured anti-PD-1 antibody titer is higher than a cutoff value.
12 The method for examining a chronic inflammatory disease according to item 11, further comprising the following steps:
1) a step of further measuring the anti-BTLA antibody titer present in the collected specimen;
2) A step of determining that the measured anti-BTLA antibody titer is a chronic inflammatory disease centered on lymphocyte infiltration when the measured anti-BTLA antibody titer is higher than a cutoff value.
13. The method for examining a chronic inflammatory disease according to item 11 or 12, further comprising the following steps:
1) a step of further measuring the anti-CTLA-4 antibody titer present in the collected specimen;
2) A step of determining that the measured anti-CTLA-4 antibody titer is a chronic inflammatory disease centered on lymphocyte infiltration when the measured anti-CTLA-4 antibody titer is higher than a cutoff value.
14 14. A test kit for use in the method for testing a chronic inflammatory disease according to any one of the preceding

items

1, 2, and 5 to 13, comprising at least a PD-1 antigen and comprising an anti-PD-1 antibody detection device.
15. 15. A test kit for use in the method for testing a chronic inflammatory disease according to item 14, further comprising a BTLA antigen and / or CTLA-4 antigen and a device for detecting an antibody against each antigen.

According to the test method of the present invention, it is possible to distinguish between acute inflammatory diseases and chronic inflammatory diseases. Although differentiation between acute inflammatory diseases and chronic inflammatory diseases is important for selection of treatment methods and prognosis prediction for patients, there is no biomarker for differentiating between acute inflammatory diseases and chronic inflammatory diseases. Confirmed that the test method of the present invention can distinguish diseases with similar clinical symptoms, such as drug-induced liver injury (Drug), primary sclerosing cholangitis (PSC), and autoimmune hepatitis (AIH). It was done. As for viral hepatitis, it was possible to distinguish between acute hepatitis B (AHB) and chronic hepatitis B (CHB). Furthermore, a significant difference was observed in the anti-PD-1 antibody titer between Crohn's disease (CD) and ulcerative colitis (UC), and was able to be differentiated. Both Crohn's disease and ulcerative colitis are autoimmune enteritis, and the symptoms are similar, but Crohn's disease is a full-thickness inflammatory finding of the gastrointestinal mucosa centered on lymphocyte infiltration. In contrast to non-caseating epithelioid cell granuloma, ulcerative colitis is characterized by a crypt abscess in which neutrophils infiltrate and aggregate in the crypt lumen of the large intestine mucosa. Crohn's disease may cause inflammation throughout the digestive tract. In ulcerative colitis, inflammation occurs only in the large intestine. On the other hand, ulcerative colitis may become cancerous, and the prognosis is different from Crohn's disease. Therefore, the treatment policy and the like differ for these diseases. With respect to these diseases, conventionally, it has been necessary to observe the progress over time, collect biopsy specimens, etc. and observe and distinguish tissue specimens, whereas according to the method of the present invention, It is excellent in that it can be distinguished using a serum sample.

It is a figure which shows the concept of an inhibitory costimulatory molecule. It is a figure which shows the measurement result of the anti- PD-1 antibody titer in serum in each disease. Example 1 It is a figure which shows the positive rate of the anti- PD-1 antibody in the serum in each disease. Example 1 It is a figure which shows the measurement result of the anti- PD-1 antibody titer before a treatment when a patient with autoimmune hepatitis is treated with the steroid preparation prednisolone (PSL) and after remission. (Example 2) It is a figure which shows the measurement result of the anti- CTLA-4 antibody titer in serum in each disease. Example 3 It is a figure which shows the positive rate of the anti- CTLA-4 antibody in the serum in each disease. Example 3 It is a figure which shows the measurement result of the anti- BTLA antibody titer in serum in each disease. (Example 4) It is a figure which shows the positive rate of the anti- BTLA antibody in the serum in each disease. (Example 4) It is a photograph figure which shows the detection result of the anti- PD-1 antibody by the immunoprecipitation method and the western blotting method. (Example 5) It is a figure which shows the correlation of the serum ALT level and anti-PD-1 antibody titer in an autoimmune hepatitis patient. (Example 12) It is a figure which shows the correlation of the serum IgG level in an autoimmune hepatitis patient, and an anti- PD-1 antibody titer. (Example 12)

The present invention relates to a method for examining a chronic inflammatory disease, characterized by measuring an antibody against an inhibitory costimulatory molecule in a collected biological specimen as a marker.

