WO2020256013A1 - Biomarker for detecting neuropsychiatric systemic lupus erythematosus - Google Patents

Biomarker for detecting neuropsychiatric systemic lupus erythematosus Download PDF

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WO2020256013A1
WO2020256013A1 PCT/JP2020/023754 JP2020023754W WO2020256013A1 WO 2020256013 A1 WO2020256013 A1 WO 2020256013A1 JP 2020023754 W JP2020023754 W JP 2020023754W WO 2020256013 A1 WO2020256013 A1 WO 2020256013A1
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npsle
biomarker
galectin
amino acid
protein
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PCT/JP2020/023754
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French (fr)
Japanese (ja)
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清志 右田
直紀 松岡
智之 浅野
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公立大学法人福島県立医科大学
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Priority to JP2021526841A priority Critical patent/JP7320863B2/en
Publication of WO2020256013A1 publication Critical patent/WO2020256013A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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

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  • the present invention is a biomarker for detecting neuropsychiatric lupus (NPSLE), a biomarker for determining the therapeutic effect on NPSLE, whether or not the test animal has NPSLE, or the risk of developing NPSLE.
  • NPSLE neuropsychiatric lupus
  • the present invention relates to a method for assisting in determining the presence or absence of NPSLE, a method for assisting in determining the therapeutic effect on NPSLE, or a kit for detecting NPSLE.
  • SLE Systemic lupus erythematosus
  • NPSLE neuropsychiatric lupus
  • NPSLE is diagnosed by combining various clinical tests such as blood, cerebrospinal fluid, and imaging findings, but diagnostic criteria have not been established, and it is extremely difficult to distinguish it from psychiatric symptoms due to side effects of steroids. Difficult to.
  • Non-Patent Document 1 describes that IL-6 and the like are increased in the cerebrospinal fluid in the NPSLE patient group as compared with the non-NPSLE and non-autoimmune disease control patient groups.
  • the biomarkers such as IL-6 described in Non-Patent Document 1 do not always have sufficient accuracy and / or accuracy in detecting NPSLE.
  • the present inventor has completed the present invention by finding that galectin 9 can be used as a biomarker for detecting NPSLE or as a biomarker for determining a therapeutic effect on NPSLE.
  • the present invention includes the following embodiments.
  • a biomarker for detecting neuropsychiatric lupus which comprises a galectin 9 protein or a peptide fragment thereof, or a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
  • the galectin 9 protein is any of the following proteins (a) to (c): (a) Protein containing the amino acid sequence shown in SEQ ID NO: 1 (b) Protein containing the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and (c) ) A protein containing an amino acid sequence having 90% or more amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 1.
  • the biomarker according to (1) is any of the following proteins (a) to (c): (a) Protein containing the amino acid sequence shown in SEQ ID NO: 1 (b) Protein containing the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID
  • the method according to (6) or (7) which comprises a step of quantifying a biomarker.
  • the method further comprising.
  • a biomarker for determining a therapeutic effect on NPSLE which comprises a galectin 9 protein or a peptide fragment thereof, or a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
  • a method for assisting determination of a therapeutic effect on NPSLE which comprises a step of detecting or quantifying the biomarker according to (9) in a sample derived from a test animal.
  • the method further comprising.
  • (12) The method according to any one of (6) to (8) and (10) and (11), wherein the test animal is a human.
  • a kit for detecting NPSLE which comprises a reagent for measuring the amount of the biomarker according to any one of (1) to (3).
  • a kit for detecting NPSLE which comprises an antibody or a fragment thereof that specifically binds to a galectin 9 protein or a peptide fragment thereof.
  • the galectin 9 protein is one of the following proteins (a) to (c): (a) Protein containing the amino acid sequence shown in SEQ ID NO: 1 (b) Protein containing the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and (c) )
  • the kit which is a protein containing an amino acid sequence having 90% or more amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 1.
  • a kit for detecting NPSLE which contains a nucleic acid that can specifically bind to a transcript of the galectin 9 gene or a fragment thereof.
  • the kit the kit: the nucleic acid is selected from the group consisting of the polynucleotides or fragments thereof shown in any of the following (d) to (h).
  • (d) (i) A nucleotide consisting of the base sequence represented by SEQ ID NO: 2 or a base sequence in which u is t in the base sequence, or a fragment thereof containing 15 or more consecutive bases.
  • a polynucleotide or fragment thereof of any of (v) to (vii) containing a modified nucleic acid and / or a modified nucleotide (g) A nucleotide containing the nucleotide sequence represented by SEQ ID NO: 2, a nucleotide sequence complementary to the nucleotide sequence in which u is t in the nucleotide sequence, or a partial sequence thereof containing 15 or more consecutive nucleotides, and ( h) A polynucleotide that hybridizes with any of the polynucleotides (d) to (g) above or a fragment thereof under highly stringent conditions. (17) The kit according to (15) or (16), wherein NPSLE has aseptic meningitis. This specification includes the disclosure of Japanese Patent Application No. 2019-112429, which is the basis of the priority of the present application.
  • the present invention may provide a marker for detecting NPSLE, or a method for detecting NPSLE using the marker. Further, the present invention may provide a marker for determining a therapeutic effect on NPSLE, or a method for determining a therapeutic effect on NPSLE using the marker.
  • FIG. 1 shows the cerebrospinal fluid galectin 9 concentrations in 18 NPSLE patients, 6 patients with other diseases (control 1), and 8 non-NPSLE SLE patients (control 2).
  • FIG. 2 shows the cerebrospinal fluid galectin 9 concentration before and after treatment in one NPSLE patient.
  • FIG. 3 shows the concentration of galectin 9 in the cerebrospinal fluid of 7 patients in the group with aseptic meningitis (AM +) and 11 patients in the group without AM (AM-) among 18 patients with NPSLE.
  • the invention relates to a biomarker for detecting neuropsychiatric lupus (NPSLE), which consists of a galectin 9 protein or peptide fragment thereof, or a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
  • NPSLE neuropsychiatric lupus
  • the present invention relates to a biomarker for determining a therapeutic effect on NPSLE, which comprises a galectin 9 protein or a peptide fragment thereof, or a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
  • systemic lupus erythematosus is a chronic recurrent autoimmune disease in which an immune complex of autoantibodies is deposited on tissues and causes tissue damage.
  • neuropsychiatric lupus NPSLE, Neuropsychiatric SLE
  • SLE central nervous system symptoms
  • neuropsychiatric SLE for example, Hanly JG et al., Arthritis Rheum., 2019. , 71 (1): 33-42).
  • Symptoms of NPSLE can be broadly divided into those of the central nervous system (psychiatric and neurological symptoms) and those of the peripheral nervous system.
  • Psychiatric symptoms include mood disorders, cognitive dysfunction, anxiety disorders, psychotic disorders, and acute confusion
  • neurological symptoms include headache, convulsions, cerebrovascular accidents, movement disorders (chorea), myelopathy, and aseptic. Examples include myelopathy and demyelinating syndrome.
  • Peripheral nervous system symptoms include multiple neuropathy, cerebral nerve neuropathy, single / multiple single neuropathy, acute inflammatory demyelinating multiple nerve root neuropathy (Guillain-Barré syndrome), autonomic neuropathy, severe myasthenia, and neuropathy. Disorders can be mentioned (see, for example, Arthritis Rheum., 1999, 42 (4): 599-608).
