WO2018016607A1 - PROCÉDÉ DE TEST DE MALADIES LIÉES AUX IgG4 - Google Patents

PROCÉDÉ DE TEST DE MALADIES LIÉES AUX IgG4 Download PDF

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WO2018016607A1
WO2018016607A1 PCT/JP2017/026359 JP2017026359W WO2018016607A1 WO 2018016607 A1 WO2018016607 A1 WO 2018016607A1 JP 2017026359 W JP2017026359 W JP 2017026359W WO 2018016607 A1 WO2018016607 A1 WO 2018016607A1
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antibody
laminin
igg4
integrin
related disease
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PCT/JP2017/026359
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English (en)
Japanese (ja)
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雅広 塩川
裕三 児玉
千葉　勉
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国立大学法人京都大学
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Publication of WO2018016607A1 publication Critical patent/WO2018016607A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/067—Pancreatitis or colitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a test method for IgG4-related diseases, a test reagent for testing IgG4-related diseases, a method for evaluating the effect of treatment on IgG4-related diseases, and a method for screening candidate substances for therapeutic agents for IgG4-related diseases. .
  • IgG4-related diseases are disease groups characterized by tissue infiltration or tumor formation of high serum IgG4 levels and IgG4-positive plasma cells.
  • IgG4-related disease is a relatively new disease concept, and the cause is unknown so far.
  • affected organs pancreas, bile duct, lacrimal gland, salivary gland, central nervous system, thyroid gland, lung, liver, gastrointestinal tract, kidney, prostate, retroperitoneum, artery, lymph node, skin, mammary gland and the like are known.
  • Lesions often have features in multiple organs and as systemic disease, but may be single organ lesions.
  • AIP Autoimmune pancreatitis
  • IgG4-related kidney disease IgG4-related lacrimal salivary glanditis and the like are known as diseases classified as IgG4-related diseases.
  • IgG4-related diseases The cause of IgG4-related diseases has not been known so far, and a marker substance that serves as an indicator of IgG4-related diseases has not been known. For this reason, it was not easy to diagnose it as an IgG4-related disease.
  • AIP is characterized by tissue infiltration in the pancreas, it is difficult to distinguish it from pancreatic cancer from the observed image of the tissue, and AIP may be erroneously diagnosed as pancreatic cancer.
  • methods for detecting autoimmune pancreatitis include a method for detecting an antibody that immunologically reacts with HSP10 (Patent Document 1) and a method for detecting an antibody that immunologically reacts with amylase ⁇ 2-A (patent 1).
  • Patent Document 1 a method for detecting an antibody that immunologically reacts with HSP10
  • Patent Document 2 a method for detecting an antibody that immunologically reacts with amylase ⁇ 2-A
  • pathogen of IgG4-related disease can be identified, testing for IgG4-related disease using the pathogen as an index, creating a model animal of IgG4-related disease using the pathogen, and developing a new therapeutic drug targeting the pathogen And so on.
  • pathogenic substances for IgG4-related diseases have not been elucidated.
  • the present inventors have surprisingly clarified that the pathogen of IgG4-related disease is an autoantibody against laminin, and that anti-laminin antibody and anti-integrin antibody in serum are indicators of IgG4-related disease. Based on this finding, the inventors have completed the following invention.
  • a test method for IgG4-related diseases Anti-laminin antibody detection step for detecting an antibody that immunologically reacts with laminin as an indicator of IgG4-related disease, and / or immunological reaction with integrin in a sample as an indicator of IgG4-related disease
  • a method comprising an anti-integrin antibody detection step of detecting an antibody to be detected.
  • the IgG4-related disease is autoimmune pancreatitis
  • the antibody in the anti-laminin antibody detection step is an antibody that immunologically reacts with laminin-511.
  • the IgG4-related disease is autoimmune pancreatitis
  • the antibody in the anti-integrin antibody detection step is an antibody that immunologically reacts with integrin ⁇ 6 ⁇ 1.
  • the IgG4-related disease is IgG4-related kidney disease, The method according to (1), wherein the antibody in the anti-laminin antibody detection step is an antibody that immunologically reacts with laminin-521.
  • the anti-laminin antibody detection step comprises detecting the antibody using laminin as an antigen.
  • the anti-integrin antibody detection step includes detecting the antibody using integrin as an antigen.
  • the specimen is a blood sample.
  • a test method for autoimmune pancreatitis As an index of autoimmune pancreatitis, an anti-laminin antibody detection step for detecting an antibody immunologically reactive with laminin-511 in a sample blood sample, and as an index of autoimmune pancreatitis in a sample blood sample And an anti-integrin antibody detection step of detecting an antibody that immunologically reacts with integrin ⁇ 6 ⁇ 1.
  • a test reagent for testing IgG4-related diseases, including laminin. The IgG4-related disease is autoimmune pancreatitis, The test reagent according to (8), wherein the laminin is laminin-511.
  • the IgG4-related disease is IgG4-related kidney disease, The test reagent according to (8), wherein the laminin is laminin-521.
  • the IgG4-related disease is autoimmune pancreatitis, The test reagent according to (12), wherein the integrin is integrin ⁇ 6 ⁇ 1.
  • a test reagent for autoimmune pancreatitis comprising laminin-511 and integrin ⁇ 6 ⁇ 1 in a form immobilized on a solid phase.
  • a method for evaluating the effect of treatment on an IgG4-related disease An anti-laminin antibody detection step of detecting an antibody that immunologically reacts with laminin in a specimen obtained from a test animal treated for an IgG4-related disease; and / or A method comprising an anti-integrin antibody detection step of detecting an antibody that immunologically reacts with an integrin in a specimen obtained from a test animal treated for an IgG4-related disease.
  • a method for screening candidate substances for therapeutic agents for IgG4-related diseases An anti-laminin antibody detection step for detecting an antibody that immunologically reacts with laminin in a specimen obtained from an animal on which a test substance has been allowed to act; And a selection step of selecting the test substance as a candidate substance for a therapeutic drug for IgG4-related disease when the antibody in the specimen decreases due to the action of the test substance.
  • a method for screening candidate substances for therapeutic agents for IgG4-related diseases An anti-integrin antibody detection step for detecting an antibody that immunologically reacts with an integrin in a sample obtained from an animal on which a test substance has been allowed to act; And a selection step of selecting the test substance as a candidate substance for a therapeutic drug for IgG4-related disease when the antibody in the specimen decreases due to the action of the test substance.
  • a method for producing a non-human model animal of an IgG4-related disease A method comprising at least one of an anti-laminin antibody administration step of administering an antibody that immunologically reacts with laminin to a non-human animal, and a laminin immunization step of immunizing a non-human animal with laminin as an antigen.
  • a method for producing a non-human animal model of an IgG4-related disease A method comprising at least one of an anti-integrin antibody administration step of administering an antibody that immunologically reacts with an integrin to a non-human animal, and an integrin immunization step of immunizing the non-human animal with an integrin as an antigen.
  • a method for obtaining an index of an IgG4-related disease A method comprising an anti-laminin antibody detection step of detecting an antibody immunologically reactive with laminin in a sample, and / or an anti-integrin antibody detection step of detecting an antibody immunologically reactive with integrin in the sample.
  • the IgG4-related disease is autoimmune pancreatitis
  • the method according to (20), wherein the antibody in the anti-laminin antibody detection step is an antibody that immunologically reacts with laminin-511.
  • the IgG4-related disease is autoimmune pancreatitis, The method according to (20) or (21), wherein the antibody in the anti-integrin antibody detection step is an antibody that immunologically reacts with integrin ⁇ 6 ⁇ 1.
  • the IgG4-related disease is IgG4-related kidney disease;
  • the method according to (20), wherein the antibody in the anti-laminin antibody detection step is an antibody that immunologically reacts with laminin-521.
  • the method according to any one of (20) to (23), wherein the anti-laminin antibody detection step comprises detecting the antibody using laminin as an antigen.
  • the IgG4-related disease is autoimmune pancreatitis, The use according to (27), wherein the laminin is laminin-511.
  • the IgG4-related disease is IgG4-related kidney disease, The use according to (27), wherein the laminin is laminin-521.
  • (31) Use of an integrin in the manufacture of a test reagent for testing an IgG4-related disease.
  • the IgG4-related disease is autoimmune pancreatitis, The use according to (31), wherein the integrin is integrin ⁇ 6 ⁇ 1.
  • a method for testing an IgG4-related disease a test reagent for testing an IgG4-related disease, a method for evaluating the effect of a treatment on an IgG4-related disease, a therapeutic agent for an IgG4-related disease, An effective means is provided as a method for screening candidate substances.
  • FIG. 1A shows H & E staining of pancreas 12 hours after IgG administration (upper row) and Gr1 staining (lower row) of control IgG-treated mice (left column) and patient IgG-treated mice (right column) in Experiment 1. ) Result.