The “biological sample” in the present invention is not particularly limited as long as the antibody as a marker in the present invention can exist, and examples thereof include whole blood, plasma, serum, spinal fluid, joint fluid, vaginal fluid, urine, Examples include body fluids, saliva, nasal discharge, tears, stool-derived materials, and the like, and it is particularly preferable to use whole blood, plasma, and serum used in clinical tests as specimens. The collected biological specimen can be appropriately preprocessed when it is examined.

In the present invention, the “suppressive costimulatory molecule” is an inhibitory costimulatory molecule that can be expressed on the surface of T cells and / or B cells. Specifically, PD-1 (Programmed death 1), CTLA- 4 (Cytotoxic T lymphocyte antigen 4) and BTLA (B and T lymphocyte attenuator). As mentioned above in the background section, expression of inhibitory costimulatory molecules such as CTLA-4 and PD-1 is observed on the surface of activated T cells. The CTLA-4 and PD-1 ligands are B7 and PD-L (PD-L1 and PD-L2), respectively, but it is known that T cells are inactivated by stimulation from CTLA-4 and PD-1. (See FIG. 1). The CD28 ligand is also B7, but CTLA-4, an inhibitory costimulatory molecule, has a 20-fold higher affinity for B7 than CD28. By maintaining the balance of co-stimulatory molecules and inhibitory co-stimulatory molecules related to T cell and / or B cell immunity, it is considered that immunity in the living body is appropriately controlled.

As described above in the Background Art section, several reports have already been made on the functions of co-stimulatory molecules and inhibitory co-stimulatory molecules related to immune functions as described above. However, detection of these substances as markers and testing for diseases has not been reported so far.

In the present invention, the “antibody against the inhibitory co-stimulatory molecule” is not particularly limited as long as it can be present in the collected biological specimen. The type of antibody may be, for example, IgG or IgM, but IgG is preferred.

In the present invention, using these antibodies as markers does not necessarily mean that these antibody titers are higher than the cutoff value. For example, among the chronic inflammatory diseases to be described later, all of them belong to autoimmune diseases such as ulcerative colitis and Crohn's disease, and have similar clinical symptoms, but have different treatment strategies. It is possible to discriminate which disease is caused by the value of the marker. Also, for autoimmune hepatitis and drug-induced liver injury, there are diseases that have similar clinical symptoms but have completely different treatment strategies, but are there any diseases depending on the above marker values? Can be identified.

The chronic inflammatory disease in the present invention is not particularly limited as long as it is a chronic inflammatory disease used in the medical field. Specific examples of chronic inflammatory diseases in the present invention include autoimmune diseases, viral chronic diseases, chronic rejection after transplantation of biological components, and those not classified into these.

Examples of autoimmune diseases include inflammatory bowel diseases (ulcerative colitis, Crohn's disease, etc.), rheumatoid arthritis, seronegative spondyloarthropathy, systemic lupus erythematosus, glomerulonephritis (nephrotic syndrome (idiopathic nephrotic syndrome) , Minimal change nephropathy, etc.), multiple sclerosis, multiple myositis, multiple chondritis, scleroderma, dermatomyositis, Wegener's granulomatosis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis , Myasthenia gravis, idiopathic sprue, sarcoidosis, Reiter syndrome, type I diabetes, Harada disease, Behcet's disease, Sjogren's syndrome, mixed connective tissue disease, Basedow disease, chronic thyroiditis, autoimmune blood disease (hemolytic anemia) Non-regenerative anemia, idiopathic thrombocytopenia, etc. Preferably, autoimmune hepatitis, ulcerative colitis, Crohn's disease and primary biliary disease Juvenile cirrhosis, rheumatoid arthritis, seronegative spondyloarthropathy, systemic lupus erythematosus, multiple sclerosis, multiple myositis, scleroderma, dermatomyositis, type I diabetes, Sjogren's syndrome, mixed connective tissue, Basedow disease, Examples include chronic thyroiditis, autoimmune hemolytic anemia, and particularly preferably autoimmune hepatitis, ulcerative colitis, Crohn's disease, primary sclerosing cholangitis, and primary biliary cirrhosis. .

Examples of viral chronic diseases include chronic hepatitis B, chronic hepatitis C and chronic active EB virus infection. Examples of chronic rejection after biological component transplantation include chronic rejection after transplantation of organs, tissues and / or cells, including chronic graft-versus-host disease (GVHD).

Furthermore, other chronic inflammatory diseases in the present invention include psoriasis-like arthritis, inflammatory skin diseases (such as lichen planus, pemphigus, bullous pemphigoid, epidermolysis bullosa, alopecia areata).

