WO2019163838A1 - Marker for multiple sclerosis - Google Patents

Marker for multiple sclerosis Download PDF

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WO2019163838A1
WO2019163838A1 PCT/JP2019/006365 JP2019006365W WO2019163838A1 WO 2019163838 A1 WO2019163838 A1 WO 2019163838A1 JP 2019006365 W JP2019006365 W JP 2019006365W WO 2019163838 A1 WO2019163838 A1 WO 2019163838A1
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ptprz
isoform
secreted
subject
multiple sclerosis
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PCT/JP2019/006365
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French (fr)
Japanese (ja)
Inventor
しのぶ 北爪
香子 作田
谷口　直之
橋本　康弘
たかし 本多
齋藤　清
正純 藤井
Original Assignee
国立研究開発法人理化学研究所
公立大学法人福島県立医科大学
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Priority to JP2020501003A priority Critical patent/JP7240679B2/en
Publication of WO2019163838A1 publication Critical patent/WO2019163838A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a test method for demyelinating diseases (particularly, multiple sclerosis), and more specifically, receptor protein tyrosine phosphatase Z (Protein Tyrosine Phosphatase Receptor-type Z; PTPRZ) in a sample derived from a subject, More preferably, the present invention relates to a test method for multiple sclerosis, which comprises the step of measuring branched O-mannose sugar chain-modified PTPRZ.
  • PTPRZ receptor protein tyrosine phosphatase Z
  • MS Multiple sclerosis
  • RRMS relapsing-remitting MS
  • PPMS primary progressive MS
  • SPMS secondary progressive MS
  • RRMS is characterized by alternating symptom worsening (recurrence) and symptom stability (remission), with repeated periods of remission and relapse for months to years.
  • PPMS is characterized in that there is a temporary stagnation period in which the disease does not progress, but the disease progresses gradually without remission.
  • SPMS is characterized in that recurrence and remission are repeated in the early stage of onset, but progresses slowly. At present, no radical cure has been established for multiple sclerosis, and symptomatic treatment is performed according to the time and symptoms of the patient.
  • Non-patent Document 7 One of the factors that make it difficult to treat multiple sclerosis is the difficulty of diagnosis. Multiple sclerosis is difficult to diagnose by a doctor because there are various symptoms and no biomarkers specific to the disease have been found. In particular, when the lesion is isolated, it is difficult to distinguish it from a brain tumor or encephalitis / encephalopathy. Currently, multiple sclerosis is diagnosed by MRI and cerebrospinal fluid, but there are only a limited number of facilities where MRI can be performed, and cerebrospinal fluid is not specific for multiple sclerosis. In some cases, it is possible to make a definitive diagnosis of multiple sclerosis only after eliminating the possibility of other diseases by trying a tissue biopsy of an affected area with risk (Non-patent Document 7).
  • Non-patent Document 2 Receptor type protein tyrosine phosphatases are classified into eight subfamilies based on structural similarity (Non-patent Document 2). Its extracellular region is composed of various domains such as immunoglobulin-like (Ig) domain, fibronectin-like (FN) domain, carbonic anhydrase-like (CAH) domain, and Meprin-A5-PTP ⁇ (MAM) domain. There are one or two tyrosine phosphatase (PTP) domains in the cell.
  • Ig immunoglobulin-like
  • FN fibronectin-like
  • CAH carbonic anhydrase-like
  • MAM Meprin-A5-PTP ⁇
  • Receptor-type protein tyrosine phosphatase Z belongs to the R5 subfamily.
  • splicing variants in mammalian PTPRZ for example, in mice, there are three isoforms (the receptor forms PTPRZ-A and PTPRZ-B, and the secreted form PTPRZ-S).
  • PTPRZ is composed of an extracellular CAH domain, an FN type III-like domain, a chondroitin sulfate chain-binding region (rich in Ser-Gly / Gly-Ser), and two intracellular PTP domains (D1 and D2). .
  • PTPRZ has sulfated polysaccharide modifications called sulfated glycosaminoglycans such as chondroitin sulfate and keratan sulfate in the extracellular region. All isoforms are expressed as chondroitin sulfate proteoglycans in brain tissue, which is the main expression site of PTPRZ (Non-Patent Documents 3 to 5).
  • PTPRZ is expressed on both neurons and glial cells (astrocytes and oligodendrocytes) in the central nervous system.
  • glial cells astrocytes and oligodendrocytes
  • PTPRZ is N-acetylglucosamine transferase GnT-IX (MGAT5B, Mannosyl (Alpha-1,6-))-Glycoprotein Beta-1,6-N-Acetyl-Glucosamineyltransferase, Isozyme Have been reported to undergo branched O-mannose modification (Non-patent Document 6).
  • An object of the present invention is to provide a novel test method for demyelinating diseases (particularly multiple sclerosis).
  • the present invention has been completed by further research based on such findings. That is, the present invention is as follows.
  • a method for testing multiple sclerosis comprising a step of measuring secretory receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, wherein the secretory PTPRZ in the sample derived from the subject If the measured amount is low compared to the amount of secreted PTPRZ in the control sample, the subject is shown to be multiple sclerosis, and the secreted PTPRZ is a secreted PTPRZ isoform. 1. A method, wherein either or both of 1 and secreted PTPRZ isoform 2.
  • a diagnostic kit for multiple sclerosis comprising either or both of an antibody specifically recognizing secretory PTPRZ and an antibody specifically recognizing a branched O-mannose sugar chain, the secretion
  • a method for examining a demyelinating state comprising a step of measuring secretory receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, wherein the amount of secreted PTPRZ in the sample derived from the subject Is lower than the amount of secreted PTPRZ in the sample of the control group, it is indicated that the subject is demyelinating, and the secreted PTPRZ is secreted PTPRZ isoform 1. And secreted PTPRZ isoform 2 or both.
  • PTPRZ secretory receptor protein tyrosine phosphatase Z
  • a method for examining a demyelinating state comprising the step of measuring branched O-mannose sugar chain-modified receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, When the measured value of the amount of branched O-mannose sugar chain-modified PTPRZ in the sample is lower than the amount of branched O-mannose sugar chain-modified PTPRZ in the sample of the control group, the subject is demyelinated
  • the branched O-mannose sugar chain-modified PTPRZ is divided into branched O-mannose sugar chain-modified PTPRZ isoform 1 and branched O-mannose sugar chain-modified PTPRZ isoform 2.
  • the present invention is as follows.
  • a test method for multiple sclerosis comprising a step of measuring receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, wherein the amount of secreted PTPRZ in the sample derived from the subject A method wherein the subject is shown to have multiple sclerosis when the measured value is low compared to the amount of PTPRZ in the sample of the control group.
  • PTPRZ receptor protein tyrosine phosphatase Z
  • the method according to [1A] wherein the PTPRZ is a branched O-mannose sugar chain-modified PTPRZ.
  • [3A] The method according to [1A] or [2A], wherein the sample derived from the subject is cerebrospinal fluid or blood.
  • [4A] The measurement of PTPRZ in spinal fluid derived from a subject is performed using an antibody that specifically recognizes the protein or an antibody that specifically recognizes a branched O-mannose sugar chain [1A] to [3A].
  • PTPRZ is any one or more selected from the group consisting of secreted PTPRZ isoform 1, secreted PTPRZ isoform 2, and secreted PTPRZ isoform 3, [1A] to [1A] 4A].
  • a diagnostic kit for multiple sclerosis comprising either or both of an antibody that specifically recognizes PTPRZ and an antibody that specifically recognizes a branched O-mannose sugar chain.
  • [7A] A method for examining a demyelination state, comprising measuring receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, and measuring the amount of secreted PTPRZ in the sample derived from the subject A method wherein the subject is shown to be demyelinated if the value is low compared to the amount of PTPRZ in the control group sample.
  • PTPRZ receptor protein tyrosine phosphatase Z
  • a test method for a demyelinating state comprising a step of measuring branched O-mannose sugar chain-modified receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, When the measured value of the amount of branched O-mannose sugar chain-modified PTPRZ in the sample is lower than the amount of branched O-mannose sugar chain-modified PTPRZ in the sample of the control group, the subject is demyelinated A method that is shown to be causing.
  • PTPRZ branched O-mannose sugar chain-modified receptor protein tyrosine phosphatase Z
  • the present invention provides a new objective index useful for the definitive diagnosis of multiple sclerosis.
  • FIG. 2 shows the results of immunohistochemical staining using Cat-315 antibody or anti-PTPRZ antibody in a multiple sclerosis patient brain section.
  • Test method for multiple sclerosis includes the step of measuring either or both of secretory receptor protein tyrosine phosphatase Z (PTPRZ) isoforms 1 and 2 in a sample derived from a subject. (Hereinafter sometimes referred to as “the inspection method of the present invention”).
  • PTPRZ secretory receptor protein tyrosine phosphatase Z
  • the multiple sclerosis that can be examined for the presence or absence of disease by the examination method of the present invention can be classified into three types as described above. According to the present invention, any of relapsing-remitting type (RRMS), primary progressive type (PPMS), and (3) secondary progressive type (SPMS) can be examined.
  • RRMS relapsing-remitting type
  • PPMS primary progressive type
  • SPMS secondary progressive type
  • the content of either or both of secreted PTPRZ isoforms 1 and 2 in a sample derived from a subject is measured as an index, and compared with the corresponding value of the control group
  • the presence or absence of multiple sclerosis in the subject can be examined.
  • PTPRZ has three types of splicing variants with different extracellular region lengths in humans. From the longest, isoform 1 (SEQ ID NO: 1), isoform 2 (SEQ ID NO: 2), isoform 3 (sequence) No. 3).
  • secreted (or also referred to as “free”) PTPRZ which is produced by cleaving the extracellular region of each isoform with a protease, is known.
  • the test method of the present invention the content of the secreted (free) PTPRZ isoform 1 and / or 2 in the sample derived from the subject is measured, and the control group (healthy person or multiple sclerosis is affected). The subject can be examined for the presence or absence of multiple sclerosis in the subject.
  • human isoforms of PTPRZ and their secreted forms will be described in detail.
  • secreted (free) PTPRZ refers to a polypeptide fragment derived from the extracellular region of each isoform in human PTPRZ.
  • PTPRZ has a site sensitive to proteases on the C-terminal side of the extracellular region. This site is cleaved by the decomposition of a protease such as a metalloprotease and becomes secreted (free) PTPRZ.
  • a “secreted” PTPR isoform can be rephrased as a “free” PTPRZ isoform.
  • PTPRZ isoform 1 consists of 2315 amino acids shown in SEQ ID NO: 1.
  • PTPRZ isoform 1 has a signal peptide at positions 1-24, a CA domain at positions 45-298, an FN domain at positions 313-401, a transmembrane domain at positions 1637-1662, and a D1 domain at positions 1717-1992 And has a D2 domain at positions 2023-2282.
  • the CS binding region corresponds to a region in which a CS chain is bonded between the FN domain and the transmembrane domain.
  • the extracellular region of PTPRZ isoform 1 consists of the amino acid sequence shown by SEQ ID NO: 4 corresponding to positions 25 to 1636 in the amino acid sequence shown by SEQ ID NO: 1.
  • the full-length or partial fragment polypeptide of SEQ ID NO: 4 corresponds to secreted PTPRZ isoform 1.
  • PTPRZ isoform 2 consists of 2308 amino acid residues shown in SEQ ID NO: 2.
  • PTPRZ isoform 2 is an isoform in which 7 amino acid residues corresponding to positions 1723 to 1729 of the D1 domain in PTPRZ isoform 1 are deleted. Therefore, the extracellular region of isoform 2 is identical to that of isoform 1 and consists of the amino acid sequence shown in SEQ ID NO: 4. Therefore, the full-length or partial fragment polypeptide of SEQ ID NO: 4 corresponds to secreted PTPRZ isoform 2.
  • PTPRZ isoform 3 consists of 1448 amino acid residues shown in SEQ ID NO: 3.
  • PTPRZ isoform 3 is an isoform in which 860 amino acid residues at positions 755 to 1614 and 7 amino acid residues corresponding to positions 1723 to 1729 are deleted, which can correspond to most of the CS binding region in PTPRZ isoform 1. is there. Therefore, the extracellular region of isoform 3 is shorter than that of isoforms 1 and 2, and consists of amino acid sequences corresponding to positions 25 to 754 and 1615 to 1636 in the amino acid sequence shown in SEQ ID NO: 1.
  • long-form of about 350 kDa derived from the extracellular domain of PTPRZ isoform 1 and PTPRZ isoform 2
  • a short-form of about 180 kDa derived from the extracellular domain of PTPRZ isoform 3.
  • long-form and its fragment polypeptide
  • the actual amino acid sequence to be detected is a full-length polypeptide of the amino acid sequence shown in SEQ ID NO: 4 (1612 amino acids) and / or a partial peptide comprising a part thereof.
  • Examples of such a partial peptide include a peptide containing 1129 or more consecutive amino acids, a peptide containing 1209 or more consecutive amino acids, and a sequence of 1290 or more amino acids.
  • Peptide, peptide containing a continuous amino acid sequence of 1371 amino acids or more, peptide containing a continuous amino acid sequence of 1451 amino acids or more, peptide containing a continuous amino acid sequence of 1532 amino acids or more, peptide containing a continuous amino acid sequence of 1580 amino acids or more examples of the peptide include a continuous amino acid sequence of 1596 amino acids or more, but are not limited thereto.
  • the content of branched O-mannose in a sample derived from a subject is measured, and compared with the corresponding value of a control group, thereby allowing multiple occurrences in the subject.
  • the presence or absence of sclerosis can be examined.
  • PTPRZ undergoes branched O-mannose modification via N-acetylglucosamine transferase GnT-IX in response to demyelination stimuli. Therefore, the content of branched O-mannose in the sample derived from the subject is considered to reflect the abundance of secreted PTPRZ isoforms 1 and 2. Therefore, the presence or absence of multiple sclerosis can also be detected by measuring the content of branched O-mannose in the sample derived from the subject and comparing it with the corresponding value of the control group.
  • any method may be used as a method for measuring secretory PTPRZ isoforms 1 and 2 in a sample derived from a subject as long as the amount of the protein can be quantified.
  • Examples include, but are not limited to, mass spectrometry, chromatography methods (liquid chromatography or gas chromatography, etc.), electrophoresis methods, immunoassay methods using antibodies, and the like.
  • the immunoassay is simple and preferable.