  • the scale bar in the observed image shows 50 ⁇ m in the upper stage and 20 ⁇ m in the lower stage.
  • FIG. 1B shows the correlation between the dose of control IgG (triangle) and patient IgG (circle) and the edema area in Experiment 1.
  • FIG. 1A shows H & E staining of pancreas 12 hours after IgG administration (upper row) and Gr1 staining (lower row) of control IgG-treated mice (left column) and patient IgG-treated mice (right column) in Experiment 1.
  • the scale bar in the observed image shows 50 ⁇ m in the upper stage and 20 ⁇ m in the lower stage.
  • FIG. 1B shows the correlation between the dose of
  • FIG. 1C shows the histopathological grade of necrosis or hemorrhage of pancreas 12 hours after IgG administration and the number of Gr1 positive cells in control IgG-treated mice (left) and patient IgG-treated mice (right) in Experiment 1.
  • FIG. 2A shows IgG in the pancreas 12 hours after IgG administration (upper row), IgG1 (middle row), IgG4 of control IgG-treated mice (left column) and patient IgG-treated mice (right column) in Experiment 1.
  • the result of immunohistochemical staining of (lower) is shown.
  • the scale bar in the observed image indicates 20 ⁇ m.
  • FIG. 2B shows the results of immunofluorescence staining of pancreatic tissue in control IgG-treated mice and patient IgG-treated mice in Experiment 1.
  • the scale bar in the observed image indicates 20 ⁇ m.
  • Control 1 in the upper part of FIG. 3A shows pancreatic H & E 12 hours after administration of mice administered with one or both of control IgG1 and control IgG4 obtained from a specific one of the 10 controls (control 1). It is an observation image of dyeing
  • the scale bar in the observed image indicates 20 ⁇ m.
  • FIG. 3A shows 12 hours after administration of a mouse administered with one or both of patient IgG1 and patient IgG4 from one specific patient (patient 9) among 10 IgG4-related disease patients.
  • 2 is an observation image of H & E staining (middle stage) and an observation image of immunohistochemical staining of Gr1 (lower stage). The scale bar in the observed image indicates 20 ⁇ m.
  • FIG. 3B shows pancreatic necrosis or 12 hours after administration of mice that received one or both of control IgG1 and control IgG4 (left) and mice that received one or both of patient IgG1 and patient IgG4 (right).
  • FIG. 3C shows pancreatic human IgG1 or human 12 hours after administration of mice administered with one or both of patient IgG1 and patient IgG4 from one particular patient (patient 1) out of 10 IgG4-related diseases It is an observation image of immunohistochemical staining of IgG4.
  • the scale bar in the observed image indicates 20 ⁇ m.
  • the upper left of FIG. 4A shows an observation image obtained by H & E staining of a pancreatic tissue section of an AIP patient collected from a boundary portion between a site with AIP lesion (left side) and a site without lesion (right side).
  • FIG. 4A shows an observation image of immunohistochemical staining for IgG4 of the same pancreatic tissue section.
  • the scale bar indicates 200 ⁇ m.
  • the lower left of FIG. 4A is an enlarged image of the lesioned acinar portion in the upper right image of FIG. 4A.
  • the lower part of FIG. 4A is an observation image obtained by enlarging the lesioned leaflet gap portion in the upper right part of FIG. 4A.
  • the lower right side of FIG. 4A is an observation image obtained by enlarging a normal acinar portion without a lesion in the upper right image of FIG. 4A.
  • the scale bar indicates 20 ⁇ m.
  • FIG. 4B shows an observation image obtained by immunofluorescent staining of a lesioned acinus in the pancreas of an AIP patient.
  • the scale bar indicates 20 ⁇ m.
  • the upper left of FIG. 4B shows an observed image of IgG4 stained with immunofluorescence in an acinus with lesions in the pancreas of an AIP patient.
  • the upper part of FIG. 4B shows an observation image of amylase stained with immunofluorescence in the same observation area as the upper left part of FIG. 4B.
  • the upper right of FIG. 4B is an image obtained by superimposing the observed image of IgG4 on the upper left of FIG. 4B and the observed image of amylase in the upper part of FIG. 4B.
  • the lower left of FIG. 4B shows an observed image of IgG4 stained with immunofluorescence in an acinus with lesions in the pancreas of an AIP patient.
  • the lower part of FIG. 4B shows an observation image of collagen IV stained with immunofluorescence in the same observation area as the lower left part of FIG. 4B.
  • the lower right of FIG. 4B is an image obtained by superimposing the observed image of IgG4 on the lower left of FIG. 4B and the observed image of collagen IV in the lower part of FIG. 4B.
  • Each image in FIG. 4C shows an observation image obtained by immunofluorescent staining of a lobular space with a lesion in the pancreas of an AIP patient.
  • the scale bar indicates 20 ⁇ m.
  • FIG. 4C shows an observed image of immunofluorescent-stained IgG4 in a lobular space with lesions in the pancreas of an AIP patient.
  • FIG. 4C shows an observation image of collagen IV stained with immunofluorescence in the same observation region as the left of FIG. 4C.
  • the right side of FIG. 4C is an image obtained by superimposing the observed image of IgG4 on the left side of FIG. 4C and the observed image of collagen IV in FIG. 4C.
  • concentration of anti-human laminin-511 active fragment antibody in AIP patient serum and control serum is shown.
  • the concentration of anti-human collagen IV antibody in AIP patient serum and control serum is shown.
  • the concentration of anti-human fibronectin antibody in AIP patient serum and control serum is shown.
  • FIG. 6 shows an observation image of a tissue section of the pancreas stained by H & E staining.
  • FIG. 6 left is an observation image of the pancreas of the control group, and
  • FIG. 6 right is an observation image of the pancreas of the test group immunized with laminin-511.
  • the concentration of anti-human laminin-521 antibody in AIP patient serum and control serum is shown.
  • the left side of FIG. 8 shows an observation image of the kidney of a mouse subcutaneously administered with IgG4 related kidney disease patient IgG
  • the right side of FIG. 8 shows an observation image of human IgG4 staining of the kidney of a mouse subcutaneously administered with control IgG.
  • the upper row shows the human IgG4 staining observation image of the whole kidney, and the lower row shows the human IgG4 staining observation image of the filamentous body.
  • the measurement results of anti-human laminin-511-E8 IgG antibody levels of serum samples from 10 AIP patients and 10 controls in the training group in Experiment 8 are shown. Results of measurement of anti-human laminin-511-E8 IgG antibody levels in serum samples from 41 AIP patients and 102 controls (20 healthy subjects, 82 non-AIP disease patients) in the validation group in Experiment 8 Indicates.
  • the upper part of FIG. 12 is a pancreatic image obtained by contrast CT of an anti-laminin 511-E8 antibody positive AIP patient.
  • FIG. 12 is a pancreatic image obtained by contrast CT of an anti-integrin ⁇ 6 ⁇ 1 antibody-positive AIP patient.
  • the arrow in each image indicates the lesion site.
  • the measurement results of anti-laminin 511-E8 antibody levels in serum before and after treatment with steroid (prednisone) of 5 patients with anti-laminin 511-E8 antibody positive AIP are shown.
  • the IgG4-related disease is a disease group characterized by tissue infiltration or tumor formation of high serum IgG4 and IgG4-positive plasma cells, specifically, autoimmune pancreatitis (AIP), IgG4-related kidney disease, Examples include IgG4-related lacrimal gland salivary gland, IgG4-related sclerosing cholangitis, IgG4-related eye disease, IgG4-related periarteritis, IgG4-related hypophysitis, IgG4-related lung disease, IgG4-related retroperitoneal fibrosis. ⁇ 2.
  • AIP autoimmune pancreatitis
  • IgG4-related kidney disease examples include IgG4-related lacrimal gland salivary gland, IgG4-related sclerosing cholangitis, IgG4-related eye disease, IgG4-related periarteritis, IgG4-related hypophysitis, IgG4-related lung disease, Ig
  • the present invention firstly A test method for IgG4-related diseases, Anti-laminin antibody detection step for detecting an antibody that immunologically reacts with laminin as an indicator of IgG4-related disease, and / or immunological reaction with integrin in a sample as an indicator of IgG4-related disease
  • the present invention relates to a method comprising an anti-integrin antibody detection step of detecting an antibody to be detected.
  • the antibody against laminin is a pathogen of an IgG4-related disease, and the antibody in a specimen obtained from a test animal suffers from an IgG4-related disease. It was completed based on the surprising finding that it is an indicator of what is being done.
  • the binding between laminin and integrin is a target of a pathogen of an IgG4-related disease, and an anti-laminin antibody in a specimen obtained from a test animal and / or It was completed based on the surprising finding that anti-integrin antibodies are indicative of having an IgG4-related disease.