In the present invention, among antibodies against inhibitory costimulatory molecules, for example, anti-PD-1 antibody, anti-CTLA-4 antibody and anti-BTLA antibody, particularly preferably anti-PD-1 antibody and / or anti-BTLA antibody are listed above. Among the diseases, in the case of a chronic inflammatory disease in which lymphocyte infiltration is the center of the disease state, it tends to be higher than the cut-off value.
Diseases that may be higher than the cutoff value include Crohn's disease, sarcoidosis, rheumatoid arthritis, seronegative spondyloarthropathy, Sjogren's syndrome, systemic lupus erythematosus, scleroderma, polymyositis, dermatomyositis, mixed Autoimmunity that has been reported to be associated with autoimmune hepatitis and primary biliary cirrhosis such as sex connective tissue disease, Basedow disease, chronic thyroiditis, type I diabetes, autoimmune hemolytic anemia, multiple sclerosis Examples include diseases and chronic viral infections such as chronic hepatitis B, chronic hepatitis C and chronic active EB virus infection.

For example, as diseases with similar symptoms, ulcerative colitis and Crohn's disease, both of which belong to autoimmune diseases, autoimmune hepatitis and primary sclerosing cholangitis, one of which is autoimmune disease, autoimmune hepatitis And drug-induced liver injury. For example, in the case of Crohn's disease, an antibody titer higher than the cut-off value is shown, and in the case of ulcerative colitis, a value similar to the cut-off value is shown. In the case of autoimmune hepatitis, the antibody titer is higher than the cut-off value, and in the case of drug-induced liver injury and primary sclerosing cholangitis, the value is comparable to the cut-off value. When multiple candidate diseases are listed as diseases with similar symptoms, first conduct a rough test using a known test method, and after narrowing down the corresponding disease to some extent, It is preferable to perform a test when differentiating the disease. Accordingly, the test method of the present invention is performed by testing using an existing test method such as C-reactive protein (CRP), amyloid A protein (SAA), antinuclear antibody, etc. It is possible to perform inspection more effectively.

As shown in the background section, CTLA4-Ig preparation (generic name: abatacept) is a T cell selective costimulatory regulator for B7 antigen for the purpose of inactivating activated T cells in rheumatoid arthritis Was conditionally approved as a drug in Japan. Such biologics such as CTLA-4 molecules are very expensive and are therefore preferably administered only for those diseases that are truly necessary.

Regardless of the administration of abatacept, if any kind of treatment is being performed for the above-mentioned diseases, the prognosis of the disease or the suitability of the treatment policy can be predicted by monitoring the antibody titer in the biological specimen. Is preferred. For example, for chronic rejection after transplantation of organs, etc., it is possible to cut antibodies against inhibitory costimulatory molecules, such as anti-PD-1 antibodies, anti-CTLA-4 antibodies and anti-BTLA antibodies, particularly preferably anti-PD-1 antibodies. Although it is considered to show a value higher than the off value, by monitoring the antibody titer along with the subsequent treatment progress, the treatment policy, such as prognosis prediction and subsequent drug dose determination, is appropriately examined, It only needs to be selectable. According to the inspection method of the present invention, such prognosis prediction and the like are possible.

Specific examination methods of the present invention, for example, examination methods for chronic inflammatory diseases in which lymphocyte infiltration is the center of pathology include the following.
1) a step of measuring the anti-PD-1 antibody titer present in the collected specimen;
2) A step of determining that the measured anti-PD-1 antibody titer is a chronic inflammatory disease centered on lymphocyte infiltration when the measured anti-PD-1 antibody titer is higher than a cutoff value.

The method for examining a chronic inflammatory disease centering on lymphocyte infiltration according to the present invention can further include the following steps.
1) a step of further measuring the antibody titer of the anti-BTLA antibody present in the collected specimen;
2) A step of determining that the antibody titer of the measured anti-BTLA antibody is a chronic inflammatory disease having lymphocyte infiltration as the center of the pathological condition when the antibody titer is higher than a cutoff value.

The method for examining a chronic inflammatory disease centering on lymphocyte infiltration according to the present invention can further include the following steps.
1) a step of further measuring the antibody titer of the anti-CTLA-4 antibody present in the collected specimen;
2) A step of determining that the measured anti-CTLA-4 antibody titer is higher than a cutoff value as a chronic inflammatory disease centered on lymphocyte infiltration.

As a method for examining chronic inflammatory diseases centering on lymphocyte infiltration according to the present invention, anti-PD-1 antibody titers existing in collected specimens, and anti-CTLA-4 antibodies for two types of diseases to be compared It can be differentiated by the difference in each antibody titer. For example, for autoimmune hepatitis and drug-induced liver injury, the anti-PD-1 antibody titer and anti-CTLA-4 antibody titer in the serum of patients with autoimmune hepatitis tended to be high, and the positive rate was also high. It was. When comparing Crohn's disease and ulcerative colitis, it was confirmed that Crohn's disease has higher antibody titer and positive rate. Thus, the measurement results of the anti-PD-1 antibody titer and further the anti-CTLA-4 antibody titer suggest that it is possible to differentiate chronic inflammatory diseases centered on lymphocyte infiltration.