  • immunoassay methods there are sandwich methods, competition methods, agglutination methods, Western blotting methods, and the like when classified based on reaction formats, and radioimmunoassay, fluorescent immunoassay, enzyme immunoassay (EIA) when classified based on labels.
  • Biotin immunoassay and the like but are not limited thereto.
  • the antibody used for the immunoassay may be either a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferable from the viewpoint of reproducibility.
  • a commercially available antibody may be used, and secreted PTPRZ isoform 1 and / or 2 or branched O-mannose sugar chain-modified PTPRZ isoform by a method known per se
  • An antibody that specifically recognizes 1 and / or 2 may be prepared and used.
  • antibody in the present specification is a concept including “antigen-binding fragment”.
  • the “antigen-binding fragment” means an antibody fragment that maintains the binding property (antigen-antibody reactivity) of the original antibody to the corresponding antigen.
  • antigen-binding fragments include, but are not limited to, Fab, F (ab ′) 2 , scFv and the like.
  • Such an antigen-binding fragment can be prepared by a method known per se.
  • Examples of commercially available antibodies used in the test method of the present invention include, but are not limited to, mouse monoclonal Cat-315 IgM (Millipore) and mouse anti-PTPRZ IgM ⁇ (122.2) (Santa cruz biotechnology).
  • an antibody is "specifically recognizes" an antigen, K D values for binding affinity of the antibody for antigen in the antigen-antibody reaction, 1 ⁇ 10 -5 M or less (preferably, 1 ⁇ 10 ⁇ 6 M or less, 1 ⁇ 10 ⁇ 7 M or less, 1 ⁇ 10 ⁇ 8 M or less, more preferably 1 ⁇ 10 ⁇ 9 M or less, most preferably 1 ⁇ 10 ⁇ 10 M or less).
  • Examples of the antibody used for the measurement of secretory PTPRZ isoform 1 and / or 2 include the following antibodies: (1) An antibody that specifically recognizes secreted PTPRZ isoform 1 (2) An antibody that specifically recognizes secreted PTPRZ isoform 2 (3) An antibody that specifically recognizes secreted PTPRZ isoform 1 and 2 .
  • PTPRZ is modified with a branched O-mannose sugar chain by GnT-IX which is a glycosyltransferase in vivo. As will be described in detail in the following examples, the present inventors have revealed that secreted PTPRZ isoforms 1 and 2 in cerebrospinal fluid are also modified with branched O-mannose sugar chains. .
  • an antibody such as a Cat-315 antibody that specifically recognizes a branched O-mannose sugar chain on PTPRZ can also be used in the test method of the present invention.
  • These antibodies can be prepared by a method known per se, or commercially available antibodies may be used. From such a viewpoint, the test method of the present invention can also be defined as follows: branched O-mannose sugar chain-modified receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject.
  • a test method for multiple sclerosis comprising a step of measuring, wherein the measured value of the amount of branched O-mannose sugar chain-modified PTPRZ in a sample derived from the subject is the branched type O in the sample of the control group
  • the amount of secreted PTPRZ isoform 1 and / or 2 in the sample derived from the subject is measured.
  • cerebrospinal fluid is used as a sample.
  • cerebrospinal fluid is known to flow into veins, blood can also be used.
  • the sample from the subject is cerebrospinal fluid.
  • the measured value of the secreted PTPRZ isoform 1 and / or 2 in the sample derived from the subject is significantly lower than the amount of the protein in the sample of the control group, multiple occurrences occur. Shown to be sclerosis.
  • a cutoff value is set in advance, and the measured value of the amount of secreted PTPRZ isoform 1 and / or 2 in the sample derived from the subject is compared. Also good. If the measured value is lower than the cutoff value, it indicates that the subject suffers from multiple sclerosis.
  • the person skilled in the art can set the cutoff value using a method known per se. For example, multiple samples are obtained from a known group of patients and a control group (a healthy person or a subject not suffering from multiple sclerosis) and the amount of secreted PTPRZ isoform 1 and / or 2 is measured and desirable A value that can distinguish both groups by sensitivity and / or specificity may be set as a cutoff value.
  • mammals such as humans, monkeys, rats, dogs, cats and the like are exemplified, and humans are particularly preferable.
  • the present invention also includes an antibody that specifically recognizes one or both of secretory PTPRZ isoforms 1 and 2, and an antibody that specifically recognizes a branched O-mannose sugar chain.
  • a kit for diagnosing multiple sclerosis (hereinafter sometimes referred to as “kit of the present invention”) is provided. By using the kit of the present invention, the amount of secreted PTPRZ isoform 1 and / or 2 in a sample derived from a subject is measured, and compared with the corresponding amount in a sample of a control group. It is possible to easily test whether or not the patient is suffering from multiple sclerosis.
  • Examples of the antibody that specifically recognizes secreted PTPRZ isoform 1 and / or 2 contained in the kit of the present invention include the following antibodies: (1) An antibody that specifically recognizes secreted PTPRZ isoform 1 (2) An antibody that specifically recognizes secreted PTPRZ isoform 2 (3) An antibody that specifically recognizes secreted PTPRZ isoform 1 and 2 (4) An antibody that specifically recognizes a branched O-mannose sugar chain. In addition, (4) an antibody that specifically recognizes a branched O-mannose sugar chain can be prepared using a method known per se, and as described above, using a commercially available antibody such as Cat-315 antibody. Also good.
  • the kit of the present invention contains one or more of the antibodies (1) to (4) as a component.
  • the kit of the present invention may contain other components other than antibodies, including instruction manuals and criteria.
  • the method for examining multiple sclerosis of the present invention may be combined with a method for treating multiple sclerosis.
  • the present invention provides a method for testing and treating multiple sclerosis comprising the following steps: (1) A step of measuring secretory receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, wherein the measured value of the amount of secreted PTPRZ in the sample derived from the subject is a control When the amount of secreted PTPRZ in the sample of the group is low, the subject is shown to have multiple sclerosis, and the secreted PTPRZ is secreted PTPRZ isoform 1 and secreted PTPRZ isoform. Or (2) a step of administering a therapeutically effective amount of a therapeutic agent for multiple sclerosis to the subject determined to suffer from multiple sclerosis in step (1). .
  • PTPRZ secretory receptor protein tyrosine phosphatase Z
  • Examples of the therapeutic agent for multiple sclerosis used in the present invention include steroids, interferon ⁇ , S1P receptor modulators, immunosuppressants, alkylating agents, anticancer antibiotics, anti-CD52 antibodies, and anti- ⁇ 4 integrins. Examples include, but are not limited to, administration of therapeutic agents for multiple sclerosis such as antibodies.
  • Steroids include, for example, amcinonide, hydrocortisone sodium succinate, prednisolone sodium succinate, prednisolone sodium methyl succinate, ciclesonide, difluprednate, betamethasone propionate, dexamethasone, deflazacote, triamcinolone acetonide, parminodide Flumetasone pivalate, prednisolone butyl acetate, budesonide, plasterone sulfate, mometasone furoate, fluocinonide, fluocinolone acetonide, fludroxycortide, flunisolide, prednisolone, alcrometasone propionate, clobetasol propionate, dexamethasone propionate, propionate Deprodon, propionate Ticazone, beclomethasone propionate, betamethasone, methylprednisolone, methylprednisolone str
  • S1P receptor modulators examples include fingolimod (fingolimod), siponimod, ponesimod, ceralifimod, and ozanimod.
  • immunosuppressant examples include azathioprine, ascomycin, everolimus, salazosulfapyridine, cyclosporine, cyclophosphamide, sirolimus, tacrolimus, bucillamine, methotrexate, leflunomide, and teriflunomide.
  • alkylating agent examples include nitrogen mustard-N-oxide hydrochloride, cyclophosphamide, ifosfamide, melphalan, thiotepa, carbocon, busulfan, nimustine hydrochloride, dacarbazine, ranimustine, carmustine, chlorambucil, bendamustine, mecloethanamine .
  • Anticancer antibiotics include, for example, actinomycin D, mitomycin C, daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride, neocartinostatin, pirarubicin hydrochloride, (epirubicin hydrochloride), idarubicin hydrochloride, chromomycin A3, (bleomycin hydrochloride) bleomycin , Pepromycin sulfate, terarubicin, dinostatin stimamarer, gemtuzumab ozogamicin, mitoxantrone hydrochloride.
  • Examples of the anti-CD52 antibody include alemtuzumab.
  • Examples of the anti- ⁇ 4 integrin antibody include natalizumab.
  • the therapeutically effective amount of the therapeutic agent for multiple sclerosis may vary depending on the type of active ingredient, the body weight and sex of the subject to be treated, the administration route, etc. can do.
  • the administration route of a therapeutic agent can select either oral or parenteral according to the kind of active ingredient.
  • Parenteral administration includes, but is not limited to, subcutaneous administration, intramuscular administration, intraperitoneal administration, and the like.
  • treatment can include not only healing of the disease but also remission of the disease and improvement of the degree of the disease.
  • Example 1 Immunohistochemical staining A paraffin-embedded section of a multiple sclerosis patient's brain was deparaffinized as follows. The sample was immersed in xylene three times, 99.5% ethanol three times, 90% ethanol once, 80% ethanol once, 70% ethanol once, five minutes, and then immersed in running water for five minutes. The mixture was autoclaved in 0.01 M citrate buffer (pH 8.5) at 121 ° C. for 5 minutes and immersed in PBS (pH 7.8).
  • Alexa fluor 546- or 488-labeled secondary antibody diluted with DAKO REAL antibody diluent and DAPI For 45 minutes at room temperature, washed 3 times for 5 minutes each in PBS-T, encapsulated in CC / Mount, and observed with FV1000-D confocal laser microscope (Olympus). Signal intensity analysis was performed with MetaMorph (Olympus) (FIG. 1), which revealed that Cat-315 positive cells and PTPRZ positive cells were in agreement, and that Cat-315 positive cells were PTPRZ positive. became.
  • Cerebrospinal fluid from a patient with multiple sclerosis was suspected to have cerebrospinal fluid from a neuromyelitis optica (NMO) patient and idiopathic normal pressure hydrocephalus (iNPH).
  • NMO neuromyelitis optica
  • iNPH idiopathic normal pressure hydrocephalus
  • a subject determined to be not iNPH as a result of diagnosis was used as a disease control for multiple sclerosis.
  • These cerebrospinal fluids were patient samples obtained at medical institutions such as Fukushima Medical University Hospital, and informed consent was obtained from all patients.
  • mice monoclonal Cat-315 IgM (Millipore), mouse anti-PTPRZ IgM ⁇ (122.2) (Santa cruz biotechnology), goat polyclonal anti-Transtyretin (TTR) antibody (Abcam).
  • Other reagents were purchased mainly from Wako.
  • the details of the chondroitinase digestion are as follows. A final concentration of 50 mM Tris-HCl (pH 8.0), 30 mM sodium acetate, 200 U / L Chondroitinase ABC (Seikagaku) was added to the sample solution, and the mixture was stirred at 37 ° C. for 1 hour.
  • Blocking was performed by immersing in 5% skim milk-0.05% Tween20-TBS (pH 7.4) (TBS-T) for 30 minutes, and then in a primary antibody diluted with TBS-T for 2 hours at room temperature or 4 ° C. Shake overnight. After washing by immersing in TBS-T three times for 5 minutes, each was reacted with an HRP-labeled secondary antibody diluted with TBS-T for 40 minutes at room temperature. After washing with TBS-T three times, light was emitted using SuperSignal West Femto (Thermo Fisher Scientific), and the signal was detected with LAS4000 mini (GE Healthcare). The signal intensity of the band was quantified using the quantification software attached to LAS4000 mini. The results are shown in Table 1.
  • Table 1 shows the signal intensity of the upper band of secreted PTPRZ isoforms 1 and 2 relative to the signal intensity of TTR detected as an internal standard of cerebrospinal fluid in samples derived from MS patients, MNO patients, and non-iNPH. It is shown as the relative value of the signal intensity of the upper band detected with the Cat-315 antibody.
  • the upper band considered to be derived from the extracellular region of PTPRZ isoforms 1 and 2 (PTPRZ isoform 2 full length is PTPRZ isoform 1 full length and The difference is only 7 amino acids, and the amino acid sequences of both extracellular regions are the same, so that they are detected as the same band (approximately 350 kDa) and the lower stage considered to be derived from the extracellular region of secreted PTPZ isoform 3.
  • a band (approximately 180 kDa) signal was detected.
  • MS multiple sclerosis
  • iNPH idiopathic normal pressure hydrocephalus
  • Mouse anti-PTPZ IgM ⁇ is a PTPRZ-specific monoclonal antibody prepared by preparing a proteoglycan fraction from rat fetal brain. Other reagents were purchased mainly from Wako.
  • Western blotting was performed according to the following procedure.
  • Laemmli Sample buffer (final concentration 20.8 mM Tris-HCl (pH 6.5), 8.3% glycerol, 1% SDS, 1% ⁇ -mercaptoethanol) was added to the chondroitinase-treated sample solution, and 5% at 99 ° C. Heated for minutes.
  • electrophoresis was performed at a constant current of 20 to 25 mA per gel for 60 to 70 minutes.
  • transfer was performed on a nitrocellulose film at a constant current of 350 mA for 45 minutes.
  • mouse anti-PTPRZ IgM ⁇ (Santa cruz, 200-fold diluted with PBS-0.1% Tween (PBS-T)
  • PBS-T PBS-T containing sc-33664)
  • PBS-T containing HRP-labeled goat anti-mouse IgM antibody SAB-110, Stressgen
  • diluted 10,000 times as a secondary antibody was washed by immersing in PBS-T three times for 5 minutes each. And at room temperature for 2 hours.
  • the signal intensity of secreted PTPRZ isoforms 1 and 2 in the cerebrospinal fluid from MS patients is the cerebrospinal fluid from the control group (idiopathic normal pressure hydrocephalus patient (including suspected cases) group) It was significantly lower than that in the middle. From this result, it was shown that secretory PTPRZ isoforms 1 and 2 can be markers for MS detection.
  • the present invention a new objective index useful for definitive diagnosis of demyelinating diseases (particularly multiple sclerosis) is provided. Therefore, the present invention is extremely useful in the medical field.

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Abstract

The present invention provides a method for testing for multiple sclerosis, the method comprising a step for measuring secretory receptor-type protein tyrosine phosphatase Z (PTPRZ) in a sample obtained from a subject. When the measurement value of the secretory PTPRZ level in the sample obtained from the subject is lower in comparison with the PTPRZ levels in samples of a control group, it is indicated that the subject is affected with multiple sclerosis. Said secretory PTPRZ is either secretory PTPRZ isoform 1 or secretory PTPRZ isoform 2, or both.