  • test animal targeted by the test method of the present invention is not particularly limited and may be a human or other non-human mammal, but is preferably a human.
  • Specimens used in the test method of the present invention include body fluids collected from test animals. Specifically, in addition to blood samples such as serum, plasma and whole blood, body fluid samples other than blood such as saliva, spinal fluid and urine may be used. Further, not only body fluids but also tissues collected from test animals, for example, tissues collected from disease sites of IgG4-related diseases to be examined (for example, pancreas, kidney, salivary gland, etc.) can be used as specimens. . The specimen is used in the examination method of the present invention in a form separated from the subject animal.
  • Laminin is a huge heterotrimeric molecule consisting of three subunit chains of ⁇ chain, ⁇ chain and ⁇ chain in the natural type. ⁇ 1 to ⁇ 5 are known as ⁇ chains, ⁇ 1 to ⁇ 3 are known as ⁇ chains, and ⁇ 1 to ⁇ 3 are known as ⁇ chains, and combinations thereof include 12 or more isoforms (see Table 1). In the present invention, laminin isoforms are not limited.
  • the laminin to which the antibody to be detected immunologically reacts is selected from one ⁇ chain selected from ⁇ 1 to ⁇ 5, one ⁇ chain selected from ⁇ 1 to ⁇ 3, and ⁇ 1 to ⁇ 3 1
  • It can consist of ⁇ chains of species, specifically, can be any of the 12 types listed in Table 1 or other isoforms, preferably ⁇ chain is ⁇ 5, ⁇ chain is “Laminin-511” in which ⁇ 1 and ⁇ chain are ⁇ 1, and “Laminin-521” in which ⁇ chain is ⁇ 5, ⁇ chain is ⁇ 2, and ⁇ chain is ⁇ 1.
  • the origin of laminin to which the antibody to be detected immunologically reacts is not particularly limited.
  • an IgG4-related disease is usually an autoimmune disease, it must be the same kind of laminin as the test animal.
  • the nucleotide sequence information of the genes encoding the ⁇ chain, ⁇ chain, and ⁇ chain of laminin from mammalian species such as humans and the amino acid sequence information of each chain can be obtained from a known database (GenBank, etc.).
  • Table 2 shows the accession numbers of the chains constituting laminin for main mammals including humans.
  • the laminin having the amino acid sequence shown in Table 2 may be further subjected to post-translational modification to form laminin.
  • the base sequence information and amino acid sequence information of laminin constituent chains of various mammals can also be obtained from known databases (GenBank, etc.).
  • laminin, laminin-511, and laminin-521 are not particularly limited to natural forms in which the ⁇ chain, the ⁇ chain, and the ⁇ chain are all full lengths, respectively. Fragments, fragments of laminin-511, fragments of laminin-521, natural laminin, natural laminin-511, and forms equivalent to natural laminin-521 are included. Therefore, the antibody to be detected may be one that immunologically reacts with a laminin fragment. Examples of the laminin fragment include those in which at least one of the ⁇ chain, ⁇ chain, and ⁇ chain constituting the natural laminin trimer is shorter than the full length.
  • the laminin fragment preferably forms a trimer, and more preferably has integrin binding activity. It can be confirmed that the laminin fragment forms a trimer by subjecting the laminin fragment to SDS-PAGE and detecting the number of bands. It can be confirmed by ELISA or the like that the laminin fragment has integrin binding activity.
  • Examples of the fragment of laminin that forms a trimer and has integrin binding activity include a fragment obtained by digesting natural laminin with a proteolytic enzyme such as elastase, and particularly preferably an E8 fragment.
  • the E8 fragment of laminin is obtained by removing the C-terminal globular (G) domains 4 and 5 from the ⁇ -chain C-terminal fragment ( ⁇ -chain E8), ⁇ -chain C-terminal fragment ( ⁇ -chain E8) and ⁇ -chain.
  • the C-terminal fragment ( ⁇ -chain E8) is a fragment that forms a trimer (Hiroyuki Ido, Aya Nakamura, Reiko Kobayashi, Shunsuke Ito, Shaoliang Li, Sugiko Futaki, and Kiyotoshi Sekiguchi, The Journal of Biological Chemist 11144-11154, 2007).
  • the E8 fragment of laminin can be obtained by enzymatic digestion of full length laminin with pancreatic elastase.
  • An example of a commercially available E8 fragment of laminin is human laminin-511-E8 (892012, manufactured by Nippi Co., Ltd.).
  • Integrin is a heterodimer molecule consisting of two subunit chains of ⁇ chain and ⁇ chain in the natural type. ⁇ 1 to ⁇ 11, ⁇ V, ⁇ X, ⁇ M, ⁇ L, ⁇ D, ⁇ E, ⁇ IIb as ⁇ chains, ⁇ 1 to ⁇ 8 as ⁇ chains, and a plurality of isoforms having different combinations thereof exist.
  • Integrin ⁇ 6 ⁇ 1, integrin ⁇ 3 ⁇ 1, and integrin ⁇ 6 ⁇ 4 are known as integrin isoforms that can be laminin ligands.
  • the integrin isoform is not limited, but is preferably “integrin ⁇ 6 ⁇ 1” in which the ⁇ chain is ⁇ 6 and the ⁇ chain is ⁇ 1.
  • the origin of the integrin to which the antibody to be detected reacts immunologically is not particularly limited.
  • an IgG4-related disease is usually an autoimmune disease, it is an integrin of the same species as the test animal.
  • the nucleotide sequence information of the genes encoding integrin ⁇ chain and ⁇ chain of mammalian species such as humans and the amino acid sequence information of each chain can be obtained from a known database (GenBank, etc.).
  • Table 3 shows the amino acid sequence and base sequence accession number of each chain constituting human integrin. Three splicing variants are known for each of ⁇ 6 chain and ⁇ 1 chain of human integrin.
  • Human integrin having the amino acid sequence shown in Table 3 that has undergone post-translational modification may also form human integrin.
  • the base sequence information and amino acid sequence information of integrin constituent chains of various mammals other than these can also be obtained from known databases (GenBank etc.).
  • the terms integrin, integrin ⁇ 6 ⁇ 1, and integrin ⁇ 3 ⁇ 1 are not particularly limited to forms in which the ⁇ chain and ⁇ chain are each composed of the full length, respectively, and are each an integrin fragment, integrin ⁇ 6 ⁇ 1 fragment, integrin It includes fragments of ⁇ 3 ⁇ 1, natural integrin, natural integrin ⁇ 6 ⁇ 1, and forms equivalent to natural integrin ⁇ 3 ⁇ 1. Therefore, the antibody to be detected may be one that immunologically reacts with the integrin fragment.
  • integrin fragments include those in which at least one of the ⁇ chain and ⁇ chain constituting the integrin dimer is shorter than the full length.
  • the integrin fragment preferably forms a dimer, and more preferably has laminin binding activity.
  • the fact that the integrin fragment forms a dimer can be confirmed by subjecting the integrin fragment to SDS-PAGE and detecting the number of bands. It can be confirmed by ELISA or the like that the integrin fragment has laminin binding activity.
  • Examples of commercially available integrins include recombinant human integrin ⁇ 6 ⁇ 1 (R & D Systems, Minnesota, USA, product number 7809-A6) and recombinant human integrin ⁇ 3 ⁇ 1 (R & D Systems, Minnesota, USA, product number 2840-A3).
  • integrin fragments include Human ITGA6 & ITGB1 (Beijing China, Sino Biological, product number CT013-H2508H-20), which is a commercially available fragment of human integrin ⁇ 6 ⁇ 1.
  • the anti-laminin antibody detection step is a step of detecting an antibody that immunologically reacts with laminin in a sample.
  • the anti-integrin antibody detection step is a step of detecting an antibody that immunologically reacts with the integrin in the specimen.
  • “detection” refers to whether or not there is an antibody that immunologically reacts with laminin or integrin in the sample, and the content of the antibody in the sample is measured. In other words, it is a concept that encompasses “quantitative”.
  • the anti-laminin antibody detection step and the anti-integrin antibody detection step may be any method that can quantitatively or qualitatively detect the antibody in the specimen, and specific embodiments are not particularly limited.
  • immunological techniques can be used, for example, antibody enzyme method (ELISA method), immunoprecipitation method (IPP method), immunoblot method (IB method), latex agglutination method, immunochromatography method, indirect fluorescent antibody Methods (IF method), radioimmunoassay methods (RIA method) and the like can be mentioned.
  • ELISA method antibody enzyme method
  • IPP method immunoprecipitation method
  • IB method immunoblot method
  • latex agglutination method immunochromatography method
  • IF method indirect fluorescent antibody Methods
  • RIA method radioimmunoassay methods
  • An ELISA method that can process many specimens is particularly preferred.
