JPWO2019163838A1 - Multiple sclerosis marker - Google Patents

Abstract

The present invention is a method for testing multiple sclerosis, which comprises a step of measuring a secretory receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, and the secretory type in the sample derived from the subject. When the measured value of the PTPRZ amount is lower than the PTPRZ amount in the sample of the control group, it is indicated that the subject has multiple sclerosis, and the secretory PTPRZ is the secretory PTPRZ isoform 1. And one or both of secretory PTPRZ isoforms 2 are provided.

Description

The present invention relates to a method for testing demyelinating diseases (particularly multiple sclerosis) and the like, and more particularly, the receptor-type protein tyrosine phosphatase Z (Protein Tyrosine Phosphase Receptor-type Z; PTPRZ) in a sample derived from a subject, More preferably, the present invention relates to a method for examining multiple sclerosis, which comprises a step of measuring a branched O-mannose sugar chain-modified PTPRZ.

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 neuropathy such as motor paralysis and sensory impairment. It is common in Caucasians in Europe and the United States, and in Northern Europe, it affects about 50 to 100 out of 100,000 people. In Japan, the number is as small as 10 out of 100,000 (Non-Patent Document 1), but it is increasing year by year, and the number of patients is more than 2.5 million worldwide.

The clinical picture of multiple sclerosis can be broadly divided into (1) relapsing-remitting MS (RRMS), (2) primary progressive MS (PPMS), and (3) secondary progressive (3) secondary sclerosis (RPMS). It is classified into three categories: Secondary progressive MS (SPMS). RRMS is characterized by alternating worsening of symptoms (recurrence) and stabilization of symptoms (remission), with repeated remission periods and recurrences of months to years. PPMS is characterized by a temporary stagnation period in which the condition does not progress, but the condition gradually progresses without remission. SPMS is characterized by repeated recurrence and remission in the early stage of onset, but gradually progressing. At present, no curative treatment has been established for multiple sclerosis, and symptomatic treatment is performed according to the time of onset and symptoms of the patient.

One of the factors that makes the treatment of multiple sclerosis difficult is the difficulty of its diagnosis. Multiple sclerosis is difficult to diagnose by a doctor because of its diverse symptoms and no biomarkers specific to the disease. In particular, when the lesion is solitary, it becomes difficult to distinguish it from brain tumors and encephalitis / encephalopathy. Currently, the diagnosis of multiple sclerosis is made by MRI and cerebrospinal fluid tests, but the facilities where MRI tests are possible are limited, and the cerebrospinal fluid tests are not specific to 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 the affected area with a risk (Non-Patent Document 7). Treatment such as administration of steroids to suppress immunity is performed for multiple sclerosis, whereas surgery, radiation therapy, and drug treatment are combined for treatment of brain tumors. Accurate diagnosis is important because the treatment approach differs for each disease. Nevertheless, at present, it is very difficult to diagnose multiple sclerosis at an early stage, and in fact, from the appearance of the first symptoms of multiple sclerosis to the definitive diagnosis of multiple sclerosis. Some survey results show that the average period exceeds 3 years.

Against this background, there is a strong demand for the establishment of a test method that can more easily diagnose multiple sclerosis.

Receptor-type protein tyrosine phosphatases are classified into eight subfamilies based on their structural similarities (Non-Patent Document 2). The extracellular space 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 proximal side of the cell membrane has phosphatase activity, and D2 has no enzymatic activity (exceptionally, D2 of the R4 RPTP subfamily has some activity). ).

The receptor-type protein tyrosine phosphatase Z (also referred to as Protein Tyrosine Phosphatase Receptor-type Z1; PTPRZ, PTPRZ1, or Ptprz, PTPζ, RPTPβ, etc.) belongs to the R5 subfamily. There are splicing variants in mammalian PTPRZ, for example in mice, which results in the presence of three isoforms (receptor types PTPRZ-A and PTPRZ-B, and secretory 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 a sulfated polysaccharide modification called sulfated glycosaminoglycan such as chondroitin sulfate or keratan sulfate in the extracellular region. In brain tissue, which is the main expression site of PTPRZ, all isoforms are expressed as chondroitin sulfate proteoglycan (Non-Patent Documents 3 to 5). PTPRZ is expressed in 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, Mannoseyl (Alpha-1,6-)-Glycoprotein Beta-1, 6-N-Aceticl-Glucosaminyltransphase, Isozyme. It has been reported that it undergoes branched O-mannose modification via (Non-Patent Document 6).