Description

多発性硬化症マーカーMultiple sclerosis marker
　本発明は、脱髄疾患（特に、多発性硬化症）の検査方法等に関し、詳細には、被検体由来の試料中の受容体型タンパク質チロシンフォスファターゼＺ（Ｐｒｏｔｅｉｎ　Ｔｙｒｏｓｉｎｅ　Ｐｈｏｓｐｈａｔａｓｅ　Ｒｅｃｅｐｔｏｒ－ｔｙｐｅ Ｚ；ＰＴＰＲＺ）、より好ましくは分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺを測定する工程を含む、多発性硬化症の検査方法に関する。 The present invention relates to a test method for demyelinating diseases (particularly, multiple sclerosis), and more specifically, receptor protein tyrosine phosphatase Z (Protein Tyrosine Phosphatase Receptor-type Z; PTPRZ) in a sample derived from a subject, More preferably, the present invention relates to a test method for multiple sclerosis, which comprises the step of measuring branched O-mannose sugar chain-modified PTPRZ.
　多発性硬化症（Ｍｕｌｔｉｐｌｅ　ｓｃｌｅｒｏｓｉｓ；ＭＳ）は、中枢神経系の白質に多発性の脱髄病変が出現し、運動麻痺や知覚障害などの神経障害を引き起こす脱髄疾患の一つである。欧米の白人に多く、北ヨーロッパでは１０万人に５０人～１００人ほどの割合で発症する。また、日本では１０万人に１０人程度と少ない（非特許文献１）が、年々増加傾向にあり、患者数は世界では２５０万人以上にのぼる。 Multiple sclerosis (MS) is one of the demyelinating diseases in which multiple demyelinating lesions appear in the white matter of the central nervous system and cause neurological disorders such as motor paralysis and sensory impairment. It is common among whites in Europe and the United States, and it affects 50 to 100 people per 100,000 people in Northern Europe. In Japan, the number is as small as about 10 in 100,000 (Non-Patent Document 1), but the number of patients is increasing year by year, and the number of patients in the world reaches 2.5 million or more.
　多発性硬化症の臨床像は、大きく分けて、（１）再発寛解型（Ｒｅｐｌａｃｉｎｇ－ｒｅｍｉｔｔｉｎｇ　ＭＳ；ＲＲＭＳ）、（２）一次進行型（Ｐｒｉｍａｒｙ　ｐｒｏｇｒｅｓｓｉｖｅ　ＭＳ；ＰＰＭＳ）、（３）二次進行型（Ｓｅｃｏｎｄａｒｙ　ｐｒｏｇｒｅｓｓｉｖｅ　ＭＳ；ＳＰＭＳ）の３つに分類される。ＲＲＭＳは、症状の悪化（再発）と症状の安定（寛解）が交互に起こり、数カ月から数年間の寛解期間と再発が繰り返されることを特徴とする。ＰＰＭＳは、病状が進行しない一時的な停滞期間があるが、寛解せず徐々に病状が進行していくことを特徴とする。ＳＰＭＳは、発症初期は再発と寛解が繰り返されるが、緩やかに進行していくことを特徴とする。現時点において、多発性硬化症も根治療法は確立されておらず、患者の発病の時期や症状に応じた対症療法が行われている。 The clinical features of multiple sclerosis can be broadly divided into (1) relapsing-remitting MS (RRMS), (2) primary progressive MS (PPMS), and (3) secondary progressive ( It is classified into three categories: secondary progressive MS (SPMS). RRMS is characterized by alternating symptom worsening (recurrence) and symptom stability (remission), with repeated periods of remission and relapse for months to years. PPMS is characterized in that there is a temporary stagnation period in which the disease does not progress, but the disease progresses gradually without remission. SPMS is characterized in that recurrence and remission are repeated in the early stage of onset, but progresses slowly. At present, no radical cure has been established for multiple sclerosis, and symptomatic treatment is performed according to the time and symptoms of the patient.
　多発性硬化症の治療を困難にする要因の一つとして、その診断の難しさが挙げられる。多発性硬化症は、症状が多様であり、該疾患に特異的なバイオマーカーも見いだされていないため、医師による診断が難しい。特に、病巣が単発性の場合、脳腫瘍や脳炎・脳症との鑑別が困難になる。現状、多発性硬化症の診断は、ＭＲＩ検査や髄液検査によって行われているが、ＭＲＩ検査が可能な施設は限られており、また髄液検査も多発性硬化症に特異的ではなく、場合によっては危険性の伴う患部の組織生検を試行することで（非特許文献７）他の疾患の可能性を排除してはじめて多発性硬化症との確定診断が可能となるものである。多発性硬化症に対しては免疫力を抑制するためにステロイド剤を投与するなどの治療を行うのに対し、脳腫瘍に対しては手術、放射線治療、薬物治療の３つを組み合わせた治療を行うなど、疾患ごとに治療アプローチが異なることからも的確な診断が重要である。それにもかかわらず、現状では多発性硬化症を早期に診断することは非常に難しく、実際に、多発性硬化症の最初の症状が現れてから多発性硬化症であると確定診断されるまでの期間は平均３年を超えるとの調査結果もある。 一 つ One of the factors that make it difficult to treat multiple sclerosis is the difficulty of diagnosis. Multiple sclerosis is difficult to diagnose by a doctor because there are various symptoms and no biomarkers specific to the disease have been found. In particular, when the lesion is isolated, it is difficult to distinguish it from a brain tumor or encephalitis / encephalopathy. Currently, multiple sclerosis is diagnosed by MRI and cerebrospinal fluid, but there are only a limited number of facilities where MRI can be performed, and cerebrospinal fluid is not specific for multiple sclerosis. In some cases, it is possible to make a definitive diagnosis of multiple sclerosis only after eliminating the possibility of other diseases by trying a tissue biopsy of an affected area with risk (Non-patent Document 7). For multiple sclerosis, treatment such as administration of steroids to suppress immunity is performed, whereas for brain tumors, treatment is a combination of surgery, radiation therapy, and drug treatment. Thus, accurate diagnosis is important because treatment approaches differ from disease to disease. Nevertheless, at present, it is very difficult to diagnose multiple sclerosis at an early stage, and in fact, from the first symptoms of multiple sclerosis to the time when multiple sclerosis is confirmed Some survey results show that the average period exceeds three years.
　かかる背景から、多発性硬化症をより簡便に診断できる検査方法の確立が強く求められている。 From this background, there is a strong demand for the establishment of a test method that can more easily diagnose multiple sclerosis.
　受容体型プロテインチロシンホスファターゼは、構造的な類似性から８つのサブファミリーに分類される（非特許文献２）。その細胞外領域は、免疫グロブリン様（Ｉｇ）ドメイン、フィブロネクチン様（ＦＮ）ドメイン、炭酸脱水酵素様（ＣＡＨ）ドメイン、Ｍｅｐｒｉｎ－Ａ５－ＰＴＰμ（ＭＡＭ）ドメインなどの、多彩なドメインで構成されており、細胞内には１つまたは２つのチロシンフォスファターゼ（ＰＴＰ）ドメインが存在する。タンデム型の受容体型プロテインチロシンホスファターゼでは細胞膜近位側のＤ１のみがホスファターゼ活性を有しており、Ｄ２には酵素活性がない（例外的にＲ４　ＲＰＴＰサブファミリーのＤ２には若干の活性が認められる）。 Receptor type protein tyrosine phosphatases are classified into eight subfamilies based on structural similarity (Non-patent Document 2). Its extracellular region is composed of various domains such as immunoglobulin-like (Ig) domain, fibronectin-like (FN) domain, carbonic anhydrase-like (CAH) domain, and Meprin-A5-PTPμ (MAM) domain. There are one or two tyrosine phosphatase (PTP) domains in the cell. In the tandem receptor protein tyrosine phosphatase, only D1 on the cell membrane proximal side has phosphatase activity, and D2 has no enzyme activity (exceptionally, some activity is observed in D2 of the R4 RPTP subfamily). ).
　受容体型タンパク質チロシンフォスファターゼＺ（Ｐｒｏｔｅｉｎ　Ｔｙｒｏｓｉｎｅ　Ｐｈｏｓｐｈａｔａｓｅ　Ｒｅｃｅｐｔｏｒ－ｔｙｐｅ Ｚ１；ＰＴＰＲＺ、ＰＴＰＲＺ１、或いはＰｔｐｒｚ、ＰＴＰζ又はＲＰＴＰβ等とも称される）は、Ｒ５サブファミリーに属する。哺乳類のＰＴＰＲＺにはスプライシングバリアントが存在し、例えばマウスにおいては、これにより３種のアイソフォーム（受容体型であるＰＴＰＲＺ－ＡおよびＰＴＰＲＺ－Ｂ、ならびに分泌型であるＰＴＰＲＺ－Ｓ）が存在する。ＰＴＰＲＺは、細胞外のＣＡＨドメイン、ＦＮタイプＩＩＩ様ドメイン、コンドロイチン硫酸鎖結合領域（Ｓｅｒ－Ｇｌｙ／Ｇｌｙ－Ｓｅｒに富む）と、細胞内の２つのＰＴＰドメイン（Ｄ１およびＤ２）で構成されている。ＰＴＰＲＺは、他のＲＰＴＰと異なり、細胞外領域にコンドロイチン硫酸やケラタン硫酸などの硫酸化グリコサミノグリカンと呼ばれる硫酸化多糖修飾を有する。ＰＴＰＲＺの主要発現部位である脳組織では、すべてのアイソフォームがコンドロイチン硫酸プロテオグリカンとして発現している（非特許文献３～５）。ＰＴＰＲＺは、中枢神経系における神経細胞およびグリア細胞（アストロサイトおよびオリゴデンドロサイト）の両方に発現している。ＰＴＰＲＺは、脱髄刺激に応答して、Ｎ－アセチルグルコサミン転移酵素ＧｎＴ－ＩＸ（ＭＧＡＴ５Ｂ、Ｍａｎｎｏｓｙｌ（Ａｌｐｈａ－１，６－）－Ｇｌｙｃｏｐｒｏｔｅｉｎ　Ｂｅｔａ－１，６－Ｎ－Ａｃｅｔｙｌ－Ｇｌｕｃｏｓａｍｉｎｙｌｔｒａｎｓｆｅｒａｓｅ，　Ｉｓｏｚｙｍｅ　Ｂとも称される）を介しての分岐型Ｏ－マンノース修飾を受けることが報告されている（非特許文献６）。 Receptor-type protein tyrosine phosphatase Z (Protein Tyrosine Phosphatase Receptor-type Z1; also referred to as PTPRZ, PTPRZ1, or Ptprz, PTPζ, RPTPβ, etc.) belongs to the R5 subfamily. There are splicing variants in mammalian PTPRZ, for example, in mice, there are three isoforms (the receptor forms PTPRZ-A and PTPRZ-B, and the secreted form PTPRZ-S). PTPRZ is composed of an extracellular CAH domain, an FN type III-like domain, a chondroitin sulfate chain-binding region (rich in Ser-Gly / Gly-Ser), and two intracellular PTP domains (D1 and D2). . Unlike other RPTPs, PTPRZ has sulfated polysaccharide modifications called sulfated glycosaminoglycans such as chondroitin sulfate and keratan sulfate in the extracellular region. All isoforms are expressed as chondroitin sulfate proteoglycans in brain tissue, which is the main expression site of PTPRZ (Non-Patent Documents 3 to 5). PTPRZ is expressed on both neurons and glial cells (astrocytes and oligodendrocytes) in the central nervous system. In response to demyelination stimulation, PTPRZ is N-acetylglucosamine transferase GnT-IX (MGAT5B, Mannosyl (Alpha-1,6-))-Glycoprotein Beta-1,6-N-Acetyl-Glucosamineyltransferase, Isozyme Have been reported to undergo branched O-mannose modification (Non-patent Document 6).
　本発明の目的は、脱髄疾患（特には多発性硬化症）の新規検査方法を提供することにある。 An object of the present invention is to provide a novel test method for demyelinating diseases (particularly multiple sclerosis).
　本発明者らは、上記課題に対して鋭意検討した結果、脱髄疾患の患者の脳脊髄液中の、ＰＴＰＲＺアイソフォーム１および２の細胞外領域に相当する分泌型（遊離型）のＰＴＰＲＺアイソフォーム１および２の含有量が、対照群のそれと比較して減少していることを見出した。特に、多発性硬化症患者の脳脊髄液中の分泌型ＰＴＰＲＺアイソフォーム１および２の含有量が顕著に減少していることを見出した。さらに驚くべきことに糖転移酵素ＧｎＴ－ＩＸによってＰＴＰＲＺが糖鎖修飾されることによって生じる、分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺ量も脳脊髄液中において対象群より減少していることを見出し、かかる知見に基づいてさらに研究を進めることによって本発明を完成するに至った。すなわち、本発明は以下の通りである。 As a result of intensive studies on the above problems, the present inventors have found that secretory (free) PTPRZ isoforms corresponding to the extracellular regions of PTPRZ isoforms 1 and 2 in the cerebrospinal fluid of patients with demyelinating diseases. It was found that the content of Forms 1 and 2 was reduced compared to that of the control group. In particular, it has been found that the contents of secreted PTPRZ isoforms 1 and 2 in the cerebrospinal fluid of patients with multiple sclerosis are significantly reduced. Furthermore, it was surprisingly found that the amount of branched O-mannose sugar chain-modified PTPRZ produced by glycosylation of PTPRZ by glycosyltransferase GnT-IX is also decreased from the subject group in cerebrospinal fluid. The present invention has been completed by further research based on such findings. That is, the present invention is as follows.
