WO2012086346A1 - Méthode de diagnostic pour des maladies inflammatoires chroniques à l'aide d'un anticorps contre des molécules co-stimulatrices inhibitrices en tant que marqueur - Google Patents

Méthode de diagnostic pour des maladies inflammatoires chroniques à l'aide d'un anticorps contre des molécules co-stimulatrices inhibitrices en tant que marqueur Download PDF

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WO2012086346A1
WO2012086346A1 PCT/JP2011/076507 JP2011076507W WO2012086346A1 WO 2012086346 A1 WO2012086346 A1 WO 2012086346A1 JP 2011076507 W JP2011076507 W JP 2011076507W WO 2012086346 A1 WO2012086346 A1 WO 2012086346A1
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antibody
chronic inflammatory
chronic
inflammatory disease
disease
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PCT/JP2011/076507
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Japanese (ja)
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康広 三宅
和秀 山本
松本　和幸
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国立大学法人 岡山大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis

Definitions

  • 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.
  • an antibody against an inhibitory costimulatory molecule 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.
  • CRP C-reactive protein
  • SAA amyloid A protein
  • 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.
  • various disease-specific markers autoantibodies
  • Non-patent Document 1 Non-patent Document 1
  • 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.
  • 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.
  • 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.
  • T cell activation plays a central role in the pathological state.
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • 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).
  • 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.
  • inhibitory costimulatory molecules such as CTLA-4 (Cytotoxic T lymphocyte antigen 4) and PD-1 is observed.
  • Patent Documents 1 and 2 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).
  • TLA selective co-stimulatory agent gene recombination including CTLA-4 part and modified Fc region derived from human IgG1
  • 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).
  • 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.
  • 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.
  • 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.
  • BTLA B and T lymphocyte attenuator
  • 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
  • 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).
  • the chronic inflammatory 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 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 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.
  • 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.
  • 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).
  • FDA drug-induced liver injury
  • PSC primary sclerosing cholangitis
  • AIH autoimmune hepatitis
  • viral hepatitis it was possible to distinguish between acute hepatitis B (AHB) and chronic hepatitis B (CHB).
  • Crohn's disease Crohn's disease
  • ulcerative colitis Crohn's disease
  • 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.
  • 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.
  • ulcerative colitis inflammation occurs only in the large intestine.
  • 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.
  • 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.
  • Example 2 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 It is a figure which shows the correlation of the serum IgG level in an autoimmune hepatitis patient, and an anti- PD-1 antibody tit
  • 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.
  • the “suppressive costimulatory molecule” is an inhibitory costimulatory molecule that can be expressed on the surface of T cells and / or B cells.
  • PD-1 Programmed death 1
  • CTLA- 4 Cytotoxic T lymphocyte antigen 4
  • BTLA B and T lymphocyte attenuator
  • 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.
  • 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.
  • using these antibodies as markers does not necessarily mean that these antibody titers are higher than the cutoff value.
  • these antibody titers are higher than the cutoff value.
  • 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.
  • 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.
  • 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 (hem
  • 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).
  • GVHD chronic graft-versus-host disease
  • 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).
  • inflammatory skin diseases such as lichen planus, pemphigus, bullous pemphigoid, epidermolysis bullosa, alopecia areata.
  • anti-PD-1 antibody for example, anti-CTLA-4 antibody and anti-BTLA antibody, particularly preferably anti-PD-1 antibody and / or anti-BTLA antibody are listed above.
  • 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.
  • autoimmune hepatitis and primary sclerosing cholangitis one of which is autoimmune disease, autoimmune hepatitis And drug-induced liver injury.
  • 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.
  • 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.
  • 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.
  • CRP C-reactive protein
  • SAA amyloid A protein
  • antinuclear antibody etc. It is possible to perform inspection more effectively.
  • 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.
  • 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.
  • 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 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 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.
  • 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.
  • 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.
  • Crohn's disease and ulcerative colitis it was confirmed that Crohn's disease has higher antibody titer and positive rate.
  • 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.
  • methods for detecting antibodies against inhibitory costimulatory molecules can be applied per se and are not particularly limited.
  • enzyme-linked immunosorbent assay enzyme-linked immunoassay: ELISA
  • radioimmunoassay radioimmunoassay
  • agglutination method turbidimetric method
  • turbidity measurement method Western blot method
  • Western blot method immunochromatography, etc.
  • 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.
  • a BTLA antigen and / or CTLA-4 antigen and a device for detecting these antibodies are included.
  • 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.
  • 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 ⁇ l of 1 ⁇ g / 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 ⁇ l of 1% bovine serum albumin (BSA). 3) 100 ⁇ l 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.
  • BSA-PBST 0.02% Tween-20
  • the anti-PD-1 antibody titer in each patient serum is shown in FIG. 2 and Table 2 below.
  • ulcerative colitis is 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.
  • 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.
  • inflammation occurs only in the large intestine.
  • 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.
  • PSL prednisolone
  • 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.
  • 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.
  • 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.
  • autoimmune hepatitis patients showed higher anti-BTLA antibody titers.
  • anti-BTLA antibody titers tended to be low.
  • the positive rate of anti-BTLA antibody based on the cut-off value was clearly higher in Crohn's disease.
  • 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.
  • 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 ⁇ g / 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.
  • mice anti-PD-1 antibody H00005133-A01: Avnova, Taiwan
  • an antigen-antibody reaction of PD-1 antigen and mouse anti-PD-1 antibody was caused.
  • an HRP-labeled anti-mouse IgG antibody RPN2124: GE Healthcare, UK
  • RPN2132 GE Healthcare, UK
  • Example 6 Relationship between anti-PD-1 antibody titer measured by ELISA and various diseases
  • anti-PD-1 antibody titer measured by ELISA described in Example 1 The sensitivity and specificity for were examined.
  • the sensitivity and specificity are as shown in Table 5 and can be distinguished from normal subjects.
  • Example 7 Relationship between anti-CTLA-4 antibody titer measured by ELISA and various diseases
  • anti-CTLA-4 antibody titer measured by ELISA described in Example 3 The sensitivity and specificity for were examined.
  • the sensitivity and specificity when the anti-CTLA-4 antibody titer is higher than the cut-off value 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
  • the anti-BTLA antibody titer measured by ELISA described in Example 4 and the sensitivity and specificity for each disease I examined the degree.
  • the sensitivity and specificity when the anti-BTLA antibody titer is higher than the cut-off value 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)
  • the case where each was greater than or equal to the cut-off value was (+) was (+)
  • autoimmune hepatitis and drug-induced liver injury were differentiated by combining various markers. .
  • Table 8 it was confirmed that the anti-PD-1 antibody showed excellent specificity by measurement.
  • both the anti-PD-1 antibody and the anti-CTLA-4 antibody are (+)
  • both the anti-PD-1 antibody and the anti-BTLA antibody are (+)
  • high specificity is exhibited. It was confirmed.
  • the anti-PD-1 antibody or anti-CTLA-4 antibody is (+)
  • the sensitivity is slightly better. However, the specificity was not high.
  • 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.
  • 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.
  • 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.
  • 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 (+).
  • Example 13 Diagnosis of pathological condition in autoimmune liver patient
  • anti-PD-1 antibody titer measured by ELISA described in Example 1 and alanine oxoglutarate aminotransferase (Alanine Aminotransferase: ALT, GPT).
  • ALT is a marker that increases when hepatocytes are damaged together with AST (GOT).
  • GAT AST
  • ALT 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.
  • anti-PD-1 antibody was correlated with disease activity.
  • 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.
  • ulcerative colitis is 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.
  • 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.
  • inflammation occurs only in the large intestine.
  • 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.
  • 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.