  • laminin or integrin as described in detail above which is the antigen of the antibody
  • the specimen is contacted with the antigen immobilized on the solid phase, and the antigen and specimen
  • the antibody can be detected by detecting an immune complex with the antibody that may be contained therein.
  • the labeling substance that can be used in the detection, the measuring instrument, and the like there are no particular limitations on the method for suppressing the non-specific reaction associated with the ELISA method, the labeling substance that can be used in the detection, the measuring instrument, and the like.
  • a solid phase for immobilizing an antigen a solid phase having an arbitrary shape such as a plate, a bead, or a tube can be used.
  • the amount of the antibody that immunologically reacts with laminin in the anti-laminin antibody detection step, and the amount of the antibody that immunologically reacts with integrin in the anti-integrin antibody detection step is the amount of the immune complex of the antibody and the antigen. It can be determined as a measured value of the label. It is also possible to prepare a calibration curve with a standard sample containing the antibody at a known concentration, and calculate the amount of the antibody from the measured value of the label using the calibration curve.
  • Determination of whether there is an antibody that immunologically reacts with laminin in the anti-laminin antibody detection process (positive / negative determination), and presence of an antibody that reacts immunologically with integrin in the anti-integrin antibody detection process
  • the determination of whether or not to perform is based on the measured value of the antibody in a specimen obtained from an individual animal subject (eg, a human suspected of suffering from an IgG4-related disease). Can be performed by comparing the measured value of the antibody in a sample obtained from an animal individual (for example, a healthy person) who does not suffer from the disease.
  • the measured value of the antibody in the sample obtained from an individual who does not suffer from an IgG4-related disease may be a value obtained by simultaneous measurement or may be a value obtained by measurement in advance. Good.
  • the measured value of the antibody in the specimen obtained from the test animal individual is significantly larger than the measured value of the antibody in the specimen obtained from the animal individual not suffering from IgG4-related disease, it is judged as positive be able to.
  • the class of the antibody that immunologically reacts with laminin in the anti-laminin antibody detection step and the antibody that immunologically reacts with integrin in the anti-integrin antibody detection step are not particularly limited, but are preferably IgG antibodies.
  • the subclass in IgG antibody is not specifically limited, For example, IgG1 antibody or IgG4 antibody can be detected. Since the present inventors have found that IgG antibodies immunologically reactive with laminin, in particular IgG1 antibodies or IgG4 antibodies, are pathogens in IgG4-related diseases, these are particularly useful as indicators of IgG4-related diseases. Useful.
  • the combination of the type of IgG4-related disease to be examined, the type of antibody that immunologically reacts with laminin, or the type of antibody that immunologically reacts with integrin is not particularly limited.
  • an antibody that immunologically reacts with laminin-511 see Experiment 4
  • an antibody that reacts immunologically with integrin ⁇ 6 ⁇ 1 see Experiment 9 are useful.
  • the antibody that immunologically reacts with laminin-511 is particularly preferably detected by a detection method using a fragment of laminin-511 (particularly, a trimer fragment having integrin binding activity) as an antigen.
  • the antibody that reacts immunologically with integrin ⁇ 6 ⁇ 1 is particularly preferably detected by a detection method using integrin ⁇ 6 ⁇ 1 as an antigen.
  • an antibody that immunologically reacts with laminin-521 is useful as an indicator of IgG4-related kidney disease (see Experiment 6).
  • laminin-511 particularly laminin-511-E8
  • AIP autoimmune pancreatitis
  • the anti-laminin antibody detection step of the IgG4-related disease testing method of the present invention can obtain an index for discriminating between AIP and pancreatic cancer, and is particularly useful.
  • an antibody that immunologically reacts with laminin, particularly laminin-511, particularly laminin-511-E8, in a sample If detected, based on the detection result, it can be determined that the test animal from which the specimen is derived has a low risk of combining a malignant tumor with IgG4-related disease, particularly AIP.
  • an antibody that immunologically reacts with integrin particularly integrin ⁇ 6 ⁇ 1
  • an anti-integrin antibody detection step based on the detection result, It can be determined that the test animal from which the specimen is derived suffers from an IgG4-related disease, particularly AIP.
  • an embodiment including the anti-integrin antibody detection step of the IgG4-related disease test method of the present invention comprises: This is particularly useful because an index for discriminating between AIP and pancreatic cancer can be obtained.
  • an anti-integrin antibody detection step if an antibody immunologically reactive with integrin, particularly integrin ⁇ 6 ⁇ 1, is detected in the sample, the detection result Based on this, it can be determined that the test animal from which the specimen is derived has a high risk of combining malignant tumors with IgG4-related diseases, particularly AIP. It is possible to determine that it is highly necessary to screen a malignant tumor throughout the body for a test animal that has been determined to have a high risk of complication of a malignant tumor.
  • the method for testing an IgG4-related disease of the present invention may include at least one of an anti-laminin antibody detection step and an anti-integrin antibody detection step, and more preferably both an anti-laminin antibody detection step and an anti-integrin antibody detection step.
  • An anti-laminin antibody-negative sample may be anti-integrin antibody-positive
  • an anti-integrin antibody-negative sample may be anti-laminin antibody-positive.
  • Test reagent for IgG4-related disease > The present invention secondly, A test reagent for testing IgG4-related diseases, including laminin, Test reagents for testing IgG4-related diseases, including integrins, or The present invention relates to a test reagent for testing an IgG4-related disease, including laminin and integrin.
  • IgG4-related diseases e.g., laminin, and integrin are as described above for the test method of the present invention.
  • test reagent of the present invention containing laminin can be used in the above-described test method of the present invention including an anti-laminin antibody detection step.
  • test reagent of the present invention containing integrin can be used in the above-described test method of the present invention including an anti-integrin antibody detection step.
  • test reagent of the present invention may contain reagents necessary for immunological measurement, for example, a buffer solution as necessary, and may be provided as a kit comprising a plurality of components.
  • the form of laminin is not particularly limited, and may be a solution state, a dry state, or a form fixed to a solid phase. May be.
  • a buffer solution or a solvent for making it into a solution state before use may be included in the test reagent of the present invention.
  • the form of integrin is not particularly limited, and may be in a solution state, a dry state, or a form fixed on a solid phase. May be.
  • a buffer solution or a solvent for making it into a solution state before use may be included in the test reagent of the present invention.
  • test reagent of the present invention containing laminin in a form immobilized on a solid phase can be in the form of a kit for testing an IgG4-related disease by ELISA.
  • An embodiment of the test reagent of the present invention containing the integrin in a form immobilized on a solid phase can also be in the form of a kit for testing an IgG4-related disease by ELISA.
  • An embodiment of the test reagent of the present invention comprising laminin and integrin in a form immobilized on a solid phase can also be in the form of a kit for testing IgG4-related diseases by ELISA.
  • the form of the solid phase may be any form such as a plate, a bead, or a tube.
  • a kit for examining an IgG4-related disease by ELISA can contain a blocking solution, a washing solution, a sample diluent, an enzyme-labeled secondary antibody, a substrate solution, and the like. ⁇ 4.
  • the present invention A method for assessing the effect of treatment on an IgG4-related disease comprising: An anti-laminin antibody detection step of detecting an antibody that immunologically reacts with laminin in a specimen obtained from a test animal treated for an IgG4-related disease; and / or The present invention relates to a method comprising an anti-integrin antibody detection step of detecting an antibody that immunologically reacts with an integrin in a specimen obtained from a test animal treated for an IgG4-related disease.
  • the above-mentioned “method for evaluating the effect of treatment on an IgG4-related disease” may be expressed as “a method for obtaining an index for evaluating the effect of treatment on an IgG4-related disease”.
  • the antibody that reacts immunologically with laminin and the antibody that reacts immunologically with integrin in the specimen are useful as indicators of IgG4-related diseases. For this reason, the antibody in the specimen obtained from the test animal treated for IgG4-related disease is reduced when the treatment is effective and cured, and when the treatment is not sufficiently effective It remains unchanged or rises from before the start of treatment. Thus, in a sample obtained from a test animal treated for IgG4-related disease. By detecting an antibody immunologically reactive with laminin and / or an antibody immunologically reactive with integrin, it is possible to evaluate the effect of treatment.
  • test animals include humans and non-human animals that have been treated for IgG4-related diseases.
  • IgG4-related diseases includes treatment of IgG4-related diseases such as steroid administration.
  • Laminin in a specimen obtained from a test animal treated with an IgG4-related disease obtained in the anti-laminin antibody detection step and / or anti-integrin antibody detection step in the method for evaluating therapeutic effect of the present invention immunologically Quantitative results of antibodies that react immunologically with reacting antibodies and / or integrins, for example, antibodies that react immunologically with laminin in a specimen obtained from the same animal individual prior to the start of the treatment and / or When the former is smaller than the latter compared with the quantitative result of the antibody that immunologically reacts with integrin, it can be evaluated that the treatment is effective. On the other hand, if the former is about the same as or larger than the latter, it can be evaluated that the treatment is not effective or sufficient. Based on the evaluation result, it is possible to determine whether to continue the treatment, change the treatment policy, or stop the treatment.