［１］被検体由来の試料中の分泌型受容体型タンパク質チロシンフォスファターゼＺ（ＰＴＰＲＺ）を測定する工程を含む、多発性硬化症の検査方法であって、該被検体由来の試料中の分泌型ＰＴＰＲＺ量の測定値が、対照群の試料中の分泌型ＰＴＰＲＺ量と比較して低い場合に、該被検体が多発性硬化症であることが示され、該分泌型ＰＴＰＲＺが、分泌型ＰＴＰＲＺアイソフォーム１及び分泌型ＰＴＰＲＺアイソフォーム２のいずれかまたは両方である、方法。
［２］前記分泌型ＰＴＰＲＺアイソフォーム１および分泌型ＰＴＰＲＺアイソフォーム２が、分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺである、［１］記載の方法。
［３］被検体由来の試料が、脳脊髄液または血液である、［１］または［２］記載の方法。
［４］被検体由来の脊髄液中の分泌型ＰＴＰＲＺの測定が、該タンパク質を特異的に認識する抗体または分岐型Ｏ－マンノース糖鎖を特異的に認識する抗体を用いて行われることを特徴とする、［１］～［３］のいずれか記載の方法。
［５］分泌型ＰＴＰＲＺを特異的に認識する抗体および分岐型Ｏ－マンノース糖鎖を特異的に認識する抗体のいずれかまたは両方を含む、多発性硬化症の診断用キットであって、該分泌型ＰＴＰＲＺが、分泌型ＰＴＰＲＺアイソフォーム１及び分泌型ＰＴＰＲＺアイソフォーム２のいずれかまたは両方である、キット。
［６］被検体由来の試料中の分泌型受容体型タンパク質チロシンフォスファターゼＺ（ＰＴＰＲＺ）を測定する工程を含む、脱髄状態の検査方法であって、該被検体由来の試料中の分泌型ＰＴＰＲＺ量の測定値が、対照群の試料中の分泌型ＰＴＰＲＺ量と比較して低い場合に、該被検体が脱髄を起こしていることが示され、該分泌型ＰＴＰＲＺが、分泌型ＰＴＰＲＺアイソフォーム１及び分泌型ＰＴＰＲＺアイソフォーム２のいずれかまたは両方である、方法。
［７］被検体由来の試料中の分岐型Ｏ－マンノース糖鎖修飾型受容体型タンパク質チロシンフォスファターゼＺ（ＰＴＰＲＺ）を測定する工程を含む、脱髄状態の検査方法であって、該被検体由来の試料中の分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺ量の測定値が、対照群の試料中の分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺ量と比較して低い場合に、該被検体が脱髄を起こしていることが示され、該分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺが、分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺアイソフォーム１および分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺアイソフォーム２のいずれかまたは両方である、方法。 [1] A method for testing multiple sclerosis, comprising a step of measuring secretory receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, wherein the secretory PTPRZ in the sample derived from the subject If the measured amount is low compared to the amount of secreted PTPRZ in the control sample, the subject is shown to be multiple sclerosis, and the secreted PTPRZ is a secreted PTPRZ isoform. 1. A method, wherein either or both of 1 and secreted PTPRZ isoform 2.
[2] The method according to [1], wherein the secretory PTPRZ isoform 1 and the secretory PTPRZ isoform 2 are branched O-mannose sugar chain-modified PTPRZ.
[3] The method according to [1] or [2], wherein the sample derived from the subject is cerebrospinal fluid or blood.
[4] The measurement of secretory PTPRZ in spinal fluid derived from a subject is performed using an antibody that specifically recognizes the protein or an antibody that specifically recognizes a branched O-mannose sugar chain. The method according to any one of [1] to [3].
[5] A diagnostic kit for multiple sclerosis comprising either or both of an antibody specifically recognizing secretory PTPRZ and an antibody specifically recognizing a branched O-mannose sugar chain, the secretion A kit in which the type PTPRZ is either or both of a secreted PTPRZ isoform 1 and a secreted PTPRZ isoform 2.
[6] A method for examining a demyelinating state, comprising a step of measuring secretory receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, wherein the amount of secreted PTPRZ in the sample derived from the subject Is lower than the amount of secreted PTPRZ in the sample of the control group, it is indicated that the subject is demyelinating, and the secreted PTPRZ is secreted PTPRZ isoform 1. And secreted PTPRZ isoform 2 or both.
[7] A method for examining a demyelinating state, comprising the step of measuring branched O-mannose sugar chain-modified receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, When the measured value of the amount of branched O-mannose sugar chain-modified PTPRZ in the sample is lower than the amount of branched O-mannose sugar chain-modified PTPRZ in the sample of the control group, the subject is demyelinated The branched O-mannose sugar chain-modified PTPRZ is divided into branched O-mannose sugar chain-modified PTPRZ isoform 1 and branched O-mannose sugar chain-modified PTPRZ isoform 2. A method that is either or both.
　また、別の一態様において、本発明は以下の通りである。
［１Ａ］被検体由来の試料中の受容体型タンパク質チロシンフォスファターゼＺ（ＰＴＰＲＺ）を測定する工程を含む、多発性硬化症の検査方法であって、該被検体由来の試料中の分泌型ＰＴＰＲＺ量の測定値が、対照群の試料中のＰＴＰＲＺ量と比較して低い場合に、該被検体が多発性硬化症であることが示される、方法。
［２Ａ］ＰＴＰＲＺが、分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺである、［１Ａ］記載の方法。
［３Ａ］被検体由来の試料が、脳脊髄液または血液である、［１Ａ］または［２Ａ］記載の方法。
［４Ａ］被検体由来の脊髄液中のＰＴＰＲＺの測定が、該タンパク質を特異的に認識する抗体または分岐型Ｏ－マンノース糖鎖を特異的に認識する抗体を用いて行われることを特徴とする、［１Ａ］～［３Ａ］のいずれか記載の方法。
［５Ａ］ＰＴＰＲＺが、分泌型ＰＴＰＲＺアイソフォーム１、分泌型ＰＴＰＲＺアイソフォーム２、および分泌型ＰＴＰＲＺアイソフォーム３からなる群から選択されるいずれか１つまたは２種以上である、［１Ａ］～［４Ａ］のいずれか記載の方法。
［６Ａ］ＰＴＰＲＺを特異的に認識する抗体および分岐型Ｏ－マンノース糖鎖を特異的に認識する抗体のいずれかまたは両方を含む、多発性硬化症の診断用キット。
［７Ａ］被検体由来の試料中の受容体型タンパク質チロシンフォスファターゼＺ（ＰＴＰＲＺ）を測定する工程を含む、脱髄状態の検査方法であって、該被検体由来の試料中の分泌型ＰＴＰＲＺ量の測定値が、対照群の試料中のＰＴＰＲＺ量と比較して低い場合に、該被検体が脱髄を起こしていることが示される、方法。
［８Ａ］被検体由来の試料中の分岐型Ｏ－マンノース糖鎖修飾型受容体型タンパク質チロシンフォスファターゼＺ（ＰＴＰＲＺ）を測定する工程を含む、脱髄状態の検査方法であって、該被検体由来の試料中の分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺ量の測定値が、対照群の試料中の分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺ量と比較して低い場合に、該被検体が脱髄を起こしていることが示される、方法。 In another aspect, the present invention is as follows.
[1A] A test method for multiple sclerosis comprising a step of measuring receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, wherein the amount of secreted PTPRZ in the sample derived from the subject A method wherein the subject is shown to have multiple sclerosis when the measured value is low compared to the amount of PTPRZ in the sample of the control group.
[2A] The method according to [1A], wherein the PTPRZ is a branched O-mannose sugar chain-modified PTPRZ.
[3A] The method according to [1A] or [2A], wherein the sample derived from the subject is cerebrospinal fluid or blood.
[4A] The measurement of PTPRZ in spinal fluid derived from a subject is performed using an antibody that specifically recognizes the protein or an antibody that specifically recognizes a branched O-mannose sugar chain [1A] to [3A].
[5A] PTPRZ is any one or more selected from the group consisting of secreted PTPRZ isoform 1, secreted PTPRZ isoform 2, and secreted PTPRZ isoform 3, [1A] to [1A] 4A].
[6A] A diagnostic kit for multiple sclerosis comprising either or both of an antibody that specifically recognizes PTPRZ and an antibody that specifically recognizes a branched O-mannose sugar chain.
[7A] A method for examining a demyelination state, comprising measuring receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, and measuring the amount of secreted PTPRZ in the sample derived from the subject A method wherein the subject is shown to be demyelinated if the value is low compared to the amount of PTPRZ in the control group sample.
[8A] A test method for a demyelinating state, comprising a step of measuring branched O-mannose sugar chain-modified receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, When the measured value of the amount of branched O-mannose sugar chain-modified PTPRZ in the sample is lower than the amount of branched O-mannose sugar chain-modified PTPRZ in the sample of the control group, the subject is demyelinated A method that is shown to be causing.
　本発明により、多発性硬化症の確定診断に有用な新たな客観的指標が提供される。 The present invention provides a new objective index useful for the definitive diagnosis of multiple sclerosis.
ヒトＰＴＰＲＺの３種類のアイソフォームの構造を示す概念図である。Ａはアイソフォーム１を、Ｂはアイソフォーム２を、Ｃはアイソフォーム３を示す。図中、ＣＡはカルボニックアンヒドラーゼ様ドメインを、ＦＮはタイプＩＩＩフィブロネクチン様ドメインを、ＣＳはコンドロイチン硫酸鎖結合領域を、ＴＭは膜貫通ドメインを、Ｄ１とＤ２はいずれもＰＴＰドメインを表す。It is a conceptual diagram which shows the structure of three types of isoforms of human PTPRZ. A shows isoform 1, B shows isoform 2, and C shows isoform 3. In the figure, CA represents a carbonic anhydrase-like domain, FN represents a type III fibronectin-like domain, CS represents a chondroitin sulfate chain binding region, TM represents a transmembrane domain, and D1 and D2 both represent PTP domains. 図２は、多発性硬化症患者脳切片におけるＣａｔ－３１５抗体、または抗ＰＴＰＲＺ抗体を用いた免疫組織化学染色の結果を示す。FIG. 2 shows the results of immunohistochemical staining using Cat-315 antibody or anti-PTPRZ antibody in a multiple sclerosis patient brain section. 図３は、多発性硬化症罹患患者では髄液中の分泌型（遊離型）ＰＴＰＲＺアイソフォーム１および分泌型（遊離型）ＰＴＰＲＺアイソフォーム２の量が突発性正常圧水頭症罹患患者（対照群）のそれと比較して顕著に減少していることを示すグラフである。なお、データは平均値＋標準誤差（ＳＥ）で示した。FIG. 3 shows that in patients suffering from multiple sclerosis, the amounts of secreted (free) PTPRZ isoform 1 and secreted (free) PTPRZ isoform 2 in cerebrospinal fluid were patients with sudden normal pressure hydrocephalus (control group). It is a graph which shows that it has decreased notably compared with that of). The data is shown as mean value + standard error (SE).
　以下、本発明について詳細に説明する。 Hereinafter, the present invention will be described in detail.
１．多発性硬化症の検査方法
　本発明は、被検体由来の試料中の分泌型受容体型タンパク質チロシンフォスファターゼＺ（ＰＴＰＲＺ）アイソフォーム１および２のいずれかまたは両方を測定する工程を含む、多発性硬化症の検査方法（以下、「本発明の検査方法」と称することがある。）を提供する。 1. Test method for multiple sclerosis The present invention includes the step of measuring either or both of secretory receptor protein tyrosine phosphatase Z (PTPRZ) isoforms 1 and 2 in a sample derived from a subject. (Hereinafter sometimes referred to as “the inspection method of the present invention”).
　本発明の検査方法により罹患の有無を検査し得る多発性硬化症は、上述した通り３つの型に分類され得る。本発明によれば、再発寛解型（ＲＲＭＳ）、一次進行型（ＰＰＭＳ）、（３）二次進行型（ＳＰＭＳ）のいずれの型も検査可能である。 The multiple sclerosis that can be examined for the presence or absence of disease by the examination method of the present invention can be classified into three types as described above. According to the present invention, any of relapsing-remitting type (RRMS), primary progressive type (PPMS), and (3) secondary progressive type (SPMS) can be examined.
　本発明の検査方法の一態様において、被検体由来の試料中の分泌型ＰＴＰＲＺアイソフォーム１および２のいずれかまたは両方の含有量を指標として測定し、対照群の対応する値と比較することにより、被検体における多発性硬化症の罹患の有無を検査することができる。ＰＴＰＲＺは、ヒトにおいては、細胞外領域の長さが異なる３種類のスプライシングバリアントが存在し、長いものからアイソフォーム１（配列番号１）、アイソフォーム２（配列番号２）、アイソフォーム３(配列番号３)となっている。さらにそれぞれの各アイソフォームの細胞外領域がプロテアーゼによって切断されて生じる分泌型（或いは「遊離型」ともいう）ＰＴＰＲＺが知られている。齧歯類では膜貫通領域以降のＣ末領域を欠失しているスプライシングバリアントも報告されているが、ヒトでは相当するバリアントは未同定である。従って、本発明の検査方法においては、被検体由来の試料中の分泌型（遊離型）ＰＴＰＲＺアイソフォーム１および／または２の含有量を測定し、対照群（健常者または多発性硬化症を罹患していない対象者）の対応する値と比較することにより、被検体における多発性硬化症の罹患の有無を検査することができる。以下、ヒトのＰＴＰＲＺの各アイソフォームおよびそれらの分泌型について詳述する。尚、本明細書において「分泌型（遊離型）ＰＴＰＲＺ」とは、ヒトＰＴＰＲＺにおける各アイソフォームの細胞外領域に由来するポリペプチド断片をいう。ＰＴＰＲＺには、細胞外領域のＣ末端側にプロテアーゼに対し感受性の高い部位が存在する。この部位がメタロプロテアーゼ等のプロテアーゼの分解により切断され、分泌型（遊離型）ＰＴＰＲＺとなる。本明細書において、「分泌型」ＰＴＰＲアイソフォームは、「遊離型」ＰＴＰＲＺアイソフォームと言い換えることができる。 In one embodiment of the test method of the present invention, the content of either or both of secreted PTPRZ isoforms 1 and 2 in a sample derived from a subject is measured as an index, and compared with the corresponding value of the control group The presence or absence of multiple sclerosis in the subject can be examined. PTPRZ has three types of splicing variants with different extracellular region lengths in humans. From the longest, isoform 1 (SEQ ID NO: 1), isoform 2 (SEQ ID NO: 2), isoform 3 (sequence) No. 3). Furthermore, secreted (or also referred to as “free”) PTPRZ, which is produced by cleaving the extracellular region of each isoform with a protease, is known. In rodents, splicing variants lacking the C-terminal region after the transmembrane region have also been reported, but the corresponding variant has not been identified in humans. Therefore, in the test method of the present invention, the content of the secreted (free) PTPRZ isoform 1 and / or 2 in the sample derived from the subject is measured, and the control group (healthy person or multiple sclerosis is affected). The subject can be examined for the presence or absence of multiple sclerosis in the subject. Hereinafter, human isoforms of PTPRZ and their secreted forms will be described in detail. In the present specification, “secreted (free) PTPRZ” refers to a polypeptide fragment derived from the extracellular region of each isoform in human PTPRZ. PTPRZ has a site sensitive to proteases on the C-terminal side of the extracellular region. This site is cleaved by the decomposition of a protease such as a metalloprotease and becomes secreted (free) PTPRZ. As used herein, a “secreted” PTPR isoform can be rephrased as a “free” PTPRZ isoform.