As a further examination method for chronic inflammatory diseases having lymphocyte infiltration as the main disease state of the present invention, it is possible to distinguish two types of diseases to be compared based on the difference in antibody titers of anti-BTLA antibodies present in the collected specimens. it can. For example, when comparing autoimmune hepatitis and drug-induced liver injury, autoimmune hepatitis tended to have a higher antibody titer and positive rate. When Crohn's disease and ulcerative colitis were compared, Crohn's disease tended to have a higher antibody titer and positive rate. Thereby, it was suggested from the measurement result of the anti-BTLA antibody that it is possible to distinguish a chronic inflammatory disease centering on lymphocyte infiltration.

In the present invention, methods for detecting antibodies against inhibitory costimulatory molecules, such as anti-PD-1 antibodies, anti-CTLA-4 antibodies, and anti-BTLA antibodies, can be applied per se and are not particularly limited. Specifically, enzyme-linked immunosorbent assay (enzyme-linked immunoassay: ELISA), radioimmunoassay (radio-RIA) method, agglutination method, turbidimetric method, turbidity measurement method, Western blot method, immunochromatography, etc. Can be mentioned. In measuring the antibody titer, the ELISA method and the RIA method are particularly preferable, and the ELISA method that can be easily tested is most preferable.

The present invention also extends to an inspection kit used in the above inspection method. The configuration of the test kit includes at least a PD-1 antigen for anti-PD-1 antibody detection, and an anti-PD-1 antibody detection device. Furthermore, in order to measure anti-BTLA antibody and / or anti-CTLA-4 antibody, a BTLA antigen and / or CTLA-4 antigen and a device for detecting these antibodies are included. As an antibody detection device, for example, when the immunoassay method is an ELISA method, a tube or a plate as a solid phase can be included. Furthermore, a buffer solution used for measurement can also be included in the kit.

In order to deepen the understanding of the present invention, the present invention will be specifically described with reference to the following examples, but it goes without saying that the present invention is not limited thereto. This study using the following clinical specimens has been approved by the Ethics Committee within Okayama University.

(Example 1) Measurement of anti-PD-1 antibody titer in sera of patients with various diseases Blood was collected from each patient shown in Table 1 below, and serum was obtained by performing treatments used in normal clinical tests.

The serum sample obtained from each of the above patients was measured for anti-PD-1 antibody titer by ELISA. The measurement was performed according to the following procedure.
1) As an antigen, 100 μl of 1 μg / ml genetically modified PD-1 (H00005133-Q01: Avnova, Taiwan) was added to each well of a 96-well microplate and allowed to stand for 1 hour to immobilize the antigen.
2) Blocking was performed by adding 300 μl of 1% bovine serum albumin (BSA).
3) 100 μl of a patient serum sample diluted 20-fold with 1% BSA-PBST (0.02% Tween-20) was added and allowed to react for 1 hour at room temperature.
4) After washing each well with PBST (0.05% Tween-20), 100 μl of 1 μg / ml peroxidase-labeled anti-human IgG antibody was added and allowed to react at room temperature for 1 hour.
5) After washing each well with PBST (0.05% Tween-20), add 100 μl of chromogenic substrate solution (2,2'-azino-bis [3-ethylbenzothiazoline-6-sulfonate]: ABTS) for 10 minutes at room temperature. Reacted.
6) Absorbance at 630 nm was measured using an ELISA reader (Model 680 Microplate Reader: Bio-Rad Laboratories).

The anti-PD-1 antibody titer in each patient serum is shown in FIG. 2 and Table 2 below. The meanings of the abbreviations shown on the horizontal axis in FIG.

Furthermore, the positive rate of anti-PD-1 antibody for each disease patient when the cut-off value is used as a reference is shown in FIG. The abbreviations shown on the horizontal axis of FIG.

From the above results, other chronic inflammatory diseases excluding primary sclerosing cholangitis (PSC) and ulcerative colitis (UC) in drug-induced liver injury (Drug) and serum derived from patients with acute hepatitis B (AHB) Compared with the patient-derived serum, anti-PD-1 antibody titer close to that of the control was shown, and the antibody positive rate was also significantly different.