Kinoshita M. et al., Neurol Sci. 2015; 36 (7): 1147-51. Tonks NK, Nat Rev Mol Cell Biol. 2006 Nov; 7 (11): 833-46. Nishiwaki T. et al., J Biochem. 1998 Mar; 123 (3): 458-67. Chow JP. Et al., J Biol Chem. 2008 Nov 7; 283 (45): 30879-89. Chow JP. Et al., Neurosci Lett. 2008 Sep 19; 442 (3): 208-12. Kanekiyo K. et al., J Neurosci. 2013 Jun 12; 33 (24): 10037-47. Nakazawa Y. et al., Jpn. J. Cli. Immunol. 2013; 36 (3): 175-9

An object of the present invention is to provide a novel test method for demyelinating diseases (particularly multiple sclerosis).

As a result of diligent studies on the above problems, the present inventors have conducted a secretory (free) PTPRZ iso corresponding to the extracellular region of PTPRZ isoforms 1 and 2 in the cerebrospinal fluid of a patient with demyelinating disease. It was found that the content of Foams 1 and 2 was reduced compared to that of the control group. In particular, it was found that the content of secretory PTPRZ isoforms 1 and 2 in the cerebrospinal fluid of patients with multiple sclerosis was significantly reduced. Even more surprisingly, it was found that the amount of branched O-mannose sugar chain-modified PTPRZ produced by sugar chain modification of PTPRZ by the glycosyltransferase GnT-IX was also reduced in the cerebrospinal fluid from the target group. The present invention has been completed by further research based on such findings. That is, the present invention is as follows.

[1] A method for testing multiple sclerosis, which comprises a step of measuring a 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 is used. When the measured amount is low compared to the amount of secretory PTPRZ in the sample of the control group, it indicates that the subject has multiple sclerosis, and the secretory PTPRZ is a secretory PTPRZ isoform. 1 and / or both of secretory PTPRZ isoforms 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 the 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 kit for diagnosing multiple sclerosis, which comprises either or both of an antibody that specifically recognizes secretory PTPRZ and an antibody that specifically recognizes a branched O-mannose sugar chain, and the secretion thereof. A kit in which the type PTPRZ is one or both of secretory PTPRZ isoform 1 and secretory PTPRZ isoform 2.
[6] A method for examining a demyelinated state, which comprises a step of measuring the secretory receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, and the amount of secretory PTPRZ in the sample derived from the subject. When the measured value of is lower than the amount of secretory PTPRZ in the sample of the control group, it is shown that the subject is demyelinated, and the secretory PTPRZ is the secretory PTPRZ isoform 1. And one or both of secretory PTPRZ isoforms 2, the method.
[7] A method for examining a demyelinated state, which comprises a step of measuring a branched O-mannose sugar chain-modified receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, which is derived from the subject. When the measured value of the branched O-mannose sugar chain-modified PTPRZ amount in the sample is lower than the branched O-mannose sugar chain-modified PTPRZ amount in the sample of the control group, the subject is demyelinated. The branched O-mannose sugar chain-modified PTPRZ is the branched O-mannose sugar chain-modified PTPRZ isoform 1 and the branched O-mannose sugar chain-modified PTPRZ isoform 2. A method that is either or both.