  • laminin and immunology in a specimen obtained from a test animal treated with an IgG4-related disease obtained in the anti-laminin antibody detection step and / or anti-integrin antibody detection step in the method for evaluating therapeutic effect of the present invention Quantification results of antibodies that react immunologically and / or antibodies that react immunologically with integrins, for example, antibodies and / or integrins that react immunologically with laminin in specimens obtained from healthy individuals of the same animal species If the former is comparable or smaller than the latter compared to the quantification result of the immunologically reactive antibody, it can be evaluated that the treatment is effective. On the other hand, if the former is larger than the latter, it can be evaluated that the treatment is not effective or sufficient.
  • Method 1 for Screening Candidate Substances for Treatment of IgG4-Related Disease comprising: An anti-laminin antibody detection step for detecting an antibody that immunologically reacts with laminin in a specimen obtained from an animal on which a test substance has been allowed to act; And a selection step of selecting the test substance as a candidate substance for a therapeutic drug for IgG4-related disease when the antibody in the specimen decreases due to the action of the test substance.
  • the antibody that immunologically reacts with laminin in the sample is useful as an indicator of IgG4-related diseases.
  • the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases.
  • the test substance is a test substance that may be a candidate for a new drug, and is not particularly limited.
  • the animal to which the test substance is allowed to act is typically an animal having a large amount of an antibody that immunologically reacts with laminin in a specimen, such as a non-human animal model of an IgG4-related disease. is there.
  • the quantification result of the antibody in the specimen obtained from the animal to which the test substance is applied obtained in the anti-laminin antibody detection step, for example,
  • the former is smaller than the latter compared with the quantification result of the antibody in the specimen obtained from the same animal individual before the test substance is allowed to act,
  • the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases.
  • the quantification result of the antibody in the specimen obtained from the animal to which the test substance is applied, obtained in the anti-laminin antibody detection step is obtained.
  • the former is comparable or smaller than the latter in comparison with the quantification result of the antibody in a sample obtained from a healthy individual of the same animal species, Judging that the antibody in the sample has decreased, the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases. ⁇ 6.
  • the present invention A method for screening candidate substances for therapeutic agents for IgG4-related diseases, comprising: An anti-integrin antibody detection step for detecting an antibody that immunologically reacts with an integrin in a sample obtained from an animal on which a test substance has been allowed to act; And a selection step of selecting the test substance as a candidate substance for a therapeutic drug for IgG4-related disease when the antibody in the specimen decreases due to the action of the test substance.
  • the antibody that immunologically reacts with the integrin in the sample is useful as an indicator of IgG4-related diseases. For this reason, when the antibody in the sample decreases due to the action of a test substance, the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases.
  • the test substance is not particularly limited and is a test substance that may be a candidate for a new drug.
  • the animal to which the test substance is allowed to act is typically an animal having a large amount of an antibody that immunologically reacts with integrin compared to a healthy individual, such as a non-human animal model of an IgG4-related disease. is there.
  • the quantification result of the antibody in the specimen obtained from the animal to which the test substance is applied obtained in the anti-integrin antibody detection step, for example,
  • the former is smaller than the latter compared with the quantification result of the antibody in the specimen obtained from the same animal individual before the test substance is allowed to act,
  • the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases.
  • the quantification result of the antibody in the specimen obtained from the animal to which the test substance is applied, obtained in the anti-integrin antibody detection step is obtained.
  • the former is comparable or smaller than the latter in comparison with the quantification result of the antibody in a sample obtained from a healthy individual of the same animal species, Judging that the antibody in the sample has decreased, the test substance can be selected as a candidate substance for a therapeutic drug for IgG4-related diseases. ⁇ 7.
  • Method 1 for producing non-human animal model of IgG4-related disease Sixth, the present invention A method for producing a non-human animal model of an IgG4-related disease comprising: The present invention relates to a method comprising at least one of an anti-laminin antibody administration step of administering an antibody that immunologically reacts with laminin to a non-human animal, and a laminin immunization step of immunizing a non-human animal with laminin as an antigen.
  • laminin, antibody, etc. in the embodiment including the anti-laminin antibody administration step or laminin immunization step of the method for producing a non-human model animal of the present invention include the anti-laminin antibody detection step of the test method of the present invention. This is the same as in the embodiment.
  • Experiment 1 and Experiment 2 described in this specification it has been confirmed that when a serum obtained from an IgG4-related disease patient or an antibody purified from the serum is administered to the mouse, the mouse develops an IgG4-related disease.
  • serum obtained from a patient with an IgG4-related disease contains an antibody that immunologically reacts with laminin. From this, it can be said that a non-human model animal of an IgG4-related disease can be produced by an anti-laminin antibody administration step in which an antibody that immunologically reacts with laminin is administered to the non-human animal.
  • non-human animals in the anti-laminin antibody administration step include mice such as BALB / c mice and B6 mice, and other non-human animals.
  • the administration route of the antibody that immunologically reacts with laminin is not particularly limited, and examples thereof include subcutaneous administration, intraperitoneal administration, and intravenous administration.
  • An antibody that immunologically reacts with laminin need not be administered to a non-human animal as a purified antibody, and may be administered to a non-human animal as serum containing the antibody, for example.
  • the antibody that immunologically reacts with laminin is selected so that it can immunologically react with laminin of the non-human animal to be administered.
  • a human-derived anti-human laminin antibody can immunologically react with laminin possessed by mice, and can cause lesions of IgG4-related diseases in mice.
  • non-human animals in the laminin immunization process include mice such as BALB / c mice and B6 mice, and other non-human animals.
  • the method in the laminin immunization process is not particularly limited.
  • a non-human animal can be immunized by administering laminin together with a suitable adjuvant such as complete Freund's adjuvant to the non-human animal by a route such as subcutaneous administration, intravenous administration, or intraperitoneal administration.
  • laminin to be administered as an antigen is selected so that an antibody against it can immunologically respond to laminin possessed by the non-human animal to be immunized by autoimmunity.
  • human laminin-511 can react immunologically with mouse antibodies against mouse laminin, and can cause lesions of IgG4-related diseases in mice. ⁇ 8.
  • a method for producing a non-human animal model of an IgG4-related disease comprising: The present invention relates to a method comprising at least one of an anti-integrin antibody administration step of administering an antibody that immunologically reacts with an integrin to a non-human animal, and an integrin immunization step of immunizing the non-human animal with an integrin as an antigen.
  • the specific form of integrin, antibody, etc. includes the anti-integrin antibody detection step of the test method of the present invention. This is the same as in the embodiment.
  • non-human animals in the anti-integrin antibody administration step include mice such as BALB / c mice and B6 mice, and other non-human animals.
  • the administration route of the antibody that immunologically reacts with the integrin in the anti-integrin antibody administration step is not particularly limited, and examples thereof include subcutaneous administration, intraperitoneal administration, and intravenous administration.
  • the antibody that immunologically reacts with the integrin need not be administered to a non-human animal as a purified antibody, and may be administered to a non-human animal as serum containing the antibody, for example.
  • the antibody that immunologically reacts with the integrin is selected so that it can react immunologically with the integrin of the non-human animal to be administered.
  • a human-derived anti-human integrin antibody (particularly, a human-derived anti-human integrin ⁇ 6 ⁇ 1 antibody) can immunologically react with an integrin possessed by a mouse, and can cause a lesion of an IgG4-related disease in the mouse.
  • non-human animals in the integrin immunization step include mice such as BALB / c mice and B6 mice, and other non-human animals.
  • the method in the integrin immunization process is not particularly limited.
  • a non-human animal can be immunized by administering the integrin together with a suitable adjuvant such as complete Freund's adjuvant to the non-human animal by a route such as subcutaneous administration, intravenous administration, or intraperitoneal administration.
  • the integrin to be administered as an antigen is selected so that the antibody against it can react immunologically with the integrin of the non-human animal to be immunized by autoimmunity.
  • human integrins can react immunologically with mouse integrins by autoimmunity with mouse antibodies against them, and can cause lesions of IgG4-related diseases in mice.
  • Experiment 1 IgG administration of IgG4-related disease patients to mice>
  • IgG purified from the serum of an IgG4-related disease patient was administered to healthy newborn mice, and the effect on the pancreas after administration was observed. As a result, it was revealed that IgG purified from the serum of patients with IgG4-related diseases showed pathogenicity to the pancreas.
  • Serum serum samples were obtained from 10 patients diagnosed with IgG4-related disease.
  • the concentration of purified IgG was measured using a Human IgG EIA Kit (MK136, manufactured by Takara).