From these results, it was confirmed that drug-induced liver injury (Drug), primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH), which have similar clinical symptoms, can be differentiated. As for viral hepatitis, it was confirmed that it is possible to differentiate between acute hepatitis B (AHB) and chronic hepatitis B (CHB). Furthermore, it was confirmed that there was a significant difference in anti-PD-1 antibody titer between Crohn's disease (CD) and ulcerative colitis (UC). Both Crohn's disease and ulcerative colitis are autoimmune enteritis, and the symptoms are similar, but Crohn's disease is a full-thickness inflammatory finding of the gastrointestinal mucosa centered on lymphocyte infiltration. In contrast to non-caseating epithelioid cell granuloma, ulcerative colitis is characterized by a crypt abscess in which neutrophils infiltrate and aggregate in the crypt lumen of the large intestine mucosa. Crohn's disease may cause inflammation throughout the digestive tract. In ulcerative colitis, inflammation occurs only in the large intestine. On the other hand, ulcerative colitis may become cancerous, and the prognosis is different from Crohn's disease. Therefore, the treatment policy etc. differ for these diseases. For these diseases, it has been necessary to observe the clinical course or to differentiate by observing a tissue sample such as a biopsy sample, whereas the method of the present invention makes it easy to use a serum sample. It is excellent in that it can be distinguished.

(Example 2) Treatment process of autoimmune hepatitis patients and measurement of anti-PD-1 antibody titer Thirty-three patients with autoimmune hepatitis were treated internally with prednisolone (PSL), which is a steroid preparation. Anti-PD-1 antibody titers were measured for the serum samples of each patient before treatment and after remission by the same method as in Example 1.

As a result, it was confirmed that the anti-PD-1 antibody titer after the treatment was lower than that before the treatment (FIG. 4). This result suggests that the therapeutic effect of the drug can be monitored by measuring the anti-PD-1 antibody titer.

(Example 3) Measurement of anti-CTLA-4 antibody titer in sera of patients with various diseases The anti-CTLA-4 antibody titer of each patient serum sample shown in Table 1 of Example 1 was measured. The anti-CTLA-4 antibody titer was measured by the same method as the ELISA method shown in Example 1, except that the PD-1 antigen was changed to CTLA-4 antigen (Avnova).

The anti-CTLA-4 antibody titer for each disease patient is shown in Table 3 and FIG. Furthermore, the positive rate of the anti-CTLA-4 antibody for each disease patient when the cut-off value is used as a reference is shown in FIG. Abbreviations shown on the horizontal axis in FIGS. 5 and 6 are shown in Table 1.

As a result, the measurement result of the anti-CTLA-4 antibody was not as remarkable as the measurement result of the anti-PD-1 antibody, but showed almost the same tendency as the result of Example 1.

(Example 4) Treatment course of autoimmune hepatitis patient and measurement of anti-BTLA antibody titer Among each patient shown in Table 1 of Example 1, drug-induced liver injury, autoimmune hepatitis, Crohn's disease and ulcerative colitis The anti-BTLA antibody titer was measured for each patient serum sample. The anti-BTLA antibody titer was measured by the same method as the ELISA method shown in Example 1, except that the PD-1 antigen was changed to BTLA antigen (Avnova).

The anti-BTLA antibody titer for each disease patient is shown in Table 4 and FIG. Furthermore, the positive rate of the anti-BTLA antibody for each disease patient when the cut-off value is used as a reference is shown in FIG. Abbreviations shown on the horizontal axis in FIGS. 7 and 8 are shown in Table 1.

As a result, among patients with drug-induced liver injury and primary sclerosing cholangitis and autoimmune hepatitis, autoimmune hepatitis patients showed higher anti-BTLA antibody titers. In patients with Crohn's disease and ulcerative colitis, anti-BTLA antibody titers tended to be low. On the other hand, the positive rate of anti-BTLA antibody based on the cut-off value was clearly higher in Crohn's disease.

From the results of the above Examples, it was confirmed that various diseases can be distinguished from the measurement results of the anti-PD-1 antibody, and various diseases can be determined by measuring the anti-BTLA antibody and / or anti-CTLA-4 antibody. It was suggested that it is possible to distinguish

(Example 5) Detection of anti-PD-1 antibody by immunoprecipitation method and western blotting method Serum samples obtained by collecting blood from each patient shown in Table 1 of Example 1 and carrying out the treatment used in normal clinical tests Then, anti-PD-1 antibody in the serum was detected by immunoprecipitation method and Western blotting method. The measurement was performed according to the following procedure.