In another aspect, the present invention is as follows.
[1A] A method for testing multiple sclerosis, which comprises a step of measuring the receptor-type protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, and the amount of secreted PTPRZ in the sample derived from the subject. A method, wherein the subject is indicated 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] 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 secretory PTPRZ isoform 1, secretory PTPRZ isoform 2, and secretory PTPRZ isoform 3, [1A] to [ 4A].
[6A] A diagnostic kit for multiple sclerosis, which comprises an antibody that specifically recognizes PTPRZ and an antibody that specifically recognizes a branched O-mannose sugar chain, or both.
[7A] A method for examining a demyelinated state, which comprises a step of measuring a receptor-type 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 value is low compared to the amount of PTPRZ in the sample of the control group, indicating that the subject is demyelinated.
[8A] A method for examining a demyelinated state, which comprises a step of measuring a branched O-mannose sugar chain-modified receptor protein tyrosine phosphatase Z (PTPRZ) in a sample derived from a subject, and is derived from the subject. When the measured value of the branched O-mannose sugar chain-modified PTPRZ amount in the sample is lower than the branched O-mannose sugar chain-modified PTPRZ amount in the sample of the control group, the subject is demyelinated. The method that is shown to be causing.

The present invention provides a new objective index useful for the definitive diagnosis of multiple sclerosis.

It is a conceptual diagram which shows the structure of three kinds of isoforms of human PTPRZ. A indicates isoform 1, B indicates isoform 2, and C indicates isoform 3. In the figure, CA represents a carbonic amphidrase-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 all represent PTP domains. FIG. 2 shows the results of immunohistochemical staining using Cat-315 antibody or anti-PTPRZ antibody in brain sections of patients with multiple sclerosis. FIG. 3 shows the amount of secretory (free) PTPRZ isoform 1 and secretory (free) PTPRZ isoform 2 in the cerebrospinal fluid in patients with multiple sclerosis suffering from idiopathic normal pressure hydrocephalus (control group). It is a graph which shows that it is remarkably decreased as compared with that of). The data is shown as mean value + standard error (SE).

Hereinafter, the present invention will be described in detail.

1. 1. Testing Method for Multiple Sclerosis The present invention comprises the step of measuring one or both of the secretory receptor protein tyrosine phosphatase Z (PTPRZ) isoforms 1 and 2 in a sample derived from a subject. (Hereinafter, it may be referred to as "the inspection method of the present invention").

Multiple sclerosis, which can be tested for morbidity by the test method of the present invention, can be classified into three types as described above. According to the present invention, any type of relapsing-remitting type (RRMS), primary progressive type (PPMS), and (3) secondary progressive type (SPMS) can be examined.

In one aspect of the test method of the present invention, by measuring the content of either or both of the secretory PTPRZ isoforms 1 and 2 in the sample derived from the subject as an index and comparing it with the corresponding value of the control group. , The presence or absence of multiple sclerosis in the subject can be examined. In humans, PTPRZ has three types of splicing variants having different extracellular space lengths, and the longest ones are Isoform 1 (SEQ ID NO: 1), Isoform 2 (SEQ ID NO: 2), and Isoform 3 (SEQ ID NO: 2). It is number 3). Further, a secretory type (also referred to as "free type") PTPRZ, which is produced by cleaving the extracellular region of each isoform by a protease, is known. Splicing variants lacking the C-terminal region after the transmembrane region have also been reported in rodents, but the corresponding variants have not been identified in humans. Therefore, in the test method of the present invention, the content of secretory (free) PTPRZ isoform 1 and / or 2 in the sample derived from the subject is measured, and the control group (healthy subject or multiple sclerosis) suffers from it. By comparing with the corresponding value of the non-subject), the presence or absence of multiple sclerosis in the subject can be examined. Hereinafter, each isoform of human PTPRZ and their secretory form will be described in detail. As used herein, the term "secretory (free) PTPRZ" refers to a polypeptide fragment derived from the extracellular region of each isoform in human PTPRZ. In PTPRZ, a site highly sensitive to protease exists on the C-terminal side of the extracellular region. This site is cleaved by degradation of proteases such as metalloproteases to become secretory (free) PTPRZ. As used herein, the "secretory" PTPR isoform can be paraphrased as the "free" PTPRZ isoform.