  • the purity of the IgG fraction was determined using Human IgA ELISA Kit (E88-102, manufactured by Ethyl Laboratories), Human IgM ELISA Kit (manufactured by E88-100, manufactured by Ethyl Laboratores), Human IgE ELISA 108-L (manufactured by Human IgE ELISA-Lit).
  • the contamination of IgA, IgM, IgE and protein was determined and confirmed using Coomassie brilliant blue staining, respectively.
  • IgG obtained from a serum sample of an IgG4-related disease patient was designated as “patient IgG”.
  • the IgG obtained from the control serum sample was designated as “control IgG”.
  • Animal test Male newborns of BALB / c mice were used as mice.
  • IgG prepared from each serum sample was administered subcutaneously to mice. 10-20 mg of human IgG was administered to 1 g of mouse body weight. 1.4. Results and discussion 1 (FIGS. 1A to 1C) Twelve hours after subcutaneous administration of patient IgG or control IgG, mouse pancreas tissue sections were collected, stained by H & E (hematoxylin & eosin) staining and immunohistochemical staining, and observed. In immunohistochemical staining, Gr1-positive cells were stained using an antibody against bone marrow cell differentiation antigen Gr1 as a primary antibody.
  • the human IgG concentration in the mouse serum was measured 12 hours after subcutaneous administration.
  • FIG. 1A shows the results of H & E staining of pancreas 12 hours after IgG administration (upper row) and Gr1 staining (lower row) of control IgG-treated mice (left column) and patient IgG-treated mice (right column).
  • the scale bar in the observed image shows 50 ⁇ m in the upper stage and 20 ⁇ m in the lower stage.
  • FIG. 1B shows the correlation between the dose of control IgG (triangle) and patient IgG (circle) and the edema area.
  • the cutoff value (21.1%) in the figure is a value obtained by adding a value twice the standard deviation (SD) to the average value of the edema area in the control IgG or PBS-administered mice.
  • FIG. 1C shows the histopathological grade of pancreatic necrosis or hemorrhage 12 hours after IgG administration and the number of Gr1-positive cells in control IgG-treated mice (left) and patient IgG-treated mice (right) ( * p ⁇ 0.05, ** p ⁇ 0.005: according to Paired Student's test).
  • FIG. 1B there was no significant difference in serum human IgG concentrations between patient IgG-administered mice and control IgG-administered mice (19.4 mg / ml and 21.4 mg / ml).
  • pancreas was damaged by administration of patient IgG to mice, and the damage was maximized 12 hours after administration as shown in FIG. 1A.
  • Administration of control IgG to mice did not cause pancreatic damage 12 hours after administration, as shown in FIG. 1A.
  • mouse pancreas tissue sections were collected, stained by immunohistochemical staining, and observed.
  • immunohistochemical staining anti-human IgG antibody, anti-human IgG1 antibody or anti-human IgG4 antibody was used as a primary antibody, and human IgG, human IgG1 or human IgG4 was stained by a conventional method.
  • mouse pancreas tissue section was stained by immunofluorescence staining and observed with a fluorescence microscope.
  • immunofluorescence staining human IgG, amylase or collagen IV was stained by a conventional method using a rabbit anti-human IgG antibody, rabbit anti-amylase antibody or rabbit anti-collagen IV antibody conjugated with fluorescein isocyanate.
  • FIG. 2A shows immunization of IgG (upper), IgG1 (middle), and IgG4 (lower) in pancreas 12 hours after IgG administration in control IgG-treated mice (left column) and patient IgG-treated mice (right column). The results of histochemical staining are shown. The scale bar in the observed image indicates 20 ⁇ m.
  • FIG. 2B shows the results of immunofluorescence staining of pancreatic tissue of control IgG-administered mice and patient IgG-administered mice.
  • the scale bar in the observed image indicates 20 ⁇ m.
  • FIG. 2B shows an observed image of immunofluorescent-stained human IgG in the pancreatic tissue of a control IgG-treated mouse. Human IgG was not observed in the mouse pancreas.
  • the second from the left in the upper part of FIG. 2B shows an observation image of human IgG stained with immunofluorescence in the pancreatic tissue of a patient IgG-administered mouse.
  • the third from the left in the upper part of FIG. 2B shows an observation image of amylase stained with immunofluorescence in the same observation region as that in the second from the left in the upper part of FIG.
  • the right end of the upper stage of FIG. 2B is an image obtained by superimposing the above observed image of human IgG and the above observed image of amylase.
  • the first from the left in the lower part of FIG. 2B shows an observation image of human IgG stained with immunofluorescence in the pancreatic tissue of a patient IgG-administered mouse.
  • the second from the left in the lower part of FIG. 2B shows an observation image of collagen IV stained with immunofluorescence in the same observation area as the first from the left in the lower part of FIG. .
  • the lower right end of FIG. 2B is an image obtained by superimposing the observed image of the above human IgG and the observed image of the above collagen IV.
  • IgG and IgG4 were observed to accumulate in the pancreatic acinar base and pancreatic tissue lobule gap in all patient IgG-treated mice, whereas in control IgG-treated mice pancreas IgG and IgG4 were not observed.
  • IgG1 was observed in 6 out of 10 mice administered with patient IgG, but IgG1 was not observed in mice administered with control IgG.
  • IgG subclasses IgG1 and IgG4 were purified from sera of patients with IgG4-related diseases and administered to healthy newborn mice, and the effect on the pancreas after administration was observed.
  • IgG1 and IgG4 purified from the serum of patients with IgG4-related diseases show pathogenicity to the pancreas, IgG1 pathogenicity is greater than that of IgG4, and administered in combination with IgG1 and IgG4 Then, it was confirmed that the pathogenicity of IgG1 was suppressed.
  • IgG1 and IgG4 were purified from the serum samples of the patients with IgG4-related diseases and the control serum samples obtained.
  • Capture Select IgG1 (Hu) affinity matrix (191303005, manufactured by Invitrogen) and Capture Select IgG4 (Hu) affinity matrix (manufactured by Invitrogen, manufactured by Invitrogen) were used.
  • the fraction of the eluted subclass (IgG1 or IgG4) was concentrated by ultrafiltration using Amicon Ultra (UFC805024, manufactured by Millipore) while thoroughly washing with PBS (pH 7.2).
  • the concentration of the purified IgG subclass was determined by using the Human IgG1 Platinum ELISA (BMS2092, eBioscience), Human IgG2 Platinum ELISA (BMS2093, eBioscience), Human IgG3 Platinum ELISA (BMS2094, eBioScientium BMS2094, eBioSc4).
  • Product was used and quantified according to the product instructions. The purity of the subclass was confirmed by determining the contamination of IgA, IgM, IgE, other IgG subclasses and proteins by the same procedure as described above.
  • IgG1 and IgG4 obtained from serum samples of patients with IgG4-related diseases were designated as “patient IgG1” and “patient IgG4”, respectively.
  • IgG1 and IgG4 obtained from the control serum samples were designated as “control IgG1” and “control IgG4”, respectively.
  • IgG1 and IgG4 prepared from each serum sample was administered subcutaneously to mice.
  • IgG1 is administered in an amount of 10 mg / mouse body weight of 1 g.
  • IgG4 is administered in an amount of 1 mg / mouse body weight of 1 g
  • IgG1 and IgG4 Were administered in an amount of 1 mg / mouse weight of 1 g of IgG4.
  • Control 1 in the upper part of FIG. 3A shows pancreatic H & E 12 hours after administration of mice administered with one or both of control IgG1 and control IgG4 obtained from a specific one of the 10 controls (control 1). It is an observation image of dyeing
  • “Patient 9” in the middle and bottom of FIG. 3A shows 12 hours of administration of a mouse administered with one or both of patient IgG1 and patient IgG4 obtained from one specific patient (patient 9) among 10 IgG4-related disease patients. It is the observation image (lower stage) of H & E staining of the pancreas after, and the observation image (lower stage) of immunohistochemical staining of Gr1. The scale bar in the observed image indicates 20 ⁇ m.
  • FIG. 3B shows pancreatic necrosis or 12 hours after administration of mice that received one or both of control IgG1 and control IgG4 (left) and mice that received one or both of patient IgG1 and patient IgG4 (right).
  • the histopathological grade of bleeding and the number of Gr1 positive cells are shown ( * p ⁇ 0.05: according to Paired Student's test).
  • both patient IgG1 and patient IgG4 are pathogenic to the pancreas, the pathogenicity of patient IgG1 is greater than the pathogenicity of patient IgG4, and patient IgG1 when administered together with patient IgG1 and patient IgG4 It was confirmed that the pathogenicity of was suppressed.