1) Protein G, which is an affinity carrier for antibody purification, was added to serum samples obtained from each patient, and IgG in the serum was adsorbed nonspecifically.
2) After washing protein G according to a conventional method, a solution containing protein G was prepared.
3) As an antigen, genetically modified PD-1 (H00005133-Q01: Avnova, Taiwan) was added to a solution containing protein G to 2.5 μg / ml and treated for 1 hour. The adsorbed serum IgG was reacted with the PD-1 antigen. When an anti-PD-1 antibody is present in serum, an antigen-antibody reaction occurs with the PD-1 antigen, and a protein G-IgG-PD-1 antigen complex is formed.
4) After washing according to a conventional method, the protein adsorbed to protein G was eluted with an eluent (50 mM Glycine Buffer, pH 2.8). The eluate contains IgG, and PD-1 antigen when anti-PD-1 antibody is present in serum.
5) After adding 2 × sample buffer (20% Glycerol, 4% SDS, 125 mM Tris-HCl / pH 6.8, 12% mercaptoethanol, 0.004% BPB (Bromophenol blue)) to the eluted protein solution, Electrophoresis by SDS-PAGE was performed according to a conventional method.
6) The migrated protein was blotted on a PVDF membrane according to a conventional method. After blocking for 1 hour, the mouse anti-PD-1 antibody (H00005133-A01: Avnova, Taiwan) as a primary antibody was treated for 1 hour and then migrated. Among the proteins, an antigen-antibody reaction of PD-1 antigen and mouse anti-PD-1 antibody was caused. After washing the membrane, an HRP-labeled anti-mouse IgG antibody (RPN2124: GE Healthcare, UK) was reacted as a secondary antibody for 1 hour. After further washing, the color was developed with ECL Western Blotting Detection System (RPN2132: GE Healthcare, UK) and detected with a luminometer.

The result of Western blotting is shown in FIG. In autoimmune hepatitis patients whose anti-PD-1 antibody was elevated by ELISA, a band was observed by Western blotting, indicating that anti-PD-1 antibody was present in the serum. On the other hand, in patients with drug-induced liver injury and healthy individuals whose anti-PD-1 antibody titers were extremely low by ELISA, no band was observed by Western blotting, and anti-PD-1 antibody was not present in the serum. Indicated. From the above, serum anti-PD-1 antibody can also be detected by Western blotting.

(Example 6) Relationship between anti-PD-1 antibody titer measured by ELISA and various diseases For patients with various diseases, anti-PD-1 antibody titer measured by ELISA described in Example 1 and each disease The sensitivity and specificity for were examined. As a result, when the anti-PD-1 antibody is higher than the cut-off value (average value in normal subjects + 2SD), the sensitivity and specificity are as shown in Table 5 and can be distinguished from normal subjects. Was confirmed.

(Example 7) Relationship between anti-CTLA-4 antibody titer measured by ELISA and various diseases For patients with various diseases, anti-CTLA-4 antibody titer measured by ELISA described in Example 3 and each disease The sensitivity and specificity for were examined. As a result, the sensitivity and specificity when the anti-CTLA-4 antibody titer is higher than the cut-off value (average value in normal subjects + 2SD) are as shown in Table 6 and can be distinguished from normal subjects. It was confirmed.

(Example 8) Relationship between anti-BTLA antibody titer measured by ELISA and various diseases For patients with various diseases, the anti-BTLA antibody titer measured by ELISA described in Example 4, and the sensitivity and specificity for each disease I examined the degree. As a result, the sensitivity and specificity when the anti-BTLA antibody titer is higher than the cut-off value (average value in normal subjects + 2SD) are as shown in Table 7 and can be distinguished from normal subjects. confirmed.

(Example 9) Differentiation of various diseases by measuring each antibody marker (1)
For each antibody titer measured by the methods described in Examples 6 to 8, the case where each was greater than or equal to the cut-off value was (+), and autoimmune hepatitis and drug-induced liver injury were differentiated by combining various markers. .
As a result, as shown in Table 8, it was confirmed that the anti-PD-1 antibody showed excellent specificity by measurement. In addition, when both the anti-PD-1 antibody and the anti-CTLA-4 antibody are (+), or both the anti-PD-1 antibody and the anti-BTLA antibody are (+), high specificity is exhibited. It was confirmed. On the other hand, when either the anti-PD-1 antibody or anti-CTLA-4 antibody is (+), or when either the anti-PD-1 antibody or anti-BTLA antibody is (+), the sensitivity is slightly better. However, the specificity was not high.

Based on the above, when it is judged that either autoimmune hepatitis or drug-induced liver injury, both diseases are identified by anti-CTLA-4 antibody and / or anti-BTLA in addition to anti-PD-1 antibody (+). It was considered possible to distinguish autoimmune hepatitis when the antibody was (+).