The PTPRZ isoform 1 consists of the 2315 amino acids set forth in SEQ ID NO: 1. The PTPRZ isoform 1 has a signal peptide at positions 1 to 24, a CA domain at positions 45 to 298, an FN domain at positions 313 to 401, a transmembrane domain at positions 1637 to 1662, and a D1 domain at positions 1717 to 1992. And has a D2 domain at positions 2023-2282. Further, the CS binding region corresponds to a region in which a CS chain is bound between the FN domain and the transmembrane domain. The extracellular region of PTPRZ isoform 1 consists of the amino acid sequence shown in SEQ ID NO: 4, which corresponds to positions 25 to 1636 in the amino acid sequence shown in SEQ ID NO: 1. The full-length or partial fragment of the polypeptide of SEQ ID NO: 4 corresponds to the secretory PTPRZ isoform 1.

The PTPRZ isoform 2 consists of the 2308 amino acid residues set forth 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 space of isoform 2 corresponds 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 of the polypeptide of SEQ ID NO: 4 corresponds to the secretory PTPRZ isoform 2.

The PTPRZ isoform 3 consists of the 1448 amino acid residues set forth in SEQ ID NO: 3. The PTPRZ isoform 3 is an isoform in which the 860 amino acid residue at positions 755 to 1614 and the 7 amino acid residue corresponding to positions 1723 to 1729 are deleted, which may correspond to most of the CS binding region in PTPRZ isoform 1. is there. Therefore, the extracellular space 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.

There are two types of secretory 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. Of these, long-form (and its fragment polypeptide) can be a marker for detection of multiple sclerosis. The amino acid sequence actually to be detected in the present invention is a full-length polypeptide of the amino acid sequence (1612 amino acids) shown in SEQ ID NO: 4 and / or a partial peptide consisting of a part thereof. Such a partial peptide includes, for example, a peptide containing a continuous amino acid of 1129 amino acids or more, a peptide containing a continuous amino acid sequence of 1209 amino acids or more, and a continuous amino acid sequence of 1290 amino acids or more of the amino acid shown in SEQ ID NO: 4. Peptides, peptides containing a continuous amino acid sequence of 1371 amino acids or more, peptides containing a continuous amino acid sequence of 1451 amino acids or more, peptides containing a continuous amino acid sequence of 1532 amino acids or more, peptides containing a continuous amino acid sequence of 1580 amino acids or more, And, but not limited to, peptides and the like containing a continuous amino acid sequence of 1596 amino acids or more are exemplified.

In addition, as another aspect of the method of the present invention, the content of branched O-mannose in the sample derived from the subject is measured and compared with the corresponding value in the control group, thereby causing multiple occurrences in the subject. The presence or absence of sclerosis can be examined. PTPRZ undergoes branched O-mannose modification via the N-acetylglucosamine transferase GnT-IX in response to demyelination stimulation. Therefore, it is considered that the content of branched O-mannose in the sample derived from the subject reflects the abundance of secretory 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 in the control group.

As the method for measuring the secretory PTPRZ isoforms 1 and 2 in the sample derived from the subject, any method may be used as long as the amount of the protein can be quantified. Examples thereof include, but are not limited to, mass spectrometry, chromatography (liquid chromatography, gas chromatography, etc.), electrophoresis, immunoassay using an antibody, and the like. In particular, the immunoassay method is simple and preferable. In the case of immunoassay, there are sandwich method, competitive method, agglutination method, Western blotting, etc. when classified based on the reaction type, and radioimmunoassay, fluorescence immunoassay, enzyme immunoassay (EIA) when classified based on the label. , Biotin immunoassay and the like, but are not limited thereto.

The antibody used for 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 secretory PTPRZ isoforms 1 and / or 2 or branched O-mannose sugar chain-modified PTPRZ isoforms may be used by a method known per se. An antibody that specifically recognizes 1 and / or 2 may be prepared and used.

The term "antibody" in the present specification is a concept including an "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).

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). ..