  • a galectin 9 protein derived from an endogenous gene of a test animal or a gene transcript thereof can be a biomarker.
  • the test animal is a human
  • the human galectin 9 protein derived from the human galectin 9 gene and the transcript (mRNA) of the human galectin 9 gene can be biomarkers of the present invention.
  • galectin 9 protein examples include a human-derived galectin 9 (human galectin 9) protein containing or consisting of the amino acid sequence shown in SEQ ID NO: 1.
  • the galectin 9 protein also includes a galectin 9 variant having an activity functionally equivalent to that of the galectin 9 protein shown in SEQ ID NO: 1 and a galectin 9 ortholog of another species. Specifically, 80% or more and 90% of the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1 As described above, galectin 9 proteins having 95% or more, 97% or more, 98% or more, or 99% or more amino acid identity are included.
  • “several pieces” means, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 4 or 2 to 3.
  • Conservative amino acid substitution is desirable for amino acid substitution.
  • “Conservative amino acid substitution” refers to a substitution between amino acids having similar properties such as charge, side chain, polarity, and aromaticity.
  • Amino acids with similar properties include, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagin, glutamine, serine, threonine, cysteine, tyrosine), non-polar amino acids.
  • sex amino acids leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine
  • branched amino acids leucine, valine, isoleucine
  • aromatic amino acids phenylalanine, tyrosine, tryptophan, histidine
  • amino acid identity refers to the sequence number when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the degree of amino acid matching between the two amino acids.
  • Amino acid identity can be calculated using a protein search system using BLAST or FASTA. For details on how to determine identity, see, for example, Altschul et al, Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215, 403-410, 1990. I want to.
  • the "galectin 9 gene” is a gene encoding the galectin 9 protein.
  • Specific examples of the galectin 9 gene include a human galectin 9 gene encoding a human galectin 9 protein containing the amino acid sequence shown in SEQ ID NO: 1. More specifically, the galectin 9 gene is a gene containing or consisting of the nucleotide sequence shown in SEQ ID NO: 2.
  • the galectin 9 gene includes a galectin 9 variant having an activity functionally equivalent to that of the galectin 9 protein encoded by the galectin 9 gene shown in SEQ ID NO: 2 and a galectin 9 gene encoding a galectin 9 ortholog of another species. Included. Specifically, 80% or more and 90% of the base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 2 or the base sequence shown in SEQ ID NO: 2 As described above, galectin 9 genes having 95% or more, 97% or more, 98% or more, or 99% or more base identity are included.
  • base identity refers to a sequence number when two base sequences are aligned and a gap is introduced as necessary so that the degree of base matching between the two is the highest.
  • hybridizing under high stringent conditions means performing hybridization and washing under low salt concentration and / or high temperature conditions. For example, incubate with probe in 6 ⁇ SSC, 5 ⁇ Denhardt reagent, 0.5% SDS, 100 ⁇ g / mL denatured fragmented salmon sperm DNA at 65 ° C-68 ° C, then in a wash solution of 2 ⁇ SSC, 0.1% SDS. It is exemplified by starting from room temperature at room temperature, lowering the salt concentration in the cleaning solution to 0.1 ⁇ SSC, and raising the temperature to 68 ° C. until no background signal is detected. The conditions for high stringent hybridization are described in Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. It can be used as a reference.
  • the nucleotide sequence information of such galectin 9 gene can be searched from public databases (GenBank, EMBL, DDBJ). For example, a gene having high base identity can be searched and obtained from a database based on the known base sequence information of the galectin 9 gene shown in SEQ ID NO: 2.
  • Transcript of galectin 9 gene means galectin 9 mRNA.
  • the mRNA may be an mRNA precursor (pre-mRNA) or a mature mRNA (mature mRNA). Normally, the mRNA precursor is immediately spliced in the nucleus to become a mature mRNA, so that the transcript of the galectin 9 gene, which is a biomarker of the present invention, may be a galectin 9 mature mRNA.
  • the term "peptide fragment” is a peptide fragment containing or consisting of a part of the amino acid sequence constituting the galectin 9 protein, and the fragment of the galectin 9 protein from the amino acid sequence constituting the fragment. Can be identified.
  • the "peptide fragment” may be 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more consecutive amino acid acid residues in the full-length amino acid sequence of the galectin 9 protein. Further, it may be a peptide consisting of 200 or less, 150 or less, 120 or less, 100 or less, or 80 or less consecutive amino acid residues.
  • a "peptide fragment” may be a peptide consisting of 10 to 200, 30 to 120, and 50 to 80 consecutive amino acid residues.
  • the "nucleic acid fragment” is a nucleic acid fragment containing or consisting of a part of the base sequence constituting galectin 9 mRNA, and is a fragment of galectin 9 mRNA from the base sequence constituting the fragment. Can be identified.
  • the "nucleic acid fragment” may be a nucleic acid consisting of 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more consecutive bases in the full-length base sequence of galectin 9 mRNA.
  • the nucleic acid may be composed of 600 or less, 450 or less, 300 or less, 200 or less, or 100 or less consecutive bases.
  • the "nucleic acid fragment” may be a nucleic acid consisting of 30 to 600, 50 to 300, or 70 to 100 consecutive bases in the full-length base sequence of galectin 9 mRNA.
  • the invention relates to the use of the biomarkers described herein to detect NPSLE.
  • the present invention relates to a method for determining (or assisting) whether or not a test animal has NPSLE or is at risk of developing NPSLE.
  • the method comprises the step of detecting or quantifying the biomarkers described herein in a sample derived from a test animal.
  • the detection or quantification step can be performed in vitro.
  • the method of the present invention further includes a step of determining whether or not the test animal has NPSLE or whether or not there is a risk of suffering from NPSLE based on the detection or quantification result obtained in the above step. Good.
  • the above-mentioned detection of NPSLE, determination of the presence or absence of NPSLE or determination of the risk of morbidity includes differentiation of NPSLE-affected patients from healthy subjects, and differentiation of NPSLE from other diseases.
  • diseases that can be differentiated from NPSLE include non-NPSLE inflammatory diseases such as SLE and Bechet's disease, and collagen diseases such as ANCA-related vasculitis, as well as encephalitis and corticosteroid-induced psychotric disorders (corticosteroid-induced psychiatric disorders). CIPDs))), neuromyelitis optica (NMO), progressive multifocal leukoencephalopathy (PML), and reverse posterior reversible leukoencephalopathy (PRES).
  • NPSLE neuromyelitis optica
  • PML progressive multifocal leukoencephalopathy
  • PRES reverse posterior reversible leukoencephalopathy
  • the detection of NPSLE and the determination of the presence or absence of NPSLE may be used to determine whether or
  • the NPSLE to be detected may be an NPSLE having either the above-mentioned central nervous system (psychiatric and neurological symptoms) or peripheral nervous system symptoms.
  • NPSLE having aseptic meningitis may be detected, or the presence or absence of morbidity or the risk of morbidity may be determined.
  • test animals in the present invention include, for example, mammals, for example, primates such as humans and red-tailed monkeys, experimental animals such as rats, mice, and rats, pigs, cows, horses, sheep, goats, etc. Domestic animals and pet animals such as dogs and cats are mentioned, and humans are preferable.
  • the test animal may be a subject who has or is suspected of having the other diseases mentioned above, such as SLE.