　ＰＴＰＲＺアイソフォーム１は、配列番号１で示す２３１５アミノ酸からなる。ＰＴＰＲＺアイソフォーム１は、１～２４位にシグナルペプチドを、４５～２９８位にＣＡドメインを、３１３～４０１位にＦＮドメインを、１６３７～１６６２位に膜貫通ドメインを、１７１７～１９９２位にＤ１ドメインを、そして２０２３～２２８２位にＤ２ドメインを有する。また、ＣＳ結合領域は、前記ＦＮドメインと膜貫通ドメイン間でＣＳ鎖結合している領域が該当する。ＰＴＰＲＺアイソフォーム１の細胞外領域は、配列番号１で示すアミノ酸配列において２５～１６３６位に相当する配列番号４で示すアミノ酸配列からなる。配列番号４の全長または部分断片のポリペプチドが、分泌型ＰＴＰＲＺアイソフォーム１に相当する。 PTPRZ isoform 1 consists of 2315 amino acids shown in SEQ ID NO: 1. PTPRZ isoform 1 has a signal peptide at positions 1-24, a CA domain at positions 45-298, an FN domain at positions 313-401, a transmembrane domain at positions 1637-1662, and a D1 domain at positions 1717-1992 And has a D2 domain at positions 2023-2282. In addition, the CS binding region corresponds to a region in which a CS chain is bonded between the FN domain and the transmembrane domain. The extracellular region of PTPRZ isoform 1 consists of the amino acid sequence shown by SEQ ID NO: 4 corresponding to positions 25 to 1636 in the amino acid sequence shown by SEQ ID NO: 1. The full-length or partial fragment polypeptide of SEQ ID NO: 4 corresponds to secreted PTPRZ isoform 1.
　ＰＴＰＲＺアイソフォーム２は、配列番号２で示す２３０８アミノ酸残基からなる。ＰＴＰＲＺアイソフォーム２は、ＰＴＰＲＺアイソフォーム１におけるＤ１ドメインの１７２３～１７２９位に相当する７アミノ酸残基が欠失したアイソフォームである。したがって、アイソフォーム２の細胞外領域は、アイソフォーム１のそれと一致し、配列番号４で示すアミノ酸配列からなる。従って、配列番号４の全長または部分断片のポリペプチドが分泌型ＰＴＰＲＺアイソフォーム２に相当する。 PTPRZ isoform 2 consists of 2308 amino acid residues shown in SEQ ID NO: 2. PTPRZ isoform 2 is an isoform in which 7 amino acid residues corresponding to positions 1723 to 1729 of the D1 domain in PTPRZ isoform 1 are deleted. Therefore, the extracellular region of isoform 2 is identical to that of isoform 1 and consists of the amino acid sequence shown in SEQ ID NO: 4. Therefore, the full-length or partial fragment polypeptide of SEQ ID NO: 4 corresponds to secreted PTPRZ isoform 2.
　ＰＴＰＲＺアイソフォーム３は、配列番号３で示す１４４８アミノ酸残基からなる。ＰＴＰＲＺアイソフォーム３は、ＰＴＰＲＺアイソフォーム１におけるＣＳ結合領域の大部分に相当し得る７５５～１６１４位の８６０アミノ酸残基と、１７２３～１７２９位に相当する７アミノ酸残基が欠失したアイソフォームである。したがって、アイソフォーム３の細胞外領域は、アイソフォーム１及び２のそれよりも短く、配列番号１で示すアミノ酸配列において２５～７５４位及び１６１５～１６３６位に相当するアミノ酸配列からなる。 PTPRZ isoform 3 consists of 1448 amino acid residues shown in SEQ ID NO: 3. PTPRZ isoform 3 is an isoform in which 860 amino acid residues at positions 755 to 1614 and 7 amino acid residues corresponding to positions 1723 to 1729 are deleted, which can correspond to most of the CS binding region in PTPRZ isoform 1. is there. Therefore, the extracellular region of isoform 3 is shorter than that of isoforms 1 and 2, and consists of amino acid sequences corresponding to positions 25 to 754 and 1615 to 1636 in the amino acid sequence shown in SEQ ID NO: 1.
　分泌型ＰＴＰＲＺには、ＰＴＰＲＺアイソフォーム１およびＰＴＰＲＺアイソフォーム２の細胞外ドメインに由来する約３５０ｋＤａのｌｏｎｇ－ｆｏｒｍと、ＰＴＰＲＺアイソフォーム３の細胞外ドメインに由来する約１８０ｋＤａのｓｈｏｒｔ－ｆｏｒｍの２種類が存在するが、このうち、ｌｏｎｇ－ｆｏｒｍ（及びこの断片ポリペプチド）が多発性硬化症の検出用マーカーとなり得る。尚、本発明において実際に検出対象となるアミノ酸配列は、配列番号４に示されるアミノ酸配列（１６１２アミノ酸）の全長ポリペプチドおよび／またはその一部分からなる部分ペプチドである。かかる部分ペプチドとしては、例えば、配列番号４に示されるアミノ酸の、連続する１１２９アミノ酸以上のアミノ酸を含むペプチド、連続する１２０９アミノ酸以上のアミノ酸配列を含むペプチド、連続する１２９０アミノ酸以上のアミノ酸配列を含むペプチド、連続する１３７１アミノ酸以上のアミノ酸配列を含むペプチド、連続する１４５１アミノ酸以上のアミノ酸配列を含むペプチド、連続する１５３２アミノ酸以上のアミノ酸配列を含むペプチド、連続する１５８０アミノ酸以上のアミノ酸配列を含むペプチド、および、連続する１５９６アミノ酸以上のアミノ酸配列を含むペプチド等が例示されるが、これらに限定されない。 There are two types of secreted PTPRZ: a long-form of about 350 kDa derived from the extracellular domain of PTPRZ isoform 1 and PTPRZ isoform 2, and a short-form of about 180 kDa derived from the extracellular domain of PTPRZ isoform 3. Among them, long-form (and its fragment polypeptide) can be a marker for detecting multiple sclerosis. In the present invention, the actual amino acid sequence to be detected is a full-length polypeptide of the amino acid sequence shown in SEQ ID NO: 4 (1612 amino acids) and / or a partial peptide comprising a part thereof. Examples of such a partial peptide include a peptide containing 1129 or more consecutive amino acids, a peptide containing 1209 or more consecutive amino acids, and a sequence of 1290 or more amino acids. Peptide, peptide containing a continuous amino acid sequence of 1371 amino acids or more, peptide containing a continuous amino acid sequence of 1451 amino acids or more, peptide containing a continuous amino acid sequence of 1532 amino acids or more, peptide containing a continuous amino acid sequence of 1580 amino acids or more, Examples of the peptide include a continuous amino acid sequence of 1596 amino acids or more, but are not limited thereto.
　また、本発明の方法の別の一態様としては、被検体由来の試料中の分岐型Ｏ－マンノースの含有量を測定し、対照群の対応する値と比較することにより、被検体における多発性硬化症の罹患の有無を検査することができる。ＰＴＰＲＺは、脱髄刺激に応答して、Ｎ－アセチルグルコサミン転移酵素ＧｎＴ－ＩＸを介しての分岐型Ｏ－マンノース修飾を受ける。従って、被検体由来の試料中の分岐型Ｏ－マンノースの含有量は、分泌型ＰＴＰＲＺアイソフォーム１および２の存在量を反映していると考えられる。従って、被検体由来の試料中の分岐型Ｏ－マンノースの含有量を測定し、対照群の対応する値と比較することによっても、多発性硬化症の罹患の有無を検出することができる。 Further, as another aspect of the method of the present invention, the content of branched O-mannose in a sample derived from a subject is measured, and compared with the corresponding value of a control group, thereby allowing multiple occurrences in the subject. The presence or absence of sclerosis can be examined. PTPRZ undergoes branched O-mannose modification via N-acetylglucosamine transferase GnT-IX in response to demyelination stimuli. Therefore, the content of branched O-mannose in the sample derived from the subject is considered to reflect the abundance of secreted PTPRZ isoforms 1 and 2. Therefore, the presence or absence of multiple sclerosis can also be detected by measuring the content of branched O-mannose in the sample derived from the subject and comparing it with the corresponding value of the control group.
　被検体由来の試料中の分泌型ＰＴＰＲＺアイソフォーム１および２の測定方法は、当該タンパク質量が定量できる方法であればいかなる方法を用いてもよい。例えば、質量分析、クロマトグラフィー法（液体クロマトグラフィーまたはガスクロマトグラフィー等）、電気泳動法、抗体を用いた免疫測定法などが挙げられるが、これらに限定されない。特に免疫測定法が簡便で好ましい。免疫測定法の場合、反応形式に基づいて分類すると、サンドイッチ法、競合法、凝集法、ウエスタンブロット法等があり、また、標識に基づいて分類すると、ラジオイムノアッセイ、蛍光イムノアッセイ、酵素イムノアッセイ（ＥＩＡ）、ビオチンイムノアッセイ等が挙げられるが、これらに限定されない。 Any method may be used as a method for measuring secretory PTPRZ isoforms 1 and 2 in a sample derived from a subject as long as the amount of the protein can be quantified. Examples include, but are not limited to, mass spectrometry, chromatography methods (liquid chromatography or gas chromatography, etc.), electrophoresis methods, immunoassay methods using antibodies, and the like. In particular, the immunoassay is simple and preferable. In the case of immunoassay methods, there are sandwich methods, competition methods, agglutination methods, Western blotting methods, and the like when classified based on reaction formats, and radioimmunoassay, fluorescent immunoassay, enzyme immunoassay (EIA) when classified based on labels. , Biotin immunoassay and the like, but are not limited thereto.
　免疫測定に用いる抗体は、ポリクローナル抗体およびモノクローナル抗体のいずれであってもよいが、再現性の観点からはモノクローナル抗体が好ましい。本発明の検査方法を実施するに際して、市販の抗体を用いてもよく、また、自体公知の方法により分泌型ＰＴＰＲＺアイソフォーム１および／または２、または分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺアイソフォーム１および／または２を特異的に認識する抗体を作製して用いてもよい。 The antibody used for the immunoassay may be either a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferable from the viewpoint of reproducibility. In carrying out the test method of the present invention, a commercially available antibody may be used, and secreted PTPRZ isoform 1 and / or 2 or branched O-mannose sugar chain-modified PTPRZ isoform by a method known per se An antibody that specifically recognizes 1 and / or 2 may be prepared and used.
　なお、本明細書における用語「抗体」は、「抗原結合断片」をも含む概念である。「抗原結合断片」とは、もとの抗体の対応抗原に対する結合性（抗原抗体反応性）を維持している抗体断片を意味する。かかる抗原結合断片の一例としては、Ｆａｂ、Ｆ（ａｂ’）２、ｓｃＦｖ等が挙げられるがこれらに限定されない。かかる抗原結合断片は、自体公知の方法により調製することができる。本発明の検査方法に用いられる市販抗体としては、例えば、マウスモノクローナルＣａｔ－３１５　ＩｇＭ（Ｍｉｌｌｉｐｏｒｅ）やマウス抗ＰＴＰＲＺ　ＩｇＭκ（１２２．２）（Ｓａｎｔａ　ｃｒｕｚ　ｂｉｏｔｅｃｈｎｏｌｏｇｙ）が挙げられるがこれらに限定されない。 The term “antibody” in the present specification is a concept including “antigen-binding fragment”. The “antigen-binding fragment” means an antibody fragment that maintains the binding property (antigen-antibody reactivity) of the original antibody to the corresponding antigen. Examples of such antigen-binding fragments include, but are not limited to, Fab, F (ab ′) 2 , scFv and the like. Such an antigen-binding fragment can be prepared by a method known per se. Examples of commercially available antibodies used in the test method of the present invention include, but are not limited to, mouse monoclonal Cat-315 IgM (Millipore) and mouse anti-PTPRZ IgMκ (122.2) (Santa cruz biotechnology).
　本明細書において、抗体が抗原を「特異的に認識する」とは、抗原抗体反応における抗体の抗原に対する結合親和性についてのＫＤ値が、１×１０－５Ｍ以下（好ましくは、１×１０－６Ｍ以下、１×１０－７Ｍ以下、１×１０－８Ｍ以下、より好ましくは１×１０－９Ｍ以下、最も好ましくは１×１０－１０Ｍ以下）であることを意味する。 As used herein, an antibody is "specifically recognizes" an antigen, K D values for binding affinity of the antibody for antigen in the antigen-antibody reaction, 1 × 10 -5 M or less (preferably, 1 × 10 −6 M or less, 1 × 10 −7 M or less, 1 × 10 −8 M or less, more preferably 1 × 10 −9 M or less, most preferably 1 × 10 −10 M or less). .
　分泌型ＰＴＰＲＺアイソフォーム１および／または２の測定に用いられる抗体は、例えば、以下の抗体が例示される：
（１）分泌型ＰＴＰＲＺアイソフォーム１を特異的に認識する抗体
（２）分泌型ＰＴＰＲＺアイソフォーム２を特異的に認識する抗体
（３）分泌型ＰＴＰＲＺアイソフォーム１および２を特異的に認識する抗体。
　また、ＰＴＰＲＺは、生体内において糖転移酵素であるＧｎＴ－ＩＸにより、分岐型Ｏ－マンノース糖鎖により修飾される。以下の実施例で詳述するように、本発明者らは、脳脊髄液中の分泌型ＰＴＰＲＺアイソフォーム１および２もまた、分岐型Ｏ－マンノース糖鎖修飾されていることを明らかにしている。従って、抗ＰＴＰＲＺ抗体の代わりに、ＰＴＰＲＺ上の分岐型Ｏ－マンノース糖鎖を特異的に認識するＣａｔ－３１５抗体等の抗体も本発明の検査方法に用いることができる。これらの抗体は、自体公知の方法により調製できるほか、市販されているものを用いてもよい。このような観点によれば、本発明の検査方法を次のように定義することもできる：被検体由来の試料中の分岐型Ｏ－マンノース糖鎖修飾型受容体型タンパク質チロシンフォスファターゼＺ（ＰＴＰＲＺ）を測定する工程を含む、多発性硬化症の検査方法であって、該被検体由来の試料中の分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺ量の測定値が、対照群の試料中の分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺ量と比較して低い場合に、該被検体が多発性硬化症であることが示される、方法。 Examples of the antibody used for the measurement of secretory PTPRZ isoform 1 and / or 2 include the following antibodies:
(1) An antibody that specifically recognizes secreted PTPRZ isoform 1 (2) An antibody that specifically recognizes secreted PTPRZ isoform 2 (3) An antibody that specifically recognizes secreted PTPRZ isoform 1 and 2 .