Antibodies used to measure secretory PTPRZ isoforms 1 and / or 2 include, for example, the following antibodies:
(1) Antibodies that specifically recognize secretory PTPRZ isoforms 1 (2) Antibodies that specifically recognize secretory PTPRZ isoforms 2 (3) Antibodies that specifically recognize secretory PTPRZ isoforms 1 and 2. ..
In addition, PTPRZ is modified with a branched O-mannose sugar chain by GnT-IX, which is a glycosyltransferase, in vivo. As detailed in the examples below, we show that secretory PTPRZ isoforms 1 and 2 in cerebrospinal fluid are also branched O-mannose glycans modified. .. Therefore, instead of the anti-PTPRZ antibody, an antibody such as Cat-315 antibody that specifically recognizes the 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 ones may be used. From this point of view, 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. It is a test method for multiple sclerosis including a step of measuring, and the measured value of the amount of branched O-mannose sugar chain-modified PTPRZ in the sample derived from the subject is the branched O in the sample of the control group. -A method in which a subject is indicated to have multiple sclerosis when it is low compared to the amount of mannose sugar chain modified PTPRZ.

In the test method of the present invention, the amount of secretory PTPRZ isoform 1 and / or 2 in a sample derived from a subject is measured. Although cerebrospinal fluid is used as a sample in the following examples, blood can also be used because it is known that cerebrospinal fluid flows into veins. In a preferred embodiment, the sample from the subject is cerebrospinal fluid.

In the test method of the present invention, when the measured value of the secretory 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, it is multiple. Shows sclerosis. In the comparison of the measured values, a cutoff value is set in advance, and the cutoff value is compared with the measured value of the amount of secretory PTPRZ isoform 1 and / or 2 in the sample derived from the subject. May be good. If the measured value is lower than the cutoff value, it indicates that the subject has multiple sclerosis.

The cutoff value can be set by a method known to those skilled in the art. For example, multiple samples are obtained from known patient and control groups (healthy or non-multiple sclerosis subjects) and the amount of secretory PTPRZ isoform 1 and / or 2 is measured and is desirable. A value that can discriminate between the two groups based on sensitivity and / or specificity may be set as the cutoff value.

Mammals such as humans, monkeys, rats, dogs, and cats are exemplified as the subject to which the test method of the present invention is applied, and humans are particularly preferable.

2. Kits for Multiple Sclerosis Testing The present invention also includes antibodies that specifically recognize either or both of secretory PTPRZ isoforms 1 and 2 and antibodies that specifically recognize branched O-mannose sugar chains. A kit for diagnosing multiple sclerosis (hereinafter, may be referred to as "kit of the present invention") including or both of them is provided. By measuring the amount of secretory PTPRZ isoform 1 and / or 2 in the sample derived from the subject using the kit of the present invention and comparing it with the corresponding amount in the sample of the control group, the subject frequently occurs. It is possible to easily test whether or not the patient has multiple sclerosis.

Antibodies that specifically recognize secretory PTPRZ isoforms 1 and / or 2 contained in the kit of the present invention include the following antibodies.
(1) Antibodies that specifically recognize secretory PTPRZ isoforms 1 (2) Antibodies that specifically recognize secretory PTPRZ isoforms 2 (3) Antibodies that specifically recognize secretory PTPRZ isoforms 1 and 2. (4) An antibody that specifically recognizes a branched O-mannose sugar chain.
(4) An antibody that specifically recognizes a branched O-mannose sugar chain can be prepared by a method known per se, and as described above, a commercially available antibody such as Cat-315 antibody can be used. May be good. In one aspect, the kit of the present invention contains one or more of the antibodies (1) to (4) above as constituents. In addition, the kit of the present invention may contain components other than antibodies, such as an instruction manual and judgment criteria.

In one aspect of the present invention, the method for testing multiple sclerosis of the present invention may be combined with a method for treating multiple sclerosis. Accordingly, the present invention provides methods of testing and treating multiple sclerosis, including the following steps:
(1) A step of measuring the 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 secretory PTPRZ in the sample of the group is low, it indicates that the subject has multiple sclerosis, and the secretory PTPRZ is secretory PTPRZ isoform 1 and secretory PTPRZ isoform. Step; and (2) Administering a therapeutically effective amount of a therapeutic agent for multiple sclerosis to a subject determined to suffer from multiple sclerosis in step (1), which is either or both of 2. ..

The therapeutic agents for multiple sclerosis used in the present invention include, for example, steroids, interferon β, S1P receptor regulators, immunosuppressants, alkylating agents, anticancer antibiotics, anti-CD52 antibodies, and anti-α4 integrin. Administration of therapeutic agents for multiple sclerosis such as antibodies can be mentioned, but is not limited thereto.