(Example 10) Differentiation of each disease by measuring each antibody marker (2)
For each antibody titer measured by the methods described in Examples 6 to 8, the case where each was greater than or equal to the cut-off value was (+), and the combination of various markers was used to differentiate autoimmune hepatitis from primary sclerosing cholangitis. went.
As a result, as shown in Table 9, it was confirmed that the anti-BTLA antibody measurement showed excellent specificity. In addition, when both anti-BTLA antibody and anti-CTLA-4 antibody are (+), or both anti-BTLA antibody and anti-PD-1 antibody are (+), high specificity may be exhibited. confirmed. On the other hand, when either the anti-BTLA antibody or the anti-CTLA-4 antibody is (+), or when either the anti-BTLA antibody or the anti-PD-1 antibody is (+), the sensitivity shows a slightly superior value. Specificity was not high.

Based on the above, when it is determined that either autoimmune hepatitis or primary sclerosing cholangitis, both diseases are identified by anti-CTLA-4 antibody and / or anti-PD in addition to anti-BTLA antibody (+). When antibody -1 was (+), it was considered possible to distinguish autoimmune hepatitis.

(Example 11) Differentiation of each disease by measuring each antibody marker (3)
With respect to various antibody titers measured by the methods described in Examples 6 to 8, the case where each was greater than or equal to the cut-off value was (+), and chronic hepatitis B and acute hepatitis B were differentiated by combining various markers. .
As a result, as shown in Table 10, it was confirmed that the anti-PD-1 antibody showed excellent specificity by measurement. In addition, when both the anti-PD-1 antibody and the anti-CTLA-4 antibody are (+), or both the anti-PD-1 antibody and the anti-BTLA antibody are (+), high specificity is exhibited. It was confirmed. On the other hand, when either the anti-PD-1 antibody or the anti-BTLA antibody was (+), the sensitivity was excellent, but the specificity was not high.

Based on the above, in the case where it is determined that the disease is either chronic hepatitis B or acute hepatitis B, both diseases are identified by anti-PD-1 antibody (+), anti-CTLA-4 antibody and / or anti-BTLA. It was considered possible to distinguish chronic hepatitis B when the antibody is (+).

(Example 12) Differentiation of each disease by measuring each antibody marker (4)
With respect to various antibody titers measured by the methods described in Examples 6 to 8, the case where each was equal to or higher than the cutoff value was defined as (+), and Crohn's disease and ulcerative colitis were differentiated by a combination of various markers.
As a result, as shown in Table 11, when the anti-PD-1 antibody, anti-CTLA-4 antibody and anti-BTLA antibody were (+), high specificity was shown. It was confirmed that it showed excellent specificity for. Furthermore, it was confirmed that the above-mentioned various antibodies exhibit superior specificity for Crohn's disease when any two or more antibodies are (+).

Based on the above, when it is judged that either Crohn's disease or ulcerative colitis, both diseases are identified as anti-PD-1 antibody (+), anti-CTLA-4 antibody and anti-BTLA antibody (+) In some cases, it was considered possible to differentiate from Crohn's disease.

(Example 13) Diagnosis of pathological condition in autoimmune liver patient For autoimmune liver patient, anti-PD-1 antibody titer measured by ELISA described in Example 1 and alanine oxoglutarate aminotransferase (Alanine Aminotransferase: ALT, GPT). ) Or the correlation with IgG (see FIGS. 10 and 11). ALT is a marker that increases when hepatocytes are damaged together with AST (GOT). However, since most of ALT is contained in hepatocytes, liver disease is suspected. From the results of FIG. 10 and FIG. 12, it was confirmed that ALT had a higher correlation than anti-PD-1 antibody titer in autoimmune liver disease patients compared to IgG. Thus, it was considered that anti-PD-1 antibody was correlated with disease activity. In chronic inflammatory diseases, serum IgG was increased nonspecifically, but it was considered that the antibody titer against inhibitory costimulatory molecules could not be evaluated by measuring serum IgG.

As detailed above, to differentiate drug-induced liver injury (Drug) and primary sclerosing cholangitis (PSC) from autoimmune hepatitis (AIH), which have similar clinical symptoms by the test method of the present invention. I was able to. As for viral hepatitis, it was possible to distinguish between acute hepatitis B (AHB) and chronic hepatitis B (CHB). Furthermore, a significant difference in anti-PD-1 antibody titer was also recognized between Crohn's disease (CD) and ulcerative colitis (UC). Both Crohn's disease and ulcerative colitis are autoimmune enteritis, and the symptoms are similar, but Crohn's disease is a full-thickness inflammatory finding of the gastrointestinal mucosa centered on lymphocyte infiltration. In contrast to non-caseating epithelioid cell granuloma, ulcerative colitis is characterized by a crypt abscess in which neutrophils infiltrate and aggregate in the crypt lumen of the large intestine mucosa. Crohn's disease may cause inflammation throughout the digestive tract. In ulcerative colitis, inflammation occurs only in the large intestine. On the other hand, ulcerative colitis may become cancerous, and the prognosis is different from Crohn's disease. Therefore, the treatment policy and the like are different for these diseases. In contrast to these diseases, which conventionally had to be differentiated by observing tissue samples such as biopsy samples, the method of the present invention is superior in that it can be differentiated using serum samples. Yes.