PTPRZ is modified with a branched O-mannose sugar chain by GnT-IX which is a glycosyltransferase in vivo. As will be described in detail in the following examples, the present inventors have revealed that secreted PTPRZ isoforms 1 and 2 in cerebrospinal fluid are also modified with branched O-mannose sugar chains. . Therefore, instead of the anti-PTPRZ antibody, an antibody such as a Cat-315 antibody that specifically recognizes a branched O-mannose sugar chain on PTPRZ can also be used in the test method of the present invention. These antibodies can be prepared by a method known per se, or commercially available antibodies may be used. From such a viewpoint, the test method of the present invention can also be defined as follows: branched O-mannose sugar chain-modified receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject. A test method for multiple sclerosis comprising a step of measuring, wherein the measured value of the amount of branched O-mannose sugar chain-modified PTPRZ in a sample derived from the subject is the branched type O in the sample of the control group A method wherein the subject is shown to have multiple sclerosis when low compared to the amount of mannose sugar chain-modified PTPRZ.
　本発明の検査方法においては、被検体由来の試料中の分泌型ＰＴＰＲＺアイソフォーム１および／または２の量を測定する。以下の実施例においては脳脊髄液を試料として用いているが、脳脊髄液は静脈に流入することが知られているため、血液も用い得る。好ましい態様において、被検体由来の試料は脳脊髄液である。 In the test method of the present invention, the amount of secreted PTPRZ isoform 1 and / or 2 in the sample derived from the subject is measured. In the following examples, cerebrospinal fluid is used as a sample. However, since cerebrospinal fluid is known to flow into veins, blood can also be used. In a preferred embodiment, the sample from the subject is cerebrospinal fluid.
　本発明の検査方法において、被検体由来の試料中の分泌型ＰＴＰＲＺアイソフォーム１および／または２の測定値が、対照群の試料中の該タンパク質量と比較して、有意に低い場合に多発性硬化症であることが示される。なお、測定値の比較においては、予めカットオフ値を設定しておき、当該カットオフ値と被検体由来の試料中の分泌型ＰＴＰＲＺアイソフォーム１および／または２の量の測定値を対比してもよい。測定値がカットオフ値より低い場合に、被検体が多発性硬化症を罹患していることが示される。 In the test method of the present invention, when the measured value of the secreted PTPRZ isoform 1 and / or 2 in the sample derived from the subject is significantly lower than the amount of the protein in the sample of the control group, multiple occurrences occur. Shown to be sclerosis. In the comparison of measured values, a cutoff value is set in advance, and the measured value of the amount of secreted PTPRZ isoform 1 and / or 2 in the sample derived from the subject is compared. Also good. If the measured value is lower than the cutoff value, it indicates that the subject suffers from multiple sclerosis.
　カットオフ値の設定は、当業者であれば自体公知の方法を用いて設定することができる。例えば、既知の患者群および対照群（健常者または多発性硬化症を罹患していない対象者）から複数の試料を得て、分泌型ＰＴＰＲＺアイソフォーム１および／または２の量を測定し、望ましい感度および／または特異度で両群を判別できる値をカットオフ値として設定すればよい。 The person skilled in the art can set the cutoff value using a method known per se. For example, multiple samples are obtained from a known group of patients and a control group (a healthy person or a subject not suffering from multiple sclerosis) and the amount of secreted PTPRZ isoform 1 and / or 2 is measured and desirable A value that can distinguish both groups by sensitivity and / or specificity may be set as a cutoff value.
　本発明の検査方法が適用される被検体としては、ヒト、サル、ラット、イヌ、ネコ等の哺乳動物が例示され、特にヒトが好ましい。 As the subject to which the test method of the present invention is applied, mammals such as humans, monkeys, rats, dogs, cats and the like are exemplified, and humans are particularly preferable.
２．多発性硬化症の検査用キット
　本発明はまた、分泌型ＰＴＰＲＺアイソフォーム１および２のいずれかまたは両方を特異的に認識する抗体および分岐型Ｏ－マンノース糖鎖を特異的に認識する抗体のいずれかまたは両方を含む、多発性硬化症の診断用キット（以下、「本発明のキット」と称することがある）を提供する。本発明のキットを用いて、被検体由来の試料中における分泌型ＰＴＰＲＺアイソフォーム１および／または２の量を測定し、対照群の試料中の対応する量と比較することで、被検体が多発性硬化症に罹患しているか否かを簡便に検査することができる。 2. Test Kit for Multiple Sclerosis The present invention also includes an antibody that specifically recognizes one or both of secretory PTPRZ isoforms 1 and 2, and an antibody that specifically recognizes a branched O-mannose sugar chain. A kit for diagnosing multiple sclerosis (hereinafter sometimes referred to as “kit of the present invention”) is provided. By using the kit of the present invention, the amount of secreted PTPRZ isoform 1 and / or 2 in a sample derived from a subject is measured, and compared with the corresponding amount in a sample of a control group. It is possible to easily test whether or not the patient is suffering from multiple sclerosis.
　本発明のキットに含まれる分泌型ＰＴＰＲＺアイソフォーム１および／または２を特異的に認識する抗体は、以下の抗体が例示される：
（１）分泌型ＰＴＰＲＺアイソフォーム１を特異的に認識する抗体
（２）分泌型ＰＴＰＲＺアイソフォーム２を特異的に認識する抗体
（３）分泌型ＰＴＰＲＺアイソフォーム１および２を特異的に認識する抗体
（４）分岐型Ｏ－マンノース糖鎖を特異的に認識する抗体。
　尚、（４）分岐型Ｏ－マンノース糖鎖を特異的に認識する抗体は、自体公知の方法を用いて調製することができるほか、上述した通り、Ｃａｔ－３１５抗体などの市販抗体を用いてもよい。一態様において、本発明のキットは、上記（１）～（４）の抗体の１つ以上を構成成分として含む。また、本発明のキットは、取扱説明書や判定基準等をはじめとする、抗体以外の他の構成成分を含んでいてよい。 Examples of the antibody that specifically recognizes secreted PTPRZ isoform 1 and / or 2 contained in the kit of the present invention include the following antibodies:
(1) An antibody that specifically recognizes secreted PTPRZ isoform 1 (2) An antibody that specifically recognizes secreted PTPRZ isoform 2 (3) An antibody that specifically recognizes secreted PTPRZ isoform 1 and 2 (4) An antibody that specifically recognizes a branched O-mannose sugar chain.
In addition, (4) an antibody that specifically recognizes a branched O-mannose sugar chain can be prepared using a method known per se, and as described above, using a commercially available antibody such as Cat-315 antibody. Also good. In one embodiment, the kit of the present invention contains one or more of the antibodies (1) to (4) as a component. In addition, the kit of the present invention may contain other components other than antibodies, including instruction manuals and criteria.
　本発明の一態様において、本発明の多発性硬化症の検査方法は、多発性硬化症の治療方法と組み合わせてもよい。従って、本発明は、以下の工程を含む、多発性硬化症の検査及び治療方法を提供する：
　（１）被検体由来の試料中の分泌型受容体型タンパク質チロシンフォスファターゼＺ（ＰＴＰＲＺ）を測定する工程であって、ここで、該被検体由来の試料中の分泌型ＰＴＰＲＺ量の測定値が、対照群の試料中の分泌型ＰＴＰＲＺ量と比較して低い場合に、該被検体が多発性硬化症であることが示され、該分泌型ＰＴＰＲＺが、分泌型ＰＴＰＲＺアイソフォーム１及び分泌型ＰＴＰＲＺアイソフォーム２のいずれかまたは両方である、工程；および
　（２）工程（１）で多発性硬化症に罹患すると判定された被検体に対し、治療有効量の多発性硬化症の治療剤を投与する工程。 In one embodiment of the present invention, the method for examining multiple sclerosis of the present invention may be combined with a method for treating multiple sclerosis. Accordingly, the present invention provides a method for testing and treating multiple sclerosis comprising the following steps:
(1) A step of measuring secretory receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, wherein the measured value of the amount of secreted PTPRZ in the sample derived from the subject is a control When the amount of secreted PTPRZ in the sample of the group is low, the subject is shown to have multiple sclerosis, and the secreted PTPRZ is secreted PTPRZ isoform 1 and secreted PTPRZ isoform. Or (2) a step of administering a therapeutically effective amount of a therapeutic agent for multiple sclerosis to the subject determined to suffer from multiple sclerosis in step (1). .
　本発明に用いられる多発性硬化症の治療剤としては、例えば、ステロイド、インターフェロンβ、Ｓ１Ｐ受容体調節薬、免疫抑制薬、アルキル化薬、抗癌性抗生物質、抗ＣＤ５２抗体、および抗α４インテグリン抗体等の多発性硬化症の治療剤の投与が挙げられるが、これらに限定されない。 Examples of the therapeutic agent for multiple sclerosis used in the present invention include steroids, interferon β, S1P receptor modulators, immunosuppressants, alkylating agents, anticancer antibiotics, anti-CD52 antibodies, and anti-α4 integrins. Examples include, but are not limited to, administration of therapeutic agents for multiple sclerosis such as antibodies.
　ステロイドとしては、例えば、アムシノニド、コハク酸ヒドロコルチゾンナトリウム、コハク酸プレドニゾロンナトリウム、メチルコハク酸プレドニゾロンナトリウム、シクレソニド、ジフルプレドナート、プロピオン酸ベタメタゾン、デキサメタゾン、デフラザコート、トリアムシノロン、トリアムシノロンアセトニド、ハルシノニド、パルミチン酸デキサメタゾン、ヒドロコルチゾン、ピバル酸フルメタゾン、ブチル酢酸プレドニゾロン、ブデソニド、硫酸プラステロン、フロ酸モメタゾン、フルオシノニド、フルオシノロンアセトニド、フルドロキシコルチド、フルニソリド、プレドニゾロン、プロピロン酸アルクロメタゾン、プロピオン酸クロベタゾール、プロピオン酸デキサメタゾン、プロピオン酸デプロドン、プロピオン酸フルチカゾン、プロピオン酸ベクロメタゾン、ベタメタゾン、メチルプレドニゾロン、メチルプレドニゾロンスレプタネート、メチルプレドニゾロンナトリウムスクシネート、リン酸デキサメタゾンナトリウム、リン酸ヒドロコルチゾンナトリウム、リン酸プレドニゾロンナトリウム、吉草酸ジフルコルトロン、吉草酸デキサメタゾン、吉草酸ベタメタゾン、吉草酸酢酸プレドニゾロン、酢酸コルチゾン、酢酸ジフロラゾン、酢酸デキサメタゾン、酢酸トリアムシノロン、酢酸パラメサゾン、酢酸ハロプレドン、酢酸フルドロコルチゾン、酢酸プレドニゾロン、酢酸メチルプレドニゾロン、酪酸クロベタゾン、酪酸ヒドロコルチゾン、酪酸プロピオン酸ヒドロコルチゾン、酪酸プロピオン酸ベタメタゾンが挙げられる。 Steroids include, for example, amcinonide, hydrocortisone sodium succinate, prednisolone sodium succinate, prednisolone sodium methyl succinate, ciclesonide, difluprednate, betamethasone propionate, dexamethasone, deflazacote, triamcinolone acetonide, parminodide Flumetasone pivalate, prednisolone butyl acetate, budesonide, plasterone sulfate, mometasone furoate, fluocinonide, fluocinolone acetonide, fludroxycortide, flunisolide, prednisolone, alcrometasone propionate, clobetasol propionate, dexamethasone propionate, propionate Deprodon, propionate Ticazone, beclomethasone propionate, betamethasone, methylprednisolone, methylprednisolone streptinate, methylprednisolone sodium succinate, dexamethasone sodium phosphate, hydrocortisone sodium phosphate, sodium prednisolone phosphate, diflucortron valerate, dexamethasone valerate, yoshiyoshi Betamethasone herbate, prednisolone acetate valerate, cortisone acetate, diflorazone acetate, dexamethasone acetate, triamcinolone acetate, paramesazone acetate, halopredon acetate, fludrocortisone acetate, prednisolone acetate, methylprednisolone acetate, clobetasone butyrate, hydrocortisone butyrate, hydrocortisone butyrate propionate Examples include betamethasone propionate.
　Ｓ１Ｐ受容体調節薬としては、例えば、フィンゴリモド（fingolimod）、シポニモド（siponimod）、ポネシモド（ponesimod）、セラリフィモド（ceralifimod）、オザニモド（ozanimod）が挙げられる。 Examples of S1P receptor modulators include fingolimod (fingolimod), siponimod, ponesimod, ceralifimod, and ozanimod.
　免疫抑制薬としては、例えば、アザチオプリン、アスコマイシン、エベロリムス、サラゾスルファピリジン、シクロスポリン、シクロホスファミド、シロリムス、タクロリムス、ブシラミン、メトトレキサート、レフルノミド、テリフルノミドが挙げられる。 Examples of the immunosuppressant include azathioprine, ascomycin, everolimus, salazosulfapyridine, cyclosporine, cyclophosphamide, sirolimus, tacrolimus, bucillamine, methotrexate, leflunomide, and teriflunomide.
　アルキル化薬としては、例えば、塩酸ナイトロジェンマスタード－Ｎ－オキシド、シクロホスファミド、イホスファミド、メルファラン、チオテパ、カルボコン、ブスルファン、塩酸ニムスチン、ダカルバジン、ラニムスチン、カルムスチン、クロラムブシル、ベンダムスチン、メクロエタナミンが挙げられる。 Examples of the alkylating agent include nitrogen mustard-N-oxide hydrochloride, cyclophosphamide, ifosfamide, melphalan, thiotepa, carbocon, busulfan, nimustine hydrochloride, dacarbazine, ranimustine, carmustine, chlorambucil, bendamustine, mecloethanamine .