Abstract

L'invention concerne une méthode de diagnostic pour des maladies inflammatoires chroniques, dont la caractéristique principale est une infiltration lymphocytaire. Spécifiquement, l'invention concerne une méthode de diagnostic qui est apte à distinguer des maladies inflammatoires chroniques, dont la caractéristique principale est une infiltration lymphocytaire, d'une inflammation aiguë et de maladies inflammatoires, dont la caractéristique principale n'est pas une infiltration lymphocytaire, ladite distinction étant impossible par des marqueurs classiques d'inflammation. La méthode de diagnostic est permise par la mesure d'un anticorps dirigé contre des molécules co-stimulatrices inhibitrices (telles que PD-1, BTLA ou CTLA-4) exprimées dans des lymphocytes T et/ou des lymphocytes B. Cette méthode de diagnostic permet la distinction de maladies inflammatoires chroniques, dont la caractéristique principale est une infiltration lymphocytaire, d'une inflammation aiguë et de maladies inflammatoires, dont la caractéristique principale n'est pas une infiltration lymphocytaire. Une différence remarquable peut être reconnue entre des maladies qui ont des caractéristiques cliniques similaires par la mesure de l'anticorps, si bien que la distinction entre de telles maladies devient possible.
PCT/JP2011/076507 2010-12-22 2011-11-17 Méthode de diagnostic pour des maladies inflammatoires chroniques à l'aide d'un anticorps contre des molécules co-stimulatrices inhibitrices en tant que marqueur WO2012086346A1 (fr)

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MOTOKO KOTANI: "Jiko Men'eki Shikkan no Hassho ni Okeru Hojo Signal no Yakuwari", MOL.MED., vol. 39, no. 10, October 2002 (2002-10-01), pages 1142 - 1148 *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10344090B2 (en) 2013-12-12 2019-07-09 Shanghai Hangrui Pharmaceutical Co., Ltd. PD-1 antibody, antigen-binding fragment thereof, and medical application thereof
US11365255B2 (en) 2013-12-12 2022-06-21 Suzhou Suncadia Biopharmaceuticals Co., Ltd. PD-1 antibody, antigen-binding fragment thereof, and medical application thereof
WO2020171158A1 (fr) * 2019-02-20 2020-08-27 株式会社パートナーファーム Puce de réaction en phase solide et procédé de mesure l'utilisant
JPWO2020171158A1 (ja) * 2019-02-20 2021-12-23 株式会社パートナーファーム 固相反応チップ及びこれを用いた測定方法
JP7205940B2 (ja) 2019-02-20 2023-01-17 株式会社パートナーファーム 固相反応チップ及びこれを用いた測定方法

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