  • the sample used in the present invention is not particularly limited, and examples thereof include tissues such as body fluids, cells, and biopsy samples.
  • body fluids include cerebrospinal fluid, blood (including serum, plasma and interstitial fluid), lymph, extracts of tissues or cells, pleural fluid, sputum, tears, nasal juice, saliva, urine, etc. Cerebrospinal fluid or blood (particularly plasma) that can be corrected with standard substances.
  • the body fluid, cells or tissue may be used as it is for the measurement of the biomarker, or may be appropriately pretreated for the measurement.
  • paraffin-embedded sections may be prepared from samples derived from test animals.
  • a protein extract or mRNA extract may be prepared from a sample derived from a test animal.
  • the biomarker measured by this method may be either a galectin 9 protein or a peptide fragment thereof, a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
  • Detection or quantification includes measuring the presence or absence of expression of a biomarker, or the magnitude of expression level or expression concentration.
  • the term "measurement" includes semi-quantitative.
  • the measurement method may be any known protein quantification method, and is not particularly limited, and examples thereof include an immunological detection method.
  • the "immunological detection method” is a method of measuring the amount of a target molecule using an antibody or antibody fragment that specifically binds to the target molecule that is an antigen.
  • the antibody can be derived from any animal, including mammals and birds. For example, mice, rats, guinea pigs, rabbits, goats, donkeys, sheep, camels, horses, chickens or humans can be mentioned.
  • the antibody used in the immunological detection method is not particularly limited, but a monoclonal antibody or a polyclonal antibody may be used.
  • monoclonal antibody refers to a group of clones of a single immunoglobulin. Each immunoglobulin constituting a monoclonal antibody contains a common framework region and a common complementarity determining region, and can recognize and bind to the same epitope of the same antigen. Monoclonal antibodies can be obtained from single cell hybridomas.
  • polyclonal antibody refers to a group of multiple immunoglobulins that recognize and bind to different epitopes of the same antigen.
  • the polyclonal antibody can be obtained from the serum of an animal after immunizing the animal with the target molecule as an antigen.
  • each class of IgG, IgM, IgA, IgE, and IgD is known as an immunoglobulin molecule, but the antibody of the present invention may be in any class. , For example, IgG.
  • the method for producing a polyclonal antibody that recognizes and binds to galectin 9 protein or a hybridoma that produces a monoclonal antibody may be carried out according to an antibody production method known in the art using galectin 9 protein or a fragment thereof as an antigen. Antibodies may also be obtained from the manufacturer. The expression level of the galectin-9 protein or a fragment thereof may be measured by a commercially available kit, and such a kit includes Quantikine TM ELISA Human Galectin-9 immunoassay. In one embodiment, the galectin-9 protein or fragment thereof comprises an epitope recognized by an antibody contained in Quantikine TM ELISA Human Galectin-9 immunoassay.
  • antibody fragment refers to a polypeptide chain or a complex thereof that is a partial fragment of a polyclonal antibody or a monoclonal antibody and has an activity substantially equivalent to the antigen-specific binding activity of the antibody. ..
  • an antibody moiety that includes at least one antigen-binding site that is, a polypeptide chain having at least one set of VL and VH, or a complex thereof.
  • Specific examples include a large number of well-characterized antibody fragments produced by cleaving immunoglobulins with various peptidases. More specific examples include Fab, F (ab') 2 , Fab'and the like. All of these antibody fragments include an antigen-binding site and have the ability to specifically bind to a target molecule that is an antigen.
  • Immunological detection methods include, for example, immunohistochemical staining, enzyme-linked immunosorbent assay (including ELISA and EIA), Western blotting, radioimmunoassay (RIA), immunoprecipitation, or flow cytometry.
  • enzyme-linked immunosorbent assay including ELISA and EIA
  • Western blotting including ELISA and EIA
  • RIA radioimmunoassay
  • immunoprecipitation or flow cytometry. The law can be mentioned.
  • a known method can be adopted as the "immunohistochemical staining method". For example, a sample derived from a test animal may be fixed in formalin, embedded in paraffin, sliced into tissue pieces, and attached to a slide glass, which may be used as a section sample. The section sample may be heat-treated to activate the antigen, and then immunohistochemical staining may be performed on the section sample using a commercially available detection system such as DakoEnVision + System (Agilent).
  • the measurement method may be any known nucleic acid quantification method, and is not particularly limited, but for example, a nucleic acid amplification method using a primer or a nucleic acid amplification method using a primer.
  • a hybridization method using a probe can be mentioned.
  • Nucleic acid amplification method refers to a method in which a specific region of a target nucleic acid is amplified by a nucleic acid polymerase using a forward / reverse primer set.
  • examples of the nucleic acid amplification method include PCR (polymerase chain reaction) methods such as RT-PCR (reverse transcription polymerase chain reaction).
  • hybridization method uses a nucleic acid fragment having a base sequence complementary to all or part of the base sequence of the target nucleic acid to be detected as a probe, and utilizes the base pairing between the nucleic acid and the probe.
  • the hybridization method several methods with different detection means are known, and examples thereof include a Northern hybridization method (Northern blot hybridization method), an in situ hybridization method, and a microarray method.
  • Nucleic acid chains such as primers and probes can be appropriately designed by a method known to those skilled in the art based on known biomarker sequence information, and can be obtained by a known production method such as chemical synthesis.
  • each of the above-mentioned measurement methods is a technique known in the art. Therefore, the specific measurement method may be performed according to a known method. For example, the method described in Green, M.R. and Sambrook, J., 2012 (described above) can be referred to.
  • the step comprises determining whether the sample is positive or negative for the biomarker from the results obtained in the detection or quantification step. If the sample is negative for biomarkers, it can be determined that the test animal does not have NPSLE or is at low risk of developing it. On the other hand, if the sample is positive for biomarkers, it can be determined that the test animal has NPSLE or is at high risk of developing it.
  • the immunohistochemical staining method when one or more cells or cell clusters are stained, it can be determined as positive, and when the stained cells do not exist, it can be determined as negative.
  • the number of stained cells exceeds a certain ratio (for example, 10%, 15% or 20%) with respect to the total number of cells, it is determined to be positive, and the number of stained cells with respect to the total number of cells is equal to or less than the certain ratio. In the case of, it may be determined as negative.
  • a certain ratio for example, 10%, 15% or 20%
  • the determination step comprises determining whether the expression level of the biomarker in the sample obtained in the detection or quantification step is high or low (eg, below a predetermined threshold). If the expression level of the biomarker in the sample is below a predetermined threshold (eg, statistically significantly lower), then the population is not affected by NPSLE or (eg, has an expression level higher than the predetermined threshold). On the other hand, it can be judged that the risk of morbidity is low. On the other hand, if the expression level of the biomarker in the sample is higher than a predetermined threshold (eg, statistically significantly higher), then the population is either suffering from NPSLE or (eg, having an expression level lower than the predetermined threshold). On the other hand, the risk of morbidity can be determined to be high.
  • a predetermined threshold eg, statistically significantly lower
  • the predetermined threshold value may be a control amount measured in a control sample (control cell or tissue, for example, control colon cell or colon tissue).
  • the control sample may be derived from a healthy individual (eg, a healthy person), a non-NPSLE SLE patient, or a patient suffering from another disease.