　抗癌性抗生物質としては、例えば、アクチノマイシンＤ、マイトマイシンＣ、塩酸ダウノルビシン、塩酸ドキソルビシン、塩酸アクラルビシン、ネオカルチノスタチン、塩酸ピラルビシン、（塩酸）エピルビシン、塩酸イダルビシン、クロモマイシンＡ３、（塩酸）ブレオマイシン、硫酸ペプロマイシン、テラルビシン、ジノスタチン・スチマラマー、ゲムツズマブオゾガマイシン、塩酸ミトキサントロンが挙げられる。 Anticancer antibiotics include, for example, actinomycin D, mitomycin C, daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride, neocartinostatin, pirarubicin hydrochloride, (epirubicin hydrochloride), idarubicin hydrochloride, chromomycin A3, (bleomycin hydrochloride) bleomycin , Pepromycin sulfate, terarubicin, dinostatin stimamarer, gemtuzumab ozogamicin, mitoxantrone hydrochloride.
　抗ＣＤ５２抗体としては、例えば、アレムツズマブ（alemtuzumab）が挙げられる。また、抗α４インテグリン抗体としては、例えば、ナタリズマブ（natalizumab）が挙げられる。 Examples of the anti-CD52 antibody include alemtuzumab. Examples of the anti-α4 integrin antibody include natalizumab.
　多発性硬化症の治療剤の治療有効量は、有効成分の種類、治療対象の体重および性別、または投与経路等によって異なり得るが、適切な投与量や投与方法は、当業者であれば適宜選択することができる。尚、治療剤の投与経路は、有効成分の種類によって経口または非経口的のいずれかを選択することができる。非経口投与には、例えば、皮下投与、筋肉内投与、腹腔内投与等が含まれるがこれらに限定されない。 The therapeutically effective amount of the therapeutic agent for multiple sclerosis may vary depending on the type of active ingredient, the body weight and sex of the subject to be treated, the administration route, etc. can do. In addition, the administration route of a therapeutic agent can select either oral or parenteral according to the kind of active ingredient. Parenteral administration includes, but is not limited to, subcutaneous administration, intramuscular administration, intraperitoneal administration, and the like.
　なお、本明細書における疾患の「治療」には、疾患の治癒のみならず、疾患の寛解および疾患の程度の改善も含まれ得る。 It should be noted that “treatment” of a disease in the present specification can include not only healing of the disease but also remission of the disease and improvement of the degree of the disease.
　以下の実施例において本発明を更に具体的に説明するが、本発明はこれらの例によってなんら限定されるものではない。 The present invention will be described more specifically in the following examples, but the present invention is not limited to these examples.
　全ての動物実験は、理化学研究所の動物実験実施規定に従って行った。ヒトの脳脊髄液および脳の免疫組織学的解析は福島県立医科大学で同大学の計画書に沿って行った。通常、脳脊髄液の採取するために腰椎穿刺を施行する場合、穿刺後に頭痛が起きることがあり、症状が強い場合は微熱、頭痛、嘔吐を伴うこともあるため、神経疾患が疑われる患者に対してのみ脳脊髄液を採取する。 All animal experiments were conducted in accordance with RIKEN animal experimentation regulations. Immunohistochemical analysis of human cerebrospinal fluid and brain was performed at Fukushima Medical University according to the university's plan. Usually, when performing lumbar puncture to collect cerebrospinal fluid, headache may occur after puncture, and patients with suspected neurological disorders may have slight fever, headache, or vomiting if symptoms are severe. Collect cerebrospinal fluid only.
［実施例１］免疫組織化学染色
　多発性硬化症患者脳のパラフィン包埋切片の脱パラフィン処理を次の通り行った。キシレンに３回、９９.５％エタノールに３回、９０％エタノールに１回、８０％エタノールに１回、７０％エタノールに１回、５分間ずつ浸し、流水中に５分間浸した。０.０１Ｍクエン酸緩衝液（ｐＨ８．５）中、１２１℃で５分間オートクレーブを行い、ＰＢＳ（ｐＨ７．８）に浸した。 [Example 1] Immunohistochemical staining A paraffin-embedded section of a multiple sclerosis patient's brain was deparaffinized as follows. The sample was immersed in xylene three times, 99.5% ethanol three times, 90% ethanol once, 80% ethanol once, 70% ethanol once, five minutes, and then immersed in running water for five minutes. The mixture was autoclaved in 0.01 M citrate buffer (pH 8.5) at 121 ° C. for 5 minutes and immersed in PBS (pH 7.8).
　多発性硬化症患者脳の切片をＰＢＳで洗浄した後、０．５％ＴｒｉｔｏｎＸ－１００－ＰＢＳに１５分間浸漬し、透過処理を行った。０．０５％Ｔｗｅｅｎ－２０－ＰＢＳ（ＰＢＳ－Ｔ）中で５分間ずつ３回振とうして洗浄した後、５％正常ヤギ血清PBS中で３０分間振とうした。ＰＢＳ－Ｔ中で５分間ずつ２回洗浄後、Ｄａｋｏ ＲＥＡＬ ａｎｔｉｂｏｄｙ ｄｉｌｕｅｎｔ（Ｄａｋｏ）で希釈した一次抗体（マウスモノクローナルＣａｔ－３１５　ＩｇＭ（Ｍｉｌｌｉｐｏｒｅ）、マウス抗ＰＴＰＲＺ　ＩｇＭκ（１２２．２）（Ｓａｎｔａ　ｃｒｕｚ　ｂｉｏｔｅｃｈｎｏｌｏｇｙ）に室温で２時間または４℃で一晩浸漬した。希釈倍率ＰＢＳ－Ｔ中で５分間ずつ３回洗浄した後、ＤＡＫＯ ＲＥＡＬ ａｎｔｉｂｏｄｙ ｄｉｌｕｅｎｔで希釈したＡｌｅｘａ ｆｌｕｏｒ ５４６－または４８８－標識二次抗体およびＤＡＰＩに室温で４５分間浸漬した。ＰＢＳ－Ｔ中で５分間ずつ３回洗浄後、ＣＣ/Ｍｏｕｎｔ中で封入し、ＦＶ１０００－Ｄ共焦点レーザー顕微鏡（Ｏｌｙｍｐｕｓ）で観察した。得られた染色図のシグナル強度の解析を、ＭｅｔａＭｏｒｐｈ（Ｏｌｙｍｐｕｓ）により行った（図１）。その結果、Ｃａｔ－３１５陽性細胞とＰＴＰＲＺ陽性細胞が一致しており、Ｃａｔ－３１５陽性細胞はＰＴＰＲＺ陽性であることが明らかとなった。 After the section of the multiple sclerosis patient brain was washed with PBS, it was immersed in 0.5% Triton X-100-PBS for 15 minutes and subjected to permeation treatment. After washing by shaking 3 times for 5 minutes each in 0.05% Tween-20-PBS (PBS-T), it was shaken in 5% normal goat serum PBS for 30 minutes. After washing twice in PBS-T for 5 minutes each, the primary antibody (mouse monoclonal Cat-315 IgM (Millipore), mouse anti-PTPRZ IgMκ (122.2) (Santa cruz biotechnology) diluted with Dako REAL antibody diluent (Dako) For 2 hours at room temperature or overnight at 4 ° C. After washing 3 times for 5 minutes each in PBS-T at a dilution ratio, Alexa fluor 546- or 488-labeled secondary antibody diluted with DAKO REAL antibody diluent and DAPI For 45 minutes at room temperature, washed 3 times for 5 minutes each in PBS-T, encapsulated in CC / Mount, and observed with FV1000-D confocal laser microscope (Olympus). Signal intensity analysis was performed with MetaMorph (Olympus) (FIG. 1), which revealed that Cat-315 positive cells and PTPRZ positive cells were in agreement, and that Cat-315 positive cells were PTPRZ positive. became.
［実施例２］
　多発性硬化症患者由来の脳脊髄液に対し、視神経脊髄炎（ｎｅｕｒｏｍｙｅｌｉｔｉｓ　ｏｐｔｉｃａ、ＮＭＯ）患者由来の脳脊髄液、および特発性正常圧水頭症（ｉｄｉｏｐａｔｈｉｃ　ｎｏｒｍａｌ　ｐｒｅｓｓｕｒｅ　ｈｙｄｒｏｃｅｐｈａｌｕｓ、ｉＮＰＨ）の疑いがあったが診断の結果ｉＮＰＨではないと判断された被検体（ｎｏｎ－ｉＮＰＨ、対照群として使用）を、多発性硬化症の疾患コントロールとして用いた。これらの脳脊髄液は、福島県立医科大学附属病院を中心とした医療機関で得られた患者試料であり、全患者からインフォームドコンセントを取得した。使用した抗体は、マウスモノクローナルＣａｔ－３１５　ＩｇＭ（Ｍｉｌｌｉｐｏｒｅ）、マウス抗ＰＴＰＲＺ　ＩｇＭκ（１２２．２）（Ｓａｎｔａ　ｃｒｕｚ　ｂｉｏｔｅｃｈｎｏｌｏｇｙ）、ヤギポリクローナル抗Ｔｒａｎｓｔｈｙｒｅｔｉｎ（ＴＴＲ）抗体（Ａｂｃａｍ）を用いた。その他の試薬は主にＷａｋｏより購入した。 [Example 2]
Cerebrospinal fluid from a patient with multiple sclerosis was suspected to have cerebrospinal fluid from a neuromyelitis optica (NMO) patient and idiopathic normal pressure hydrocephalus (iNPH). A subject determined to be not iNPH as a result of diagnosis (non-iNPH, used as a control group) was used as a disease control for multiple sclerosis. These cerebrospinal fluids were patient samples obtained at medical institutions such as Fukushima Medical University Hospital, and informed consent was obtained from all patients. The antibodies used were mouse monoclonal Cat-315 IgM (Millipore), mouse anti-PTPRZ IgMκ (122.2) (Santa cruz biotechnology), goat polyclonal anti-Transtyretin (TTR) antibody (Abcam). Other reagents were purchased mainly from Wako.
　ＭＳ患者（ｎ＝３）、ＮＭＯ患者（ｎ＝３）、ｎｏｎ－ｉＮＰＨ患者（ｎ＝３、コントロール）由来の脳脊髄液１０μＬにコンドロイチナーゼ消化を行い、ウェスタンブロッティングを行った。なお、コンドロイチナーゼ消化の詳細は次の通りである。試料溶液に、終濃度５０ｍＭ Ｔｒｉｓ－ＨＣｌ（ｐＨ８．０）、３０ｍＭ酢酸ナトリウム、２００Ｕ／Ｌ Ｃｈｏｎｄｒｏｉｔｉｎａｓｅ ＡＢＣ（Ｓｅｉｋａｇａｋｕ）を加え、３７℃で１時間攪拌した。 Chondroitinase digestion was performed on 10 μL of cerebrospinal fluid from MS patients (n = 3), NMO patients (n = 3), non-iNPH patients (n = 3, control), and Western blotting was performed. The details of the chondroitinase digestion are as follows. A final concentration of 50 mM Tris-HCl (pH 8.0), 30 mM sodium acetate, 200 U / L Chondroitinase ABC (Seikagaku) was added to the sample solution, and the mixture was stirred at 37 ° C. for 1 hour.
　ウェスタンブロッティングは以下の手順にて行った。試料溶液にＬａｅｍｍｌｉのＳａｍｐｌｅ　ｂｕｆｆｅｒ（終濃度２０．８ｍＭ　Ｔｒｉｓ－ＨＣｌ（ｐＨ６．５）、８．３％グリセロール、１％ＳＤＳ、１％βメルカプトエタノール）を加え、９９℃で５分加熱した。３－１０％グラジエントポリアクリルアミドゲル（Ａｔｔｏ）を用いてゲル１枚あたり２０－２５ｍＡの定電流で６０－７０分間泳動した。セミドライ式の転写装置を用いて、ニトロセルロース膜に１５０ｍＡの定電流で９０分間転写を行った。５％スキムミルク－０．０５％Ｔｗｅｅｎ２０－ＴＢＳ（ｐＨ７．４）（ＴＢＳ－Ｔ）に３０分間浸漬してブロッキングを行った後、ＴＢＳ－Ｔで希釈した一次抗体中で室温で２時間または４℃で一晩振とうした。ＴＢＳ－Ｔ中に５分間ずつ３回浸漬して洗浄したのち、ＴＢＳ－Ｔで希釈したＨＲＰ標識二次抗体と室温で４０分間反応させた。ＴＢＳ－Ｔで３回洗浄後、ＳｕｐｅｒＳｉｇｎａｌ　Ｗｅｓｔ　Ｆｅｍｔｏ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）を用いて発光させ、シグナルをＬＡＳ４０００　ｍｉｎｉ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）で検出した。なお、バンドのシグナル強度の定量は、ＬＡＳ４０００　ｍｉｎｉ付属の定量ソフトを用いて行った。結果を表１に示す。表１は、ＭＳ患者由来、ＭＮＯ患者由来、ｎｏｎ－ｉＮＰＨ由来の試料において、脳脊髄液の内部標準として検出したＴＴＲのシグナル強度に対する分泌型ＰＴＰＲＺアイソフォーム１および２の上段のバンドのシグナル強度あるいはＣａｔ－３１５抗体で検出される上部のバンドのシグナル強度の相対値として示したものである。 Western blotting was performed according to the following procedure. Laemmli Sample buffer (final concentration 20.8 mM Tris-HCl (pH 6.5), 8.3% glycerol, 1% SDS, 1% β-mercaptoethanol) was added to the sample solution, and heated at 99 ° C. for 5 minutes. The gel was run for 60-70 minutes at a constant current of 20-25 mA per gel using a 3-10% gradient polyacrylamide gel (Atto). Using a semi-dry type transfer device, transfer was performed on a nitrocellulose membrane at a constant current of 150 mA for 90 minutes. Blocking was performed by immersing in 5% skim milk-0.05% Tween20-TBS (pH 7.4) (TBS-T) for 30 minutes, and then in a primary antibody diluted with TBS-T for 2 hours at room temperature or 4 ° C. Shake overnight. After washing by immersing in TBS-T three times for 5 minutes, each was reacted with an HRP-labeled secondary antibody diluted with TBS-T for 40 minutes at room temperature. After washing with TBS-T three times, light was emitted using SuperSignal West Femto (Thermo Fisher Scientific), and the signal was detected with LAS4000 mini (GE Healthcare). The signal intensity of the band was quantified using the quantification software attached to LAS4000 mini. The results are shown in Table 1. Table 1 shows the signal intensity of the upper band of secreted PTPRZ isoforms 1 and 2 relative to the signal intensity of TTR detected as an internal standard of cerebrospinal fluid in samples derived from MS patients, MNO patients, and non-iNPH. It is shown as the relative value of the signal intensity of the upper band detected with the Cat-315 antibody.