The above method makes it possible to distinguish various diseases with similar clinical symptoms at a relatively early stage, and at the early stage, the need for more appropriate treatment policy, such as drug selection, dosage determination, and surgical operation. Etc. can be determined. This not only reduces the mental and economic burden on the patient, but can also contribute to the medical economy.

*: P <0.05
**: P <0.01
***: P <0.001
****: P <0.0001

Claims

A method for examining a chronic inflammatory disease, comprising measuring an antibody against an inhibitory costimulatory molecule in a collected biological specimen as a marker.
The inhibitory costimulatory molecule is PD-1 (Programmed Cell Death protein 1) expressed on the surface of T cells and / or B cells, and is measured using an anti-PD-1 antibody as a marker. The test | inspection method of the chronic inflammatory disease of description.
The chronic inhibitory molecule according to claim 1, wherein the inhibitory costimulatory molecule is BTLABT (B 発現 and T lymphocyte attenuator) expressed on the surface of T cells and / or B cells, and the anti-BTLA antibody is measured as a marker. Test method for inflammatory diseases.
The inhibitory costimulatory molecule is CTLA-4A (Cytotoxic T-Lymphocyte Antigen 4) expressed on the surface of T cells and / or B cells, and the anti-CTLA-4 antibody is measured as a marker, 2. The method for examining a chronic inflammatory disease according to 1.
The method for examining a chronic inflammatory disease according to claim 1, wherein an anti-PD-1 antibody and an anti-BTLA antibody are each measured as a marker as an antibody against an inhibitory costimulatory molecule.
6. The method for examining a chronic inflammatory disease according to claim 4 or 5, wherein an anti-CTLA-4 antibody is further measured as an antibody against the inhibitory costimulatory molecule, respectively, as a marker.
The method for examining a chronic inflammatory disease according to any one of claims 1 to 6, wherein the chronic inflammatory disease is an autoimmune disease, a viral chronic disease, or a chronic rejection reaction after transplantation of a biological component.
The chronic inflammatory according to claim 7, wherein the autoimmune disease is any one selected from autoimmune hepatitis, Crohn's disease, ulcerative colitis, primary sclerosing cholangitis and primary biliary cirrhosis. Disease testing method.
The method for examining a chronic inflammatory disease according to claim 7, wherein the viral chronic disease is chronic hepatitis B or chronic hepatitis C.
The method for examining a chronic inflammatory disease according to claim 7, wherein the chronic rejection after the transplantation of biological components is a chronic rejection after transplantation of an organ, tissue and / or cell.
The test for a chronic inflammatory disease according to any one of claims 1, 2, and 5 to 10, wherein the chronic inflammatory disease is a chronic inflammatory disease having a lymphocyte infiltration as a main pathological condition, and includes the following steps: Method:
1) a step of measuring the anti-PD-1 antibody titer present in the collected specimen;
2) A step of determining that the measured anti-PD-1 antibody titer is a chronic inflammatory disease centered on lymphocyte infiltration when the measured anti-PD-1 antibody titer is higher than a cutoff value.
The method for examining a chronic inflammatory disease according to claim 11, further comprising the following steps:
1) a step of further measuring the anti-BTLA antibody titer present in the collected specimen;
2) A step of determining that the measured anti-BTLA antibody titer is a chronic inflammatory disease centered on lymphocyte infiltration when the measured anti-BTLA antibody titer is higher than a cutoff value.
The method for examining a chronic inflammatory disease according to claim 11 or 12, further comprising the following steps:
1) a step of further measuring the anti-CTLA-4 antibody titer present in the collected specimen;
2) A step of determining that the measured anti-CTLA-4 antibody titer is a chronic inflammatory disease centered on lymphocyte infiltration when the measured anti-CTLA-4 antibody titer is higher than a cutoff value.
The test kit used in the test method for chronic inflammatory disease according to any one of claims 1, 2, and 5 to 13, comprising at least a PD-1 antigen and a device for detecting an anti-PD-1 antibody.
Furthermore, the test kit used for the test | inspection method of the chronic inflammatory disease of Claim 14 containing the device for antibody detection with respect to each antigen with BTLA antigen and / or CTLA-4 antigen.