Examples of steroids include amcinonide, sodium hydrocortisone succinate, prednisolone sodium succinate, prednisolone sodium methylsuccinate, cyclesonide, difluprednisolone, betamethasone propionate, dexamethasone, defrazacoat, triamsinolone, triamsinolone, acetonide, and halcinonide. , Pivalate flumethasone, butylacetate prednisolone, budesonide, sulphate plasterone, furoate mometasone, fluosinone, fluosinolone acetnide, fludroxycortide, flunisolide, prednisolone, propronate alcromethasone, propionate clobetazol, propionate dexamethasone, propionate dexamethasone Deprodon, fluticazone propionate, bechrometasone propionate, betamethasone, methylprednisolone, methylprednisolone sleptate, methylprednisolone sodium succinate, dexamethasone phosphate, hydrocortisone sodium phosphate, prednisolone sodium phosphate, diflucortron valerate, yoshi Dexamethasone herbate, betamethazone valerate, prednisolone valerate, cortisone acetate, diflorazone acetate, dexamethasone acetate, triamcinolone acetate, paramesazone acetate, halopredonate acetate, fludrocortisone acetate, prednisolone acetate, methylprednisolone acetate, clobetazone butyrate Examples include hydrocortisone acid and betamethasone propionate butyrate.

Examples of the S1P receptor regulator include fingolimod, siponimod, ponesimod, ceralifimod, and ozanimod.

Immunosuppressive agents include, for example, azathioprine, ascomycin, everolimus, salazosulfapyridine, cyclosporine, cyclophosphamide, silolimus, tacrolimus, bushiramine, methotrexate, leflunomide, teriflunomide.

Examples of alkylating agents include nitrogen mustard hydrochloride-N-oxide, cyclophosphamide, ifosfamide, melphalan, thiotepa, carbocon, busulfan, nimustine hydrochloride, dacarbazine, lanimustine, carmustine, chlorambucil, bendamustine, and mecloetanamine. ..

Examples of anticancer antibiotics include actinomycin D, mitomycin C, daunorubicin hydrochloride, doxorubicin hydrochloride, acralubicin hydrochloride, neocultinostatin, pyrarubicin hydrochloride, epirubicin (hydrochloride), idarubicin hydrochloride, chromomycin A3, and bleomycin (hydrochloride). , Pepromycin sulfate, teralubicin, dinostatin stimalamer, gemtuzumab ozogamicin, mitoxantrone hydrochloride.

Examples of the anti-CD52 antibody include alemtuzumab. In addition, examples of the anti-α4 integrin antibody include natalizumab.

The therapeutically effective amount of the therapeutic agent for multiple sclerosis may vary depending on the type of active ingredient, body weight and sex of the subject to be treated, administration route, etc., but an appropriate dose and administration method can be appropriately selected by those skilled in the art. can do. The administration route of the therapeutic agent can be selected from oral or parenteral depending on the type of the active ingredient. Parenteral administration includes, but is not limited to, for example, subcutaneous administration, intramuscular administration, intraperitoneal administration, and the like.

It should be noted that the "treatment" of a disease herein may include not only cure of the disease, but also remission of the disease and improvement of the degree of the disease.

The present invention will be described in more detail in the following examples, but the present invention is not limited to these examples.

All animal experiments were conducted in accordance with RIKEN's animal experiment implementation regulations. Immunohistochemical analysis of human cerebrospinal fluid and brain was performed at Fukushima Medical University according to the university's plan. Usually, when lumbar puncture is performed to collect cerebrospinal fluid, headache may occur after the puncture, and if the symptoms are severe, it may be accompanied by slight fever, headache, and vomiting. Cerebrospinal fluid is collected only for the patient.

[Example 1] Immunohistochemical staining The paraffin-embedded sections of the brains of patients with multiple sclerosis were deparaffinized as follows. It was soaked in xylene three times, in 99.5% ethanol three times, in 90% ethanol once, once in 80% ethanol, once in 70% ethanol for 5 minutes each, and soaked in running water for 5 minutes. It was autoclaved at 121 ° C. for 5 minutes in 0.01 M citrate buffer (pH 8.5) and immersed in PBS (pH 7.8).