　表１に示される通り、多発性硬化症患者由来の脳脊髄液においては、ＰＴＰＲＺアイソフォーム１および２の細胞外領域に由来すると考えられる上段バンド（ＰＴＰＲＺアイソフォーム２全長はＰＴＰＲＺアイソフォーム１全長と７アミノ酸の違いしかなく、また、両者の細胞外領域のアミノ酸配列は同一であるため、同じバンドとして検出される。約３５０ｋＤａ）および分泌型ＰＴＰＺアイソフォーム３の細胞外領域に由来すると考えられる下段バンド（約１８０ｋＤａ）のシグナルが検出された。このうち、多発性硬化症患者由来の脳脊髄液では、対照と比較して、上段のバンドのシグナル量が著しく減少していることが分かった。また、対応する分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺのシグナル量も同様に減少していた。興味深いことに、ＮＭＯ患者の検体でもＭＳ患者の検体と同レベルまで低下している検体があった。ＮＭＯはアストロサイトのアクアポリン４が自己抗体によって攻撃されることで炎症が起こる疾患だが、炎症が進むことで脱髄を起こすと考えられている。従って、一部のＮＭＯ患者の検体において分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺの減少がみられたことは、当該患者においてＭＳと同様に脱髄が起こっていることを示している可能性が考えられる。以上より、脳脊髄液中の分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺの量は脱髄の程度を反映するバイオマーカーとして利用し得ることが示された。 As shown in Table 1, in cerebrospinal fluid derived from a patient with multiple sclerosis, the upper band considered to be derived from the extracellular region of PTPRZ isoforms 1 and 2 (PTPRZ isoform 2 full length is PTPRZ isoform 1 full length and The difference is only 7 amino acids, and the amino acid sequences of both extracellular regions are the same, so that they are detected as the same band (approximately 350 kDa) and the lower stage considered to be derived from the extracellular region of secreted PTPZ isoform 3. A band (approximately 180 kDa) signal was detected. Among these, in the cerebrospinal fluid derived from a patient with multiple sclerosis, it was found that the signal amount of the upper band was remarkably reduced as compared with the control. Similarly, the signal amount of the corresponding branched O-mannose sugar chain-modified PTPRZ also decreased. Interestingly, some samples from NMO patients were down to the same level as those from MS patients. NMO is a disease that causes inflammation when astrocyte aquaporin 4 is attacked by autoantibodies, but is thought to cause demyelination as inflammation progresses. Therefore, a decrease in branched O-mannose sugar chain-modified PTPRZ in some NMO patient specimens may indicate that demyelination is occurring in the patient as in MS. Conceivable. From the above, it was shown that the amount of branched O-mannose sugar chain-modified PTPRZ in cerebrospinal fluid can be used as a biomarker reflecting the degree of demyelination.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
［実施例３］髄液中の分泌型ＰＴＰＲＺアイソフォーム１および２のＭＳ検出用マーカーとしての検証
　以下の実験を通じて、分泌型ＰＴＰＲＺ（分泌型ＰＴＰＲＺアイソフォーム１および２（即ち、分泌型ＰＴＰＲＺ ｌｏｎｇ－ｆｏｒｍ））がＭＳ検出用マーカーとして機能し得ることを検証した。 [Example 3] Verification of secretory PTPRZ isoforms 1 and 2 in cerebrospinal fluid as a marker for MS detection Through the following experiment, secreted PTPRZ (secreted PTPRZ isoforms 1 and 2 (ie, secreted PTPRZ long- It was verified that form)) can function as a marker for MS detection.
　多発性硬化症（ＭＳ）患者群（２４名）、および特発性正常圧水頭症（ｉＮＰＨ）患者（疑い例含む）群（２５名）のそれぞれに由来する髄液中の分泌型ＰＴＰＲＺアイソフォーム１および２の量をウェスタンブロッティングで測定した。 Secreted PTPRZ isoform 1 in cerebrospinal fluid from each of the multiple sclerosis (MS) patient group (24 patients) and the idiopathic normal pressure hydrocephalus (iNPH) patient group (including suspected cases) (25 patients) The amount of 2 and 2 was measured by Western blotting.
　抗体は、抗ＰＴＰＲＺ抗体であるマウス抗ＰＴＰζ ＩｇＭκ (ｓｃ―３３６６４、Ｓａｎｔａ ｃｒｕｚ)、及びヤギ抗マウスＩｇＭ抗体-ＨＲＰ(ＳＡＢ－１１０、Ｓｔｒｅｓｓｇｅｎ)を使用した。マウス抗ＰＴＰＺ ＩｇＭκはラット胎児脳からプロテオグリカン画分を調製して作製されたＰＴＰＲＺ特異的モノクローナル抗体である。その他の試薬は、主にＷａｋｏ社より購入した。 The antibodies used were anti-PTPRZ antibodies, mouse anti-PTPζ IgMκ (sc-33664, Santa cruz) and goat anti-mouse IgM antibody-HRP (SAB-110, Stressgen). Mouse anti-PTPZ IgMκ is a PTPRZ-specific monoclonal antibody prepared by preparing a proteoglycan fraction from rat fetal brain. Other reagents were purchased mainly from Wako.
　上記各患者から採取した１０μＬの髄液を、まずコンドロイチナーゼ消化に供した。具体的には、試料溶液に、終濃度１００ｍＭ Ｔｒｉｓ－ＨＣｌ(ｐＨ７．５)、６０ｍＭ 酢酸ナトリウム、４ｍＵコンドロイチナーゼＡＢＣ（Ｓｉｇｍａ、２９０５）を添加し、３７℃にて１時間インキュベートした。 10 μL of cerebrospinal fluid collected from each patient was first subjected to chondroitinase digestion. Specifically, a final concentration of 100 mM Tris-HCl (pH 7.5), 60 mM sodium acetate, 4 mU chondroitinase ABC (Sigma, 2905) was added to the sample solution and incubated at 37 ° C. for 1 hour.
　その後、ウェスタンブロッティングを行った。ウェスタンブロッティングは以下の手順で行った。コンドロイチナーゼ処理した試料溶液にＬａｅｍｍｌｉのＳａｍｐｌｅ　ｂｕｆｆｅｒ（終濃度２０．８ｍＭ　Ｔｒｉｓ－ＨＣｌ（ｐＨ６．５）、８．３％グリセロール、１％ＳＤＳ、１％βメルカプトエタノール）を加え、９９℃で５分間加熱した。３～１０％グラジエントポリアクリルアミドゲル（Ａｔｔｏ社、ＮＰＧ－３１０Ｌ）を用いてゲル１枚あたり２０ｍＡ～２５ｍＡの定電流で６０分間～７０分間泳動した。ウェット式の転写装置を用いて、ニトロセルロース膜に３５０ｍＡの定電流で４５分間転写を行った。１％ＢＳＡ－ＰＢＳ（ｐＨ７．４）に４℃で１晩浸漬してブロッキングを行った後、ＰＢＳ－０．１％Ｔｗｅｅｎ（ＰＢＳ－Ｔ）で２００倍希釈したマウス抗ＰＴＰＲＺ　ＩｇＭκ（Ｓａｎｔａ　ｃｒｕｚ、ｓｃ－３３６６４）を一次抗体として含む１．５ｍＬのＰＢＳ－Ｔ中で室温にて２時間振とうした。続いて、ＰＢＳ－Ｔ中に５分間ずつ３回浸漬して洗浄した後、二次抗体として１０，０００倍希釈したＨＲＰ標識化ヤギ抗マウスＩｇＭ抗体（ＳＡＢ－１１０、Ｓｔｒｅｓｓｇｅｎ）を含むＰＢＳ－Ｔと室温で２時間反応させた。ＴＢＳ－Ｔで３回洗浄後、ＳｕｐｅｒＳｉｇｎａｌ　Ｗｅｓｔ　Ｆｅｍｔｏ　ｍａｘｉｍｕｍ　ｓｅｎｓｉｔｉｖｉｔｙ　ｓｕｂｓｔｒａｔｅ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社、　３４０９６）を用いて発色させて、得られたシグナルをＡＴＴＯ　Ｅｚ－Ｃａｐｔｕｒｅ　ＭＧ（ＡＴＴＯ）で検出した。なお、バンドのシグナル強度の定量は、付属の定量ソフトＣＳ　Ａｎａｌｙｚｅｒを用いて行った。結果を図３に示す。 After that, Western blotting was performed. Western blotting was performed according to the following procedure. Laemmli Sample buffer (final concentration 20.8 mM Tris-HCl (pH 6.5), 8.3% glycerol, 1% SDS, 1% β-mercaptoethanol) was added to the chondroitinase-treated sample solution, and 5% at 99 ° C. Heated for minutes. Using a 3 to 10% gradient polyacrylamide gel (Atto, NPG-310L), electrophoresis was performed at a constant current of 20 to 25 mA per gel for 60 to 70 minutes. Using a wet type transfer device, transfer was performed on a nitrocellulose film at a constant current of 350 mA for 45 minutes. After blocking by immersion in 1% BSA-PBS (pH 7.4) overnight at 4 ° C., mouse anti-PTPRZ IgMκ (Santa cruz, 200-fold diluted with PBS-0.1% Tween (PBS-T) The mixture was shaken in 1.5 mL of PBS-T containing sc-33664) as a primary antibody at room temperature for 2 hours. Subsequently, PBS-T containing HRP-labeled goat anti-mouse IgM antibody (SAB-110, Stressgen) diluted 10,000 times as a secondary antibody was washed by immersing in PBS-T three times for 5 minutes each. And at room temperature for 2 hours. After washing 3 times with TBS-T, the color was developed using SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher Scientific, 34096), and the resulting signal was detected with ATTO Ez-CaptureTO (ATTO Ez-CaptureTO). The signal intensity of the band was quantified using the attached quantification software CS Analyzer. The results are shown in FIG.
　図３に示される通り、ＭＳ患者由来の髄液中の分泌型ＰＴＰＲＺアイソフォーム１および２のシグナル強度は、コントロール群（特発性正常圧水頭症患者（疑い例を含む）群）由来の髄液中のそれと比較して、有意に低かった。この結果から、分泌型ＰＴＰＲＺアイソフォーム１および２がＭＳ検出用マーカーとなり得ることが示された。 As shown in FIG. 3, the signal intensity of secreted PTPRZ isoforms 1 and 2 in the cerebrospinal fluid from MS patients is the cerebrospinal fluid from the control group (idiopathic normal pressure hydrocephalus patient (including suspected cases) group) It was significantly lower than that in the middle. From this result, it was shown that secretory PTPRZ isoforms 1 and 2 can be markers for MS detection.
　本発明によれば、脱髄疾患（特には多発性硬化症）の確定診断に有用な新たな客観的指標が提供される。従って、本発明は医療分野において極めて有益である。 According to the present invention, a new objective index useful for definitive diagnosis of demyelinating diseases (particularly multiple sclerosis) is provided. Therefore, the present invention is extremely useful in the medical field.
　本出願は、日本で出願された特願２０１８－０２８３２９（出願日：２０１８年２月２０日）を基礎としており、その内容は本明細書に全て包含されるものである。 This application is based on Japanese Patent Application No. 2018-028329 (filing date: February 20, 2018) filed in Japan, the contents of which are incorporated in full herein.

Claims (5)

  1. 　被検体由来の試料中の分泌型受容体型タンパク質チロシンフォスファターゼＺ（ＰＴＰＲＺ）を測定する工程を含む、多発性硬化症の検査方法であって、該被検体由来の試料中の分泌型ＰＴＰＲＺ量の測定値が、対照群の試料中の分泌型ＰＴＰＲＺ量と比較して低い場合に、該被検体が多発性硬化症であることが示され、該分泌型ＰＴＰＲＺが、分泌型ＰＴＰＲＺアイソフォーム１及び分泌型ＰＴＰＲＺアイソフォーム２のいずれかまたは両方である、方法。 A test method for multiple sclerosis comprising a step of measuring secretory receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, and measuring the amount of secreted PTPRZ in the sample derived from the subject When the value is low compared to the amount of secreted PTPRZ in the sample of the control group, the subject is shown to have multiple sclerosis, and the secreted PTPRZ is expressed as secreted PTPRZ isoform 1 and secreted. A method which is either or both of type PTPRZ isoform 2.
  2. 　前記分泌型ＰＴＰＲＺアイソフォーム１および分泌型ＰＴＰＲＺアイソフォーム２が、分岐型Ｏ－マンノース糖鎖修飾型ＰＴＰＲＺである、請求項１記載の方法。 The method according to claim 1, wherein the secretory PTPRZ isoform 1 and the secretory PTPRZ isoform 2 are branched O-mannose sugar chain-modified PTPRZ.
  3. 　被検体由来の試料が、脳脊髄液または血液である、請求項１または２記載の方法。 The method according to claim 1 or 2, wherein the sample derived from the subject is cerebrospinal fluid or blood.
  4. 　被検体由来の脊髄液中の分泌型ＰＴＰＲＺの測定が、該タンパク質を特異的に認識する抗体または分岐型Ｏ－マンノース糖鎖を特異的に認識する抗体を用いて行われることを特徴とする、請求項１～３のいずれか一項記載の方法。 The measurement of secretory PTPRZ in spinal fluid derived from a subject is performed using an antibody that specifically recognizes the protein or an antibody that specifically recognizes a branched O-mannose sugar chain, The method according to any one of claims 1 to 3.
  5. 　分泌型ＰＴＰＲＺを特異的に認識する抗体および分岐型Ｏ－マンノース糖鎖を特異的に認識する抗体のいずれかまたは両方を含む、多発性硬化症の診断用キットであって、該分泌型ＰＴＰＲＺが、分泌型ＰＴＰＲＺアイソフォーム１及び分泌型ＰＴＰＲＺアイソフォーム２のいずれかまたは両方である、キット。 A diagnostic kit for multiple sclerosis comprising either or both of an antibody specifically recognizing secretory PTPRZ and an antibody specifically recognizing a branched O-mannose sugar chain, wherein the secretory PTPRZ comprises A kit, which is either or both of secretory PTPRZ isoform 1 and secreted PTPRZ isoform 2.
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Citations (2)

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