  • the "healthy individual” refers to a healthy individual of the same species as the test individual, which is not affected by NPSLE, and includes an individual who has been affected by NPSLE but has been treated with NPSLE.
  • the median value, average value, upper limit level, lower limit level, and a certain range of values of the expression level in these control samples or the expression level in a plurality of individuals can be used as a predetermined threshold value.
  • the above value or range multiplied by an appropriate coefficient for example, 0.3 or more, 0.5 or more, 0.8 or more, 0.9 or more, or 3 or less, 2 or less, 1.5 or less, or 1.1 or less
  • the threshold value can be appropriately set according to the accuracy of prediction and the like, and can be determined by, for example, ROC (receiver operating characteristic curve) analysis.
  • statically significant means that when the risk rate (significance level) of the obtained value is small, specifically, p ⁇ 0.05 (less than 5%) and p ⁇ 0.01 (less than 1%). ) Or p ⁇ 0.001 (less than 0.1%).
  • a known test method capable of determining the presence or absence of significance may be appropriately used, and is not particularly limited. For example, the Student's t-test method, the multiple comparison test method, and the log rank test method can be used.
  • the term "high risk of morbidity" for NPSLE refers to a period of time (eg, within a week, within a few weeks, within a month, within 2 months, within 3 months), although not currently suffering from NPSLE. Within, within 6 months, within 1 year, within 2 years, or within 5 years), it is intended to be more likely to have NPSLE.
  • “low risk of morbidity” of NPSLE means that the patient is not currently suffering from NPSLE and has a certain period of time (for example, within 1 week, within several weeks, within 1 month, within 2 months, within 3 months, It is intended that the likelihood of contracting NPSLE within 6 months, 1 year, 2 years, or 5 years) is low.
  • the method of determining (or assisting) whether or not the test animal of the present invention has NPSLE or is at risk of developing NPSLE is combined with another NPSLE diagnostic method in order to further improve the accuracy. May be good.
  • Other such NPSLE diagnostic methods include imaging tests (CT, MRI, SPECT, PET, etc., for example, presence or absence of abnormal findings in head MRI), electroencephalography (presence or absence of electroencephalogram abnormalities), and spinal fluid test (medullary fluid).
  • General spinal fluid tests such as cell number, spinal protein and sugar content, measurement of cytokines such as spinal fluid IL-6), and autoantibodies (anti-cardiolipin antibody, lupus anticogrant, anti-ribosomal P antibody, anti-Ro / SS-A antibody, anti-Sm antibody, anti-ds-DNA antibody, etc.) detection and the like.
  • Diagnostic criteria for such diagnostic methods are known to those skilled in the art, for example, Hiroyuki Yamashita, 2017, Journal of the Japanese Society of Internal Medicine 106, No. 8, pp. 1555 to 1563, Masaru Nishimura et al., 2011, General Hospital Psychiatry. See Medicine, 23 (1), pp. 42-51, etc.
  • the presence or absence or risk of morbidity of NPSLE can be determined, and based on the result, the treatment policy (for example, the presence or absence of concomitant drug use, steroid drug (and concomitant drug if used)). Type, dose, dosing interval, and dosing period) can be determined.
  • the present invention determines that a subject has or is at high risk of developing NPSLE, steroids may be administered to the test animal to treat and / or prevent NPSLE. Therefore, the present invention also provides a method for treating and / or preventing NPSLE, which comprises administering a steroid agent to a test animal which is determined to have NPSLE or is at high risk of developing NPSLE by the method of the present invention. To do.
  • steroids examples include, but are not limited to, corticosteroids such as prednisolone, methylprednisolone, betamethasone, dexamethasone, triamcinolone and hydrocortisone, and their solvents.
  • Steroids can be used alone or in combination. Steroids can be administered by routes such as injection, intravenous administration, or oral administration.
  • the dose of steroids is not limited, but NPSLE often uses high doses of steroids (for example, 1.0 mg / kg body weight / day), and the dose is reduced by about 10% every two weeks while observing the symptoms. Often.
  • the dosage is, for example, 0.005 to 100 mg / kg body weight / day, 0.05 to 10 mg / kg body weight / day, 0.1 to 5.0 mg / kg body weight / day, 0.25 to 2.0 mg / kg body weight / day, or 0.5 to 1.0 mg / body weight. It may be kg / day.
  • the number of administrations is not limited, but is 3 times a day, 2 times a day, once a day, once every 2 days, once every 3 days, once a week, once every two weeks, It may be once a month or the like.
  • the administration period may be, but is not limited to, 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, 6 months, 1 year, or more.
  • Steroidal pulse therapy with high doses may be given for several days.
  • the dose in steroid pulse therapy may be 250 mg to 1000 mg / day in terms of methylprednisolone, and may be intravenous drip infusion.
  • the duration of steroid pulse therapy may be, but is not limited to, 1 day, 2 days, 3 days, or longer.
  • Steroids may be used in combination with other drugs, and examples of other drugs include immunosuppressants.
  • immunosuppressants include, but are not limited to, azathioprine, cyclophosphamide, mycophenolate mofetil, mizoribine and the like.
  • the dose of the immunosuppressant is not limited, but is, for example, 0.005 to 5000 mg / kg body weight / day, 0.05 to 1000 mg / kg body weight / day, 0.1 to 100 mg / kg body weight / day, or 1.0 to 10 mg / kg body weight / day. It may be there.
  • the number of administrations is not limited, but is 3 times a day, 2 times a day, once a day, once every 2 days, once every 3 days, once a week, once every two weeks, It may be once a month or the like.
  • the administration period may be, but is not limited to, 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, 6 months, 1 year, or more.
  • azathioprine can be administered at 0.5 to 2.5 mg / kg body weight / day once daily
  • cyclophosphamide can be administered at 500 to 750 mg / kg body weight / day for 2 to 3 weeks by intravenous injection. It can be administered 7 to 10 times in total every other time
  • mycophenolate mofetil can be administered 1000 mg to 2000 mg / kg body weight / day, intravenously or orally twice daily
  • mizoribine can be administered 150 to 150 times. It can be administered at 300 mg / kg body weight / day orally once to three times a day every day.
  • the steroid and the immunosuppressant may be administered at the same time or at different time points. When administered at the same time, it may be administered simultaneously as a composition containing a steroid agent and an immunosuppressive agent, or the steroid agent and the immunosuppressive agent may be administered separately.
  • the order of administration is not limited, and the immunosuppressant may be administered after the steroid is administered, or the immunosuppressant may be administered before the steroid is administered. Inhibitors may be administered.
  • the invention relates to the use of the biomarkers described herein to determine a therapeutic effect on NPSLE (eg, a therapeutic method or agent, etc.).
  • the invention relates to a method of determining (or assisting) a therapeutic effect on NPSLE (eg, a therapeutic method or drug).
  • the method comprises the step of detecting or quantifying the biomarkers described herein in a sample derived from a test animal. This detection or quantification step is as described for the step in the method of determining whether or not the patient has NPSLE or the risk of suffering from NPSLE.
  • the present method may further include a step of determining whether or not there is a therapeutic effect on NPSLE based on the detection or quantification result obtained in the above step.
  • This determination step includes determining whether or not there is a therapeutic effect on NPSLE.
  • This determination step is also as described in the method for determining whether or not the patient has NPSLE or whether or not the patient is at risk of developing NPSLE.