A section of the brain of a multiple sclerosis patient was washed with PBS and then immersed in 0.5% Triton X-100-PBS for 15 minutes for permeabilization. After washing with shaking in 0.05% Tween-20-PBS (PBS-T) for 5 minutes each 3 times, it was shaken in 5% normal goat serum PBS for 30 minutes. Primary antibody (mouse monoclonal Cat-315 IgM (Millipore), mouse anti-PTPRZ IgMκ (122.2) (Santa cruz biotechnology) diluted with Dako REAL antibody room temperature (Dako) after washing twice in PBS-T for 5 minutes each. Was washed 3 times in PBS-T at room temperature for 2 hours or overnight at 4 ° C., and then diluted with DAKO REAL antibody diluent Alexa fluoro 546- or 488-labeled secondary antibody and DAPI. Was immersed in PBS-T for 45 minutes at room temperature. After washing in PBS-T three times for 5 minutes each, the mixture was sealed in CC / Mountain and observed with an FV1000-D confocal laser microscope (Olympus). Intensity analysis was performed by MetaMorph (Room temperature) (Fig. 1). As a result, Cat-315-positive cells and PTPRZ-positive cells were in agreement, and it was clarified that Cat-315-positive cells were PTPRZ-positive. It was.

[Example 2]
There was a suspicion of cerebrospinal fluid derived from patients with multiple sclerosis, cerebrospinal fluid derived from patients with neuromyelitis optica (NMO), and idiopathic normal pressure hydrocephalus (iNPH). A subject (non-iNPH, used as a control group) judged not to be 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 centered on 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), and goat polyclonal anti-Transthyretin (TTR) antibody (Abcam). Other reagents were mainly purchased from Wako.

10 μL of cerebrospinal fluid derived from MS patients (n = 3), NMO patients (n = 3), and non-iNPH patients (n = 3, control) was digested with chondroitinase and Western blotting was performed. The details of chondroitinase digestion are as follows. To the sample solution, a final concentration of 50 mM Tris-HCl (pH 8.0), 30 mM sodium acetate, and 200 U / L Johnsoninase ABC (Seikagaku) was added, and the mixture was stirred at 37 ° C. for 1 hour.

Western blotting was performed according to the following procedure. Laemmli's 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 the mixture was heated at 99 ° C. for 5 minutes. A 3-10% gradient polyacrylamide gel (Atto) was run for 60-70 minutes at a constant current of 20-25 mA per gel. Using a semi-dry transfer device, transfer was performed on the nitrocellulose membrane at a constant current of 150 mA for 90 minutes. Immersed in 5% skim milk-0.05% Tween20-TBS (pH 7.4) (TBS-T) for 30 minutes for blocking, then in a primary antibody diluted with TBS-T for 2 hours or 4 ° C. at room temperature. I shook it overnight. After immersing in TBS-T three times for 5 minutes each for washing, the cells were reacted with HRP-labeled secondary antibody diluted in 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 secretory PTPRZ isoforms 1 and 2 with respect 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 a relative value of the signal intensity of the upper band detected by the Cat-315 antibody.

As shown in Table 1, in cerebrospinal fluid derived from patients with multiple sclerosis, the upper band considered to be derived from the extracellular space of PTPRZ isoforms 1 and 2 (the total length of PTPRZ isoform 2 is the total length of PTPRZ isoform 1). Since there are only 7 amino acid differences and the amino acid sequences of both extracellular regions are the same, they are detected as the same band. Approximately 350 kDa) and the lower row considered to be derived from the extracellular region of secretory PTPZ isoform 3. A band (about 180 kDa) signal was detected. Of these, it was found that in the cerebrospinal fluid derived from patients with multiple sclerosis, the signal amount in the upper band was significantly reduced as compared with the control. In addition, the signal amount of the corresponding branched O-mannose sugar chain-modified PTPRZ was also decreased. Interestingly, some samples of NMO patients were reduced to the same level as those of MS patients. NMO is a disease in which inflammation occurs when astrocyte aquaporin 4 is attacked by autoantibodies, but it is thought that demyelination occurs as inflammation progresses. Therefore, the decrease in branched O-mannose sugar chain-modified PTPRZ in the samples of some NMO patients may indicate that demyelination occurs in the patients 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.