  • FIG. 3C shows pancreatic human IgG1 or human 12 hours after administration of mice administered with one or both of patient IgG1 and patient IgG4 from one particular patient (patient 1) out of 10 IgG4-related diseases It is an observation image of immunohistochemical staining of IgG4.
  • the scale bar in the observed image indicates 20 ⁇ m.
  • Human IgG1 staining intensity of pancreas in mice administered with both patient IgG1 and patient IgG4 (upper right of FIG. 3C) is greater than human IgG1 staining intensity of pancreas in mice administered only with patient IgG1 (upper left of FIG. 3C) It was confirmed to be small.
  • the human IgG4 staining intensity of the pancreas in the mouse administered with both patient IgG1 and patient IgG4 is the human IgG4 staining intensity of the pancreas in the mouse administered only with patient IgG4 (upper in FIG. 3C) It was comparable.
  • mice administered with one or both of patient IgG1 and patient IgG4 edema formation, acinar necrosis, hemorrhage and infiltration of Gr1-positive polymorphonuclear leukocytes were observed in the pancreas.
  • no abnormality was observed in the pancreas in mice administered with one or both of control IgG1 and control IgG4.
  • IgG1 and IgG4 are pathogenic, and IgG1 is more pathogenic than IgG4.
  • the pathogenicity of IgG1 was markedly suppressed when IgG4 was administered simultaneously, suggesting that IgG4 inhibits the action of IgG1 (FIG. 3B).
  • the amount of IgG1 staining in mice administered only with IgG1 is significantly greater than when IgG1 and IgG4 are administered in combination (FIG. 3C).
  • the amount of IgG4 staining in mice administered with IgG4 alone was similar to that obtained when IgG1 and IgG4 were administered in combination (FIG. 3C).
  • FIG. 4A shows an observation image obtained by H & E staining of a pancreatic tissue section of an AIP patient collected from a boundary portion between a site with AIP lesion (left side) and a site without lesion (right side).
  • the upper right of FIG. 4A shows an observation image of immunohistochemical staining for IgG4 of the same pancreatic tissue section.
  • the scale bar indicates 200 ⁇ m.
  • the lower left of FIG. 4A is an enlarged image of the lesioned acinar portion in the upper right image of FIG. 4A.
  • the lower part of FIG. 4A is an observation image obtained by enlarging the lesioned leaflet gap portion in the upper right part of FIG. 4A.
  • the lower right side of FIG. 4A is an observation image obtained by enlarging a normal acinar portion without a lesion in the upper right image of FIG. 4A.
  • the scale bar indicates 20 ⁇ m.
  • IgG4 positive plasma cells were stained, and IgG4 was linearly stained at the base of the acinus and the lobule space. On the other hand, such staining was not observed in the pancreatic tissue without lesion.
  • FIG. 4B shows an observation image obtained by immunofluorescent staining of an acinus having a lesion in the pancreas of an AIP patient.
  • the scale bar indicates 20 ⁇ m.
  • FIG. 4B upper left, shows an observed image of IgG4 stained with immunofluorescence in the acinus of the AIP patient with a lesion in the pancreas.
  • the upper part of FIG. 4B shows an observation image of amylase stained with immunofluorescence in the same observation area as the upper left part of FIG. 4B.
  • the upper right of FIG. 4B is an image obtained by superimposing the observed image of IgG4 on the upper left of FIG. 4B and the observed image of amylase in the upper part of FIG. 4B. From this result, it can be seen that IgG4 does not coexist with amylase and exists at different sites.
  • FIG. 4B lower left, shows an observed image of immunofluorescent-stained IgG4 in an acinus having a lesion in the pancreas of an AIP patient.
  • the lower part of FIG. 4B shows an observation image of collagen IV stained with immunofluorescence in the same observation area as the lower left part of FIG. 4B.
  • the lower right of FIG. 4B is an image obtained by superimposing the observed image of IgG4 on the lower left of FIG. 4B and the observed image of collagen IV in the lower part of FIG. 4B.
  • a portion where red IgG4 and green collagen IV overlap is shown in yellow. From this result, it can be seen that IgG4 is present at the base of the acinar together with collagen IV.
  • FIG. 4C shows an observation image obtained by immunofluorescent staining of a lobular space with a lesion in the pancreas of an AIP patient.
  • the scale bar indicates 20 ⁇ m.
  • FIG. 4C left shows an observed image of IgG4 stained with immunofluorescence in the lobular space with lesions in the pancreas of an AIP patient.
  • FIG. 4C shows an observation image of collagen IV stained with immunofluorescence in the same observation region as the left of FIG. 4C.
  • the right side of FIG. 4C is an image obtained by superimposing the observed image of IgG4 on the left side of FIG. 4C and the observed image of collagen IV in FIG. 4C.
  • On the right side of FIG. 4C a portion where red IgG4 and green collagen IV overlap is shown in yellow. From this result, it can be seen that IgG4 exists in the extracellular matrix expressing collagen IV.
  • the present inventors speculated that the antibody contained in the serum of the AIP patient recognizes an extracellular matrix protein expressed in the pancreas as an antigen.
  • the inventors of the present invention individually fixed an extracellular matrix protein expressed in the pancreas known from the literature as an antigen candidate on a solid phase, and in the serum of an AIP patient, Whether or not an antibody that binds to an extracellular matrix protein was contained was confirmed by ELISA (Enzyme-Linked Immunosorbent Assay) method.
  • Human laminin-511-E8 (892012, manufactured by Nippi Co., Ltd.) (Laminin-511 active fragment) Human collagen IV (ab7536, abcam) Human fibronectin GFP (green fluorescent protein) (MB-0752, VECTOR LABORATORIES) Human Notch1 (ab68580, abcam) Human Annexin A2 (ab93005, abcam) Human SDF2L1 (TP313102, OriGene Technologies) Human Bile salt-activated lipidase (ab167963, abcam) 4.2. Serum samples Serum samples were obtained from 9 patients diagnosed with AIP.
  • ELISA coating buffer (ELISA Coating Buffer), ELISA wash solution (ELISA Wash Solution), ELISA blocking buffer (ELISA Blocking Buffer), and conjugate diluent (Conjugate Diluent) were all prepared according to the instructions of the kit.
  • HRP-conjugated secondary antibody (1) Secondary antibody conjugated with HRP (horseradish peroxidase) (abcam6759, rabbit anti-human IgG H & L (HRP), polyclonal) is conjugated diluent of the kit (Conjugate Diluent) Diluted 1: 2000.
  • Enzyme substrate reaction (1) A TMB (3,3 ′, 5,5′-tetramethylbenzidine) solution was prepared according to the manufacturer's recommended conditions.
  • FIG. 5A anti-human laminin-511 active fragment antibody
  • FIG. 5B anti-human collagen IV antibody
  • FIG. 5C anti-human fibronectin antibody
  • FIG. 5D anti-GFP antibody
  • n 40 for the AIP patient serum sample and for the control serum sample.
  • the vertical axis represents the relative absorbance at 450 nm, which is proportional to the serum concentration of the antibody against each antigen candidate.
  • the cut-off value is a value obtained by adding a value twice the standard deviation (SD) to the average value of the serum concentration of the antibody against each antigen candidate in the control serum sample. When the serum concentration of the antibody against the antigen candidate is higher than the cutoff value, it can be determined that the antibody against the antigen candidate is positive.
  • anti-human laminin-511 antibody was positive in 20 of 40 cases in AIP patient serum, whereas no positive case was found in 40 cases in control serum. This confirms that AIP can be detected using the serum concentration of anti-human laminin-511 antibody as an index. Further, in one of the AIP patient sera positive for anti-human laminin-511 antibody, the blood IgG concentration and blood IgG4 concentration were normal values with no substantial difference from the control serum. The method of discriminating AIP using the serum concentration of ⁇ 511 antibody as an index may be able to discriminate AIP with higher sensitivity than the method using blood IgG concentration and blood IgG4 concentration as an index.
  • mice were treated in the same procedure and schedule except that 120 ⁇ g of ovalbumin (OVA) emulsified with CFA was used instead of the emulsion of laminin-511-E8.
  • OVA ovalbumin
  • Sample sera are blood serum (pre-immune serum) collected from mice prior to the first dose and blood serum collected after sacrifice. Sample serum was stored at ⁇ 80 ° C. until analysis. Tissue sections of mouse pancreas were collected 28 days after the third immunization and stained with H & E staining for observation.
  • FIG. 6 shows an observation image of a tissue section of the pancreas stained by H & E staining.
  • FIG. 6 left is an observation image of a control group
  • FIG. 6 right is an observation image of a test group immunized with a human laminin-511 active fragment (i511 laminin).
  • i511 laminin a human laminin-511 active fragment
  • Human recombinant laminin 521 (BLA-LN521-02, Bio Lamina) was used as human laminin-521.
  • IgG4-related kidney disease patient IgG The serum concentration of anti-human laminin-521 antibody may be effective as an indicator of discrimination for IgG4-related renal diseases.
  • Experiment 7 Administration of IgG4 Related Kidney Disease Patient IgG to Mice> IgG was prepared from the serum of an IgG4-related renal disease patient who was confirmed to be positive for anti-human laminin-521 antibody in Experiment 6 by the same procedure as in Experiment 1. This IgG was designated as “IgG4-related kidney disease patient IgG”.
  • IgG was prepared from the serum of an IgG4-related disease patient who has no kidney disease by the same procedure as in Experiment 1. This IgG was designated as “IgG4 related disease patient IgG without renal disease”.
  • IgG4-related kidney disease patient IgG, control IgG, and IgG4-related disease patient IgG without kidney disease were each subcutaneously administered to male newborns of BALB / c mice in the same procedure as in Experiment 1.
  • mice kidneys were collected in the same manner as in Experiment 1, and human IgG4 was stained and observed by immunohistochemical staining.
  • FIG. 8 shows the observation image of the kidney of the mouse subcutaneously administered with IgG4 related kidney disease patient IgG on the left side of FIG. 8, and shows the human IgG4 staining observation image of the kidney of the mouse subcutaneously administered with the control IgG on the right side of FIG.
  • the upper row shows the human IgG4 staining observation image of the whole kidney
  • the lower row shows the human IgG4 staining observation image of the filamentous body.
  • Pierson syndrome is caused by a genetic abnormality of laminin ⁇ 2 chain.
  • Pierson's syndrome includes renal failure (mesangial proliferating filamentous nephritis), neuropathy (muscular weakness), and miosis.
  • Anti-human laminin-521 antibody-positive IgG4-related kidney disease patients who have obtained the above IgG4-related kidney disease patient IgG have been confirmed to have mesangial proliferative fibronephritis by renal biopsy, and muscle weakness has also been confirmed. Therefore, Pierson's syndrome matches symptoms and pathology.
  • this IgG4-related renal disease patient has an autoantibody against laminin ⁇ 2 chain, which is presumed to cause symptoms of IgG4-related renal disease.
  • Experiment 8 Measurement of anti-laminin 511-E8 autoantibodies in serum of AIP patients> 8.1. Samples Serum samples from 51 in-treatment AIP patients who met the diagnostic criteria for IgG4-RD 2011 or the diagnostic criteria for AIP were obtained. Control serum samples were obtained from 25 healthy individuals and 86 disease patients (49 histologically confirmed cancer patients and 39 non-cancer disease patients). Serum samples from 10 of 51 AIP patients and serum samples from 10 of 112 controls were used as training groups, and the rest were used as validation groups.
  • Human laminin-511-E8 was diluted in the ELISA coating buffer of the kit to obtain a 2 ⁇ g / ml concentration solution, and this solution was added to the microtiter plate of the kit so as to be 100 ⁇ l per well. Incubate for time to coat. The plate was washed five times with 0.05% Tween 20-containing Tris buffered saline (washing solution), then coated with 1% bovine serum albumin-containing Tris buffered saline, and 100 ⁇ L of 1:20 diluted serum. And incubated at room temperature for 30 minutes.
  • the plate After washing 5 times with the washing solution, the plate was incubated with 100 ⁇ L of goat anti-human IgG antibody conjugated with HRP (horseradish peroxidase) (1: 4000; ab6759, Abcam) for 1 hour at room temperature. . After washing with the washing solution three times, the plate was incubated with TMB (3,3 ′, 5,5′-tetramethylbenzidine) for 3 minutes, and the absorbance at 450 nm was measured to quantify the amount of bound substance. Control wells not coated with human laminin-511-E8 were used as negative controls in the ELISA for each serum sample.
  • HRP horseradish peroxidase
  • the cut-off value (optical density unit [OD]) is a value obtained by adding three times the standard deviation (SD) to the mean value of anti-human laminin-511-E8 IgG antibody level in the serum sample from the control. is there. Serum samples from 6 of 10 AIP patients were positive for IgG antibodies to human laminin-511-E8, whereas all 10 controls had IgG antibodies to human laminin-511-E8. Negative.
  • FIG. 9B shows the measurement results of anti-human laminin-511-E8 IgG antibody levels of serum samples from 41 AIP patients in the validation group and 102 controls (20 healthy subjects, 82 disease patients other than AIP). Shown in The cut-off value (optical density unit [OD]) is a value obtained by adding 3 times the standard deviation (SD) to the mean value of anti-human laminin-511-E8 IgG antibody level in the serum sample from the control. is there. Serum samples from 20 of 41 AIP patients had anti-human laminin-511-E8 IgG antibody (positive), whereas only 2 out of 102 controls Anti-human laminin-511-E8 IgG antibody was positive.
  • SD standard deviation
  • the IgG antibody positive against human laminin-511-E8 was 26/51 (51.0%) in the serum of AIP patients, whereas it was 2 in the control serum. / 112 (1.8%) (P ⁇ 0.001).
  • integrin ⁇ 6 ⁇ As the integrin ⁇ 6 ⁇ 1, recombinant human integrin ⁇ 6 ⁇ 1 (R & D Systems, Minnesota, USA, product number 7809-A6) was used.
  • FIG. 10 shows the measurement results of the anti-integrin ⁇ 3 ⁇ 1 IgG antibody level, respectively.
  • the cut-off value (optical density unit [OD]) is the mean value of anti-integrin ⁇ 6 ⁇ 1 IgG antibody level or anti-integrin ⁇ 3 ⁇ 1 IgG antibody level in the serum sample from the control, which is 3 times the standard deviation (SD). It is the added value.
  • AIP patients were divided into 26 anti-laminin 511-E8 antibody positive AIP patients (anti-integrin ⁇ 6 ⁇ 1 antibody negative) and 4 anti-integrin ⁇ 6 ⁇ 1 antibody positive AIP patients (anti-laminin 511-E8 antibody negative).
  • the 21 anti-laminin 511-E8 antibody negative anti-integrin ⁇ 6 ⁇ 1 antibody negative AIP patients were classified, and the clinical features of each group of AIP patients are summarized in Table 5 below. There were no subjects who were positive for both anti-laminin 511-E8 antibody and anti-integrin ⁇ 6 ⁇ 1 antibody.
  • pancreatic images Comparison of pancreatic images>
  • the upper part of FIG. 12 is a pancreatic image by contrast-enhanced CT of an anti-laminin 511-E8 antibody positive AIP patient.
  • the lower part of FIG. 12 is a pancreatic image obtained by contrast CT of an anti-integrin ⁇ 6 ⁇ 1 antibody-positive AIP patient.
  • the arrow in each image indicates the lesion site.
  • FIG. 13 shows the serum anti-laminin 511-E8 antibody levels of five anti-laminin 511-E8 antibody positive AIP patients before and after steroid treatment.
  • Steroid treatment reduced the anti-laminin 511-E8 antibody titer in the serum of each AIP patient below the cut-off value.
  • pancreatic imaging findings also improved with the reduction of the anti-laminin 511-E8 antibody titer in the serum of each AIP patient. This confirms that anti-laminin 511-E8 antibody in serum is also useful as an indicator of the effect of AIP treatment.

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Abstract

La présente invention vise à trouver une substance pathogène responsable d'une maladie liée aux IgG et à fournir un procédé de test d'une maladie liée aux IgG4 et autres à l'aide de la substance pathogène. La présente invention concerne par conséquent un procédé de test d'une maladie liée aux IgG4, ledit procédé comprenant une étape de détection d'anticorps anti-laminine consistant à détecter, en tant qu'indicateur d'une maladie liée aux IgG4, un anticorps capable de réagir immunologiquement avec la laminine dans un échantillon et/ou une étape de détection d'anticorps anti-intégrine consistant à détecter, en tant qu'indicateur d'une maladie liée aux IgG4, un anticorps capable de réagir immunologiquement avec l'intégrine dans l'échantillon. La présente invention concerne également : un réactif de test destiné à être utilisé dans le test d'une maladie liée aux IgG 4, qui contient de la laminine; et un réactif de test destiné à être utilisé dans le test d'une maladie liée aux IgG 4, qui contient de l'intégrine.
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JP2002514660A (ja) * 1998-05-13 2002-05-21 ジェネンテック・インコーポレーテッド 肝疾患の診断及び治療
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JP2012050369A (ja) * 2010-08-31 2012-03-15 Kanazawa Medical Univ ＩｇＧ４関連疾患診断用マーカー及びその利用
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WO2002042769A1 (fr) * 2000-11-22 2002-05-30 Matsuura, Eiji Procede de detection d'un anticorps anti-laminine-1 et sa mise en application
JP2012050369A (ja) * 2010-08-31 2012-03-15 Kanazawa Medical Univ ＩｇＧ４関連疾患診断用マーカー及びその利用
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