  • the determination step includes determining whether the sample is positive or negative for the biomarker from the results obtained in the detection or quantification step. If the sample is negative for the biomarker, it can be determined to be therapeutically effective or high. On the other hand, if the sample is positive for the biomarker, it can be determined that the therapeutic effect is ineffective or low.
  • the criteria for determining positive and negative are as described.
  • the determination step also includes determining whether the expression level of the biomarker in the sample obtained in the detection or quantification step is higher or lower (for example, than a predetermined threshold value).
  • a predetermined threshold for example, statistically significantly lower
  • the therapeutic effect is effective or high (for example, for a population having an expression level higher than the predetermined threshold).
  • the expression level of the biomarker in the sample is higher than a predetermined threshold (for example, statistically significantly higher)
  • there is no therapeutic effect for example, for a population having an expression level lower than the predetermined threshold. It can be judged to be low.
  • the predetermined threshold value is also as described above. However, the threshold in this embodiment may include the expression level in the test animal before treatment.
  • the method may also include the step of treating NPSLE before, during, and / or after the detection or quantification step.
  • the treatment step may include administering to the test animal a steroid (and optionally an immunosuppressant).
  • a steroid and optionally an immunosuppressant.
  • the types, doses, administration periods, etc. of steroids and immunosuppressants are as described in the above-mentioned treatment and / or prevention methods.
  • the treatment process may be changed according to the result of the above determination process. For example, if it is determined in the determination step that there is no therapeutic effect, a drug (for example, an immunosuppressant) for increasing and / or administering the drug may be added.
  • a drug for example, an immunosuppressant
  • an immunosuppressant for increasing and / or administering the drug
  • kits In one aspect, the invention also provides a kit for detecting NPSLE, which comprises a reagent for measuring the amount of biomarker described herein.
  • the reagent for measuring the amount of biomarker examples include the above-mentioned antibody or antibody fragment, or probe or primer.
  • the kit includes known immunohistochemical staining, ELISA, Western blot, or reagents for RT-PCR, such as labeling reagents, buffers, chromogenic substrates, secondary antibodies, blocking agents, and instruments and controls required for testing. And at least one of the instructions and the like may be further included.
  • Example 1 Measurement of galectin 9 concentration in different patient groups> (Materials and methods) As a sample, among patients with rheumatoid arthritis who visited the Department of Internal Medicine for Rheumatoid Arthritis, Fukushima Prefectural Medical University between June 2004 and January 2019, lupus puncture was performed and the spinal fluid was discharged at -80 ° C. Twenty-six cases of systemic lupus erythematosus (SLE) and 6 cases of other diseases that had been cryopreserved were used. Galectin 9 concentrations in cerebrospinal fluid samples were measured by enzyme-linked immunosorbent assay (ELISA) using Quantikine TM ELISA Human Galectin-9 immunoassay (R & D systems) according to the manufacturer's protocol.
  • ELISA enzyme-linked immunosorbent assay
  • NPSLE neuropsychiatric lupus
  • control 2 patients who were judged to be negatively involved in NPSLE.
  • NPSLE made a comprehensive diagnosis based on the serological evaluation of SLE activity, spinal fluid examination, diagnostic imaging (MRI, SPECT), and clinical course, and made a comprehensive diagnosis (specifically, 1997 ACR).
  • a definitive diagnosis of SLE is made according to the diagnostic criteria, and in addition to symptoms such as headache and fever, SPECT shows a decrease in cerebral blood flow, MRI shows a highly absorbed region in the brain parenchyma, and the Ig index and IL-6 of the spinal fluid.
  • Example 2 Measurement of galectin 9 concentration before and after NPSLE treatment> (Materials and methods) Galectin 9 levels were measured before and after NPSLE treatment in a 19-year-old male patient (mainly fever, headache, and rash) at the time of onset. The course of treatment is as follows.
  • Cerebrospinal fluid examination revealed an increase in meningeal pressure and an increase in the number of lymphocyte-dominant cells, but viral and bacterial infections were negative, indicating aseptic meningitis.
  • Brain SPECT examination revealed decreased blood flow throughout the brain.
  • NPSLE was diagnosed after a series of courses, and the dose of prednisolone was increased to 60 mg / kg body weight / day. After the start of treatment, symptoms such as headache and fever improved, and prednisolone 50 mg / day from July 2 and prednisolone 45 mg / day from July 16 did not relapse even after tapering, and again on July 28.
  • the number of cells in the cerebrospinal fluid decreased to within the standard value.
  • the galectin 9 concentration in the cerebrospinal fluid sample before and after the treatment was measured in the same manner as in Example 1.
  • FIG. 2 shows that treatment with NPSLE significantly reduces the concentration of galectin 9 in the sample. This suggests that galectin 9 can be used to determine if treatment for NPSLE was successful.
  • Example 3 Measurement of galectin 9 concentration in NPSLE patients with aseptic meningitis> (Materials and methods) Aseptic meningitis (AM) is one of the neurological symptoms of NPSLE. Eighteen patients in the NPSLE group in Example 1 were classified into two groups, an AM group (7 patients) and an AM-less group (11 patients), based on the presence or absence of aseptic meningitis, and the cerebrospinal fluid in both groups. Medium galectin 9 concentrations were compared.
  • AM Aseptic meningitis

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Abstract

In one embodiment, the present invention addresses the problem of providing: a maker for detecting NPSLE; or a method for detecting NPSLE using the marker.　In one embodiment, the present invention relates to a biomarker for detecting NPSLE or a biomarker for determining a therapeutic effect on NPSLE, wherein the biomarker comprises galectin-9 protein or a peptide fragment thereof or a transcript of galectin-9 gene or a nucleotide fragment thereof. In one embodiment, the present invention relates to a method for assisting the determination as to whether or not a subject animal is affected by NPSLE or as to the presence or absence of NPSLE incidence risk, a method for assisting the determination of a therapeutic effect on NPSLE, or a kit for detecting NPSLE, in each of which the biomarker is used.

Description

神経精神ループスを検出するためのバイオマーカーBiomarker for detecting neuropsychiatric lupus
　一実施形態において、本発明は、神経精神ループス（NPSLE）を検出するためのバイオマーカー、NPSLEに対する治療効果の判定用バイオマーカー、被検動物がNPSLEに罹患しているか否か、又はNPSLE罹患リスクの有無の判定を補助する方法、NPSLEに対する治療効果の判定を補助する方法、又はNPSLEを検出するためのキットに関する。 In one embodiment, the present invention is a biomarker for detecting neuropsychiatric lupus (NPSLE), a biomarker for determining the therapeutic effect on NPSLE, whether or not the test animal has NPSLE, or the risk of developing NPSLE. The present invention relates to a method for assisting in determining the presence or absence of NPSLE, a method for assisting in determining the therapeutic effect on NPSLE, or a kit for detecting NPSLE.
　全身性エリテマトーデス（SLE）は、自己抗体の免疫複合体が組織に沈着して組織の損傷を引き起こす、慢性の再発性自己免疫疾患である。SLEのうち、中枢神経症状を呈する神経精神ループス（NPSLE）は、SLEの最重症病型であり、予後不良である。通常のSLEではステロイド剤による治療が行われるが、NPSLEではステロイド剤の増量や免疫抑制剤との併用が行われ、また治療開始が遅れると致死的である。 Systemic lupus erythematosus (SLE) is a chronic recurrent autoimmune disease in which immune complexes of autoantibodies deposit in tissues and cause tissue damage. Among SLEs, neuropsychiatric lupus (NPSLE), which presents with central nervous system symptoms, is the most severe form of SLE and has a poor prognosis. In normal SLE, treatment with steroids is performed, but in NPSLE, the dose of steroids is increased or used in combination with immunosuppressants, and it is fatal if the start of treatment is delayed.
　NPSLEは、現在、血液、髄液、画像所見等様々な臨床検査を組み合わせて診断が行われているが、診断基準は確立されておらず、またステロイドの副作用による精神症状などとの鑑別が非常に難しい。 Currently, NPSLE is diagnosed by combining various clinical tests such as blood, cerebrospinal fluid, and imaging findings, but diagnostic criteria have not been established, and it is extremely difficult to distinguish it from psychiatric symptoms due to side effects of steroids. Difficult to.
　非特許文献1には、NPSLE患者群において、非NPSLE及び非自己免疫疾患対照患者群に対して、髄液中においてIL-6等が増加していることが記載されている。しかしながら、非特許文献1に記載されたIL-6等のバイオマーカーは、NPSLEの検出について、必ずしも十分な精度及び／又は正確度を有するものとは言えない。 Non-Patent Document 1 describes that IL-6 and the like are increased in the cerebrospinal fluid in the NPSLE patient group as compared with the non-NPSLE and non-autoimmune disease control patient groups. However, the biomarkers such as IL-6 described in Non-Patent Document 1 do not always have sufficient accuracy and / or accuracy in detecting NPSLE.
　一実施形態において、本発明は、NPSLEを検出するためのマーカー、又は当該マーカーを用いて、NPSLEを検出する方法を提供することを課題とする。 In one embodiment, it is an object of the present invention to provide a marker for detecting NPSLE, or a method for detecting NPSLE using the marker.
　本発明者は、ガレクチン9がNPSLEを検出するためのバイオマーカー、又はNPSLEに対する治療効果の判定用バイオマーカーとして用い得ることを見出し、本発明を完成させた。 The present inventor has completed the present invention by finding that galectin 9 can be used as a biomarker for detecting NPSLE or as a biomarker for determining a therapeutic effect on NPSLE.
　本発明は、以下の実施形態を包含する。
（１）ガレクチン9タンパク質若しくはそのペプチド断片、又はガレクチン9遺伝子の転写産物若しくはその核酸断片からなる、神経精神ループス（NPSLE）を検出するためのバイオマーカー。
（２）前記ガレクチン9タンパク質が、以下の(a)～(c)のいずれかのタンパク質：
　(a)配列番号1で示されるアミノ酸配列を含むタンパク質
　(b)配列番号1で示されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列を含むタンパク質、及び
　(c)配列番号1で示されるアミノ酸配列に対して90％以上のアミノ酸同一性を有するアミノ酸配列を含むタンパク質、
である、（１）に記載のバイオマーカー。
（３）前記ガレクチン9遺伝子が、（２）に示すタンパク質をコードする、（１）又は（２）に記載のバイオマーカー。
（４）NPSLEを検出するための、（１）～（３）のいずれかに記載のバイオマーカーの使用。
（５）全身性エリテマトーデス（SLE）が、NPSLEであるか否かを判別するための、（１）～（３）のいずれかに記載のバイオマーカーの使用。
（６）被検動物由来の試料において、（１）～（３）のいずれかに記載のバイオマーカーを検出又は定量する工程を含む、該被検動物がNPSLEに罹患しているか否か、又はNPSLE罹患リスクの有無の判定を補助する方法。
（７）被験動物がSLEに罹患している、（６）に記載の方法。
（８）バイオマーカーを定量する工程を含む（６）又は（７）に記載の方法であって、
　定量されたバイオマーカーの量を閾値と比較する工程、
　定量されたバイオマーカーの量が閾値以上である場合に、前記被検動物がNPSLEに罹患しているか、又は罹患リスクが高いと判定する工程、
をさらに含む、前記方法。
（９）ガレクチン9タンパク質若しくはそのペプチド断片、又はガレクチン9遺伝子の転写産物若しくはその核酸断片からなる、NPSLEに対する治療効果の判定用バイオマーカー。
（１０）被検動物由来の試料において、（９）に記載のバイオマーカーを検出又は定量する工程を含む、NPSLEに対する治療効果の判定を補助する方法。
（１１）前記バイオマーカーを定量する工程を含む（１０）に記載の方法であって、
　定量されたバイオマーカーの量を閾値と比較する工程、
　定量されたバイオマーカーの量が閾値以下である場合に、NPSLEに対する治療効果があると判定する工程、
をさらに含む、前記方法。
（１２）前記被験動物がヒトである、（６）～（８）及び（１０）並びに（１１）のいずれかに記載の方法。
（１３）前記試料が髄液である、（６）～（８）及び（１０）～（１２）のいずれかに記載の方法。
（１４）（１）～（３）のいずれかに記載のバイオマーカーの量を測定するための試薬を含む、NPSLEを検出するためのキット。
（１５）ガレクチン9タンパク質又はそのペプチド断片と特異的に結合する抗体又はその断片を含む、NPSLEを検出するためのキットであって、
　前記ガレクチン9タンパク質が、以下の(a)～(c)のいずれかのタンパク質：
　　(a)配列番号1で示されるアミノ酸配列を含むタンパク質
　　(b)配列番号1で示されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列を含むタンパク質、及び
　　(c)配列番号1で示されるアミノ酸配列に対して90％以上のアミノ酸同一性を有するアミノ酸配列を含むタンパク質
である、前記キット。
（１６）ガレクチン9遺伝子の転写産物又はその断片と特異的に結合可能な核酸を含む、NPSLEを検出するためのキットであって、
　前記核酸が、以下の(d)～(h)のいずれかに示すポリヌクレオチド又はその断片からなる群から選択される、前記キット：
　　(d)
　　　(i)配列番号2で表される塩基配列若しくは当該塩基配列においてｕがｔである塩基配列からなるポリヌクレオチド、又は15以上の連続した塩基を含むその断片、
　　　(ii)(i)のポリヌクレオチド又はその断片の塩基配列において1又は数個の塩基の欠失、置換、付加又は挿入を含むポリヌクレオチド又はその断片、
　　　(iii)(i)のポリヌクレオチド又はその断片の塩基配列に対して90％以上の塩基同一性を有する塩基配列を含むポリヌクレオチド又はその断片、
　　　(iv)修飾核酸及び／又は修飾ヌクレオチドを含む(i)～(iii)のいずれかのポリヌクレオチド又はその断片、
　　(e)配列番号2で表される塩基配列、当該塩基配列においてｕがｔである塩基配列、又は15以上の連続した塩基を含むそのいずれかの部分配列を含むポリヌクレオチド、
　　(f)
　　　(v)配列番号2で表される塩基配列若しくは当該塩基配列においてｕがｔである塩基配列に相補的な塩基配列からなるポリヌクレオチド、又は15以上の連続した塩基を含むその断片、
　　　(vi)(v)のポリヌクレオチド又はその断片の塩基配列において1又は数個の塩基の欠失、置換、付加又は挿入を含むポリヌクレオチド又はその断片、
　　　(vii)(v)のポリヌクレオチド又はその断片の塩基配列に対して90％以上の塩基同一性を有する塩基配列を含むポリヌクレオチド又はその断片、
　　　(viii)修飾核酸及び／又は修飾ヌクレオチドを含む(v)～(vii)のいずれかのポリヌクレオチド又はその断片、
　　(g)配列番号2で表される塩基配列若しくは当該塩基配列においてｕがｔである塩基配列に相補的な塩基配列、又は15以上の連続した塩基を含むその部分配列を含むポリヌクレオチド、及び
　　(h)前記(d)～(g)のいずれかのポリヌクレオチド又はその断片と高ストリンジェントな条件でハイブリダイズするポリヌクレオチド。
（１７）NPSLEが無菌性髄膜炎を有する、（１５）又は（１６）に記載のキット。
　本明細書は本願の優先権の基礎となる日本国特許出願番号2019-112429号の開示内容を包含する。 The present invention includes the following embodiments.
(1) A biomarker for detecting neuropsychiatric lupus (NPSLE), which comprises a galectin 9 protein or a peptide fragment thereof, or a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
(2) The galectin 9 protein is any of the following proteins (a) to (c):
(a) Protein containing the amino acid sequence shown in SEQ ID NO: 1 (b) Protein containing the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and (c) ) A protein containing an amino acid sequence having 90% or more amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 1.
The biomarker according to (1).
(3) The biomarker according to (1) or (2), wherein the galectin 9 gene encodes the protein shown in (2).
(4) Use of the biomarker according to any one of (1) to (3) for detecting NPSLE.
(5) Use of the biomarker according to any one of (1) to (3) for determining whether systemic lupus erythematosus (SLE) is NPSLE.
(6) Whether or not the test animal suffers from NPSLE, which comprises the step of detecting or quantifying the biomarker according to any one of (1) to (3) in the sample derived from the test animal. A method to assist in determining the risk of developing NPSLE.
(7) The method according to (6), wherein the test animal has SLE.
(8) The method according to (6) or (7), which comprises a step of quantifying a biomarker.
The step of comparing the amount of quantified biomarker to the threshold,
A step of determining that the test animal has NPSLE or is at high risk of morbidity when the amount of the quantified biomarker is equal to or higher than the threshold value.
The method, further comprising.
(9) A biomarker for determining a therapeutic effect on NPSLE, which comprises a galectin 9 protein or a peptide fragment thereof, or a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
(10) A method for assisting determination of a therapeutic effect on NPSLE, which comprises a step of detecting or quantifying the biomarker according to (9) in a sample derived from a test animal.
(11) The method according to (10), which comprises a step of quantifying the biomarker.
The step of comparing the amount of quantified biomarker to the threshold,
A step of determining that there is a therapeutic effect on NPSLE when the amount of quantified biomarker is below the threshold value.
The method, further comprising.
(12) The method according to any one of (6) to (8) and (10) and (11), wherein the test animal is a human.
(13) The method according to any one of (6) to (8) and (10) to (12), wherein the sample is cerebrospinal fluid.
(14) A kit for detecting NPSLE, which comprises a reagent for measuring the amount of the biomarker according to any one of (1) to (3).
(15) A kit for detecting NPSLE, which comprises an antibody or a fragment thereof that specifically binds to a galectin 9 protein or a peptide fragment thereof.
The galectin 9 protein is one of the following proteins (a) to (c):
(a) Protein containing the amino acid sequence shown in SEQ ID NO: 1 (b) Protein containing the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and (c) ) The kit, which is a protein containing an amino acid sequence having 90% or more amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 1.
(16) A kit for detecting NPSLE, which contains a nucleic acid that can specifically bind to a transcript of the galectin 9 gene or a fragment thereof.
The kit: the kit: the nucleic acid is selected from the group consisting of the polynucleotides or fragments thereof shown in any of the following (d) to (h).
(d)
(i) A nucleotide consisting of the base sequence represented by SEQ ID NO: 2 or a base sequence in which u is t in the base sequence, or a fragment thereof containing 15 or more consecutive bases.
(ii) A polynucleotide or fragment thereof containing a deletion, substitution, addition or insertion of one or several bases in the base sequence of the polynucleotide or fragment thereof of (i).
(iii) A polynucleotide or fragment thereof containing a base sequence having 90% or more base identity with respect to the base sequence of the polynucleotide or fragment thereof of (i).
(iv) A polynucleotide or fragment thereof according to any one of (i) to (iii) containing a modified nucleic acid and / or a modified nucleotide.
(e) A nucleotide containing the base sequence represented by SEQ ID NO: 2, a base sequence in which u is t in the base sequence, or a partial sequence thereof containing 15 or more consecutive bases.
(f)
(v) A polynucleotide consisting of the base sequence represented by SEQ ID NO: 2 or a base sequence complementary to the base sequence in which u is t in the base sequence, or a fragment thereof containing 15 or more consecutive bases.
(vi) A polynucleotide or fragment thereof containing the deletion, substitution, addition or insertion of one or several bases in the base sequence of the polynucleotide or fragment thereof of (v).
(vii) A polynucleotide or fragment thereof containing a base sequence having 90% or more base identity with respect to the base sequence of the polynucleotide or fragment thereof of (v).
(viii) A polynucleotide or fragment thereof of any of (v) to (vii) containing a modified nucleic acid and / or a modified nucleotide.
(g) A nucleotide containing the nucleotide sequence represented by SEQ ID NO: 2, a nucleotide sequence complementary to the nucleotide sequence in which u is t in the nucleotide sequence, or a partial sequence thereof containing 15 or more consecutive nucleotides, and ( h) A polynucleotide that hybridizes with any of the polynucleotides (d) to (g) above or a fragment thereof under highly stringent conditions.
(17) The kit according to (15) or (16), wherein NPSLE has aseptic meningitis.
This specification includes the disclosure of Japanese Patent Application No. 2019-112429, which is the basis of the priority of the present application.
　本発明により、NPSLEを検出するためのマーカー、又は当該マーカーを用いてNPSLEを検出する方法が提供され得る。また、本発明により、NPSLEに対する治療効果を判定するためのマーカー、又は当該マーカーを用いてNPSLEに対する治療効果を判定する方法が提供され得る。 INDUSTRIAL APPLICABILITY The present invention may provide a marker for detecting NPSLE, or a method for detecting NPSLE using the marker. Further, the present invention may provide a marker for determining a therapeutic effect on NPSLE, or a method for determining a therapeutic effect on NPSLE using the marker.
図1は、NPSLE患者18例、他の疾患の患者（対照1）6例、及び非NPSLEのSLE患者（対照2）8例の髄液中ガレクチン9濃度を示す。FIG. 1 shows the cerebrospinal fluid galectin 9 concentrations in 18 NPSLE patients, 6 patients with other diseases (control 1), and 8 non-NPSLE SLE patients (control 2). 図2は、NPSLE患者1例の、治療前後の髄液中ガレクチン9濃度を示す。FIG. 2 shows the cerebrospinal fluid galectin 9 concentration before and after treatment in one NPSLE patient. 図3は、NPSLE患者18例のうち、無菌性髄膜炎（aseptic meningitis; AM）有り群（AM+）7例、及びAM無し群（AM-）11例の髄液中ガレクチン9濃度を示す。FIG. 3 shows the concentration of galectin 9 in the cerebrospinal fluid of 7 patients in the group with aseptic meningitis (AM +) and 11 patients in the group without AM (AM-) among 18 patients with NPSLE.
(バイオマーカー)
　一態様において、本発明は、ガレクチン9タンパク質若しくはそのペプチド断片、又はガレクチン9遺伝子の転写産物若しくはその核酸断片からなる、神経精神ループス（NPSLE）を検出するためのバイオマーカーに関する。 (Biomarker)
In one aspect, the invention relates to a biomarker for detecting neuropsychiatric lupus (NPSLE), which consists of a galectin 9 protein or peptide fragment thereof, or a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
　一態様において、本発明は、ガレクチン9タンパク質若しくはそのペプチド断片、又はガレクチン9遺伝子の転写産物若しくはその核酸断片からなる、NPSLEに対する治療効果の判定用バイオマーカーに関する。 In one aspect, the present invention relates to a biomarker for determining a therapeutic effect on NPSLE, which comprises a galectin 9 protein or a peptide fragment thereof, or a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
　本明細書において、「全身性エリトマトーデス（Systemic Lupus Erythematosus、SLE）」とは、自己抗体の免疫複合体が組織に沈着して組織の損傷を引き起こす、慢性の再発性自己免疫疾患である。本明細書において、「神経精神ループス（NPSLE、Neuropsychiatric SLE）」は、SLEのうち、中枢神経症状を呈するものを指し、神経精神SLEとも呼ばれる（例えば、Hanly JG et al.,Arthritis Rheum., 2019, 71(1):33-42を参照されたい）。 In the present specification, "systemic lupus erythematosus (SLE)" is a chronic recurrent autoimmune disease in which an immune complex of autoantibodies is deposited on tissues and causes tissue damage. In the present specification, "neuropsychiatric lupus (NPSLE, Neuropsychiatric SLE)" refers to SLE that exhibits central nervous system symptoms, and is also called neuropsychiatric SLE (for example, Hanly JG et al., Arthritis Rheum., 2019. , 71 (1): 33-42).
　NPSLEの症状は、大きく中枢神経系（精神症状及び神経症状）、末梢神経系の症状に分けられる。精神症状としては、気分障害、認知機能障害、不安障害、精神病性障害、及び急性錯乱状態が挙げられ、神経症状としては頭痛、痙攣、脳血管障害、運動障害（舞踏病）、脊髄症、無菌性髄膜炎、及び脱髄性症候群が挙げられる。末梢神経系の症状としては、多発ニューロパチー、脳神経のニューロパチー、単発／多発単ニューロパチー、急性炎症性脱髄性多発神経根ニューロパチー（ギラン・バレー症候群）、自律神経障害、重症筋無力症、及び神経叢障害が挙げられる（例えば、Arthritis Rheum., 1999, 42(4):599-608を参照されたい）。 Symptoms of NPSLE can be broadly divided into those of the central nervous system (psychiatric and neurological symptoms) and those of the peripheral nervous system. Psychiatric symptoms include mood disorders, cognitive dysfunction, anxiety disorders, psychotic disorders, and acute confusion, and neurological symptoms include headache, convulsions, cerebrovascular accidents, movement disorders (chorea), myelopathy, and aseptic. Examples include myelopathy and demyelinating syndrome. Peripheral nervous system symptoms include multiple neuropathy, cerebral nerve neuropathy, single / multiple single neuropathy, acute inflammatory demyelinating multiple nerve root neuropathy (Guillain-Barré syndrome), autonomic neuropathy, severe myasthenia, and neuropathy. Disorders can be mentioned (see, for example, Arthritis Rheum., 1999, 42 (4): 599-608).
　本発明では、被験動物の内在遺伝子に由来するガレクチン9タンパク質又はその遺伝子転写産物がバイオマーカーとなり得る。例えば、前記被験動物がヒトであれば、ヒトガレクチン9遺伝子に由来するヒトガレクチン9タンパク質及びヒトガレクチン9遺伝子の転写産物(mRNA)が本発明のバイオマーカーとなり得る。 In the present invention, a galectin 9 protein derived from an endogenous gene of a test animal or a gene transcript thereof can be a biomarker. For example, if the test animal is a human, the human galectin 9 protein derived from the human galectin 9 gene and the transcript (mRNA) of the human galectin 9 gene can be biomarkers of the present invention.
　ガレクチン9タンパク質の具体例として、配列番号1で示されるアミノ酸配列を含む、又はからなるヒト由来のガレクチン9(ヒトガレクチン9)タンパク質が挙げられる。 Specific examples of the galectin 9 protein include a human-derived galectin 9 (human galectin 9) protein containing or consisting of the amino acid sequence shown in SEQ ID NO: 1.
　また、ガレクチン9タンパク質には、配列番号1で示されるガレクチン9タンパク質と機能的に同等の活性を有するガレクチン9バリアントや他生物種のガレクチン9オルソログも包含される。具体的には、配列番号1で示されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、あるいは配列番号1で示されるアミノ酸配列に対して80%以上、90％以上、95％以上、97％以上、98％以上又は99％以上のアミノ酸同一性を有するガレクチン9タンパク質が包含される。 In addition, the galectin 9 protein also includes a galectin 9 variant having an activity functionally equivalent to that of the galectin 9 protein shown in SEQ ID NO: 1 and a galectin 9 ortholog of another species. Specifically, 80% or more and 90% of the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1 As described above, galectin 9 proteins having 95% or more, 97% or more, 98% or more, or 99% or more amino acid identity are included.
　本明細書において「数個」とは、例えば、2～10個、2～7個、2～5個、2～4個又は2～3個をいう。また、アミノ酸の置換は、保存的アミノ酸置換が望ましい。「保存的アミノ酸置換」とは、電荷、側鎖、極性、芳香族性等の性質の類似するアミノ酸間の置換をいう。性質の類似するアミノ酸は、例えば、塩基性アミノ酸(アルギニン、リジン、ヒスチジン)、酸性アミノ酸(アスパラギン酸、グルタミン酸)、無電荷極性アミノ酸(グリシン、アスパラギン、グルタミン、セリン、トレオニン、システイン、チロシン)、無極性アミノ酸(ロイシン、イソロイシン、アラニン、バリン、プロリン、フェニルアラニン、トリプトファン、メチオニン)、分枝鎖アミノ酸(ロイシン、バリン、イソロイシン)、芳香族アミノ酸(フェニルアラニン、チロシン、トリプトファン、ヒスチジン)等に分類することができる。 In the present specification, "several pieces" means, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 4 or 2 to 3. Conservative amino acid substitution is desirable for amino acid substitution. “Conservative amino acid substitution” refers to a substitution between amino acids having similar properties such as charge, side chain, polarity, and aromaticity. Amino acids with similar properties include, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagin, glutamine, serine, threonine, cysteine, tyrosine), non-polar amino acids. It can be classified into sex amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched amino acids (leucine, valine, isoleucine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine), etc. it can.
　本明細書において「アミノ酸同一性」とは、2つのアミノ酸配列を整列(アラインメント)し、必要に応じてギャップを導入して、両者のアミノ酸一致度が最も高くなるようにしたときに、配列番号1で示されるアミノ酸配列を含むガレクチン9タンパク質の全アミノ酸残基に対する2つのアミノ酸配列間で同一アミノ酸残基の割合(％)をいう。アミノ酸同一性は、BLASTやFASTAによるタンパク質