[Example 3] Verification of secretory PTPRZ isoforms 1 and 2 in cerebrospinal fluid as markers for MS detection Through the following experiments, secretory PTPRZ (secretory PTPRZ isoforms 1 and 2 (that is, secretory PTPRZ long-) It was verified that form)) could function as a marker for MS detection.

Secretory PTPRZ isoform 1 in the cerebrospinal fluid derived from each of the multiple sclerosis (MS) patient group (24 patients) and the idiopathic normal pressure hydrocephalus (iNPH) patient (including suspected cases) group (25 patients). And 2 quantities were measured by Western blotting.

As the antibody, mouse anti-PTPζ IgMκ (sc-33664, Santa cruz), which is an anti-PTPRZ antibody, and goat anti-mouse IgM antibody-HRP (SAB-110, Stressgen) were used. Mouse anti-PTPZ IgMκ is a PTPRZ-specific monoclonal antibody prepared by preparing a proteoglycan fraction from rat fetal brain. Other reagents were mainly purchased from Wako.

10 μL of cerebrospinal fluid collected from each of the above patients was first subjected to chondroitinase digestion. Specifically, a final concentration of 100 mM Tris-HCl (pH 7.5), 60 mM sodium acetate, and 4 mU chondroitinase ABC (Sigma, 2905) were added to the sample solution, and the mixture was incubated at 37 ° C. for 1 hour.

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. A 3-10% gradient polyacrylamide gel (Atto, NPG-310L) was run for 60 to 70 minutes at a constant current of 20 mA to 25 mA per gel. Using a wet transfer device, transfer was performed on the nitrocellulose membrane at a constant current of 350 mA for 45 minutes. Mouse anti-PTPRZ IgMκ (Santa cruz,) diluted 200-fold with PBS-0.1% Tween (PBS-T) after blocking by immersing in 1% BSA-PBS (pH 7.4) overnight at 4 ° C. 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 after being washed by immersing it in PBS-T three times for 5 minutes each. Was reacted 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 Co., Ltd., 34096), and the obtained signal was detected with ATTO Ez-Capture. The signal intensity of the band was quantified using the attached quantification software CS Analyzer. The results are shown in FIG.

As shown in FIG. 3, the signal intensity of secretory PTPRZ isoforms 1 and 2 in the cerebrospinal fluid derived from MS patients is the cerebrospinal fluid derived from the control group (the group of patients with idiopathic normal pressure hydrocephalus (including suspected cases)). It was significantly lower than that of the inside. From this result, it was shown that secretory PTPRZ isoforms 1 and 2 can be markers for MS detection.

INDUSTRIAL APPLICABILITY According to the present invention, a new objective index useful for a definitive diagnosis of demyelinating disease (particularly multiple sclerosis) is provided. Therefore, the present invention is extremely useful in the medical field.

This application is based on Japanese Patent Application No. 2018-028329 (filed on February 20, 2018) filed in Japan, the contents of which are incorporated herein by reference in its entirety.

Claims (5)

A method for testing multiple sclerosis, which comprises the step of measuring the 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 secretory PTPRZ in the sample of the control group, it indicates that the subject has multiple sclerosis, and the secretory PTPRZ is secreted PTPRZ isoform 1 and secreted. A method that is either or both of the type PTPRZ isoform 2. 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. The method according to claim 1 or 2, wherein the sample derived from the subject is cerebrospinal fluid or blood. The measurement of secretory PTPRZ in the 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. A kit for diagnosing multiple sclerosis, comprising either or both an antibody that specifically recognizes secretory PTPRZ and an antibody that specifically recognizes a branched O-mannose sugar chain, wherein the secretory PTPRZ is , A kit which is one or both of secretory PTPRZ isoform 1 and secretory PTPRZ isoform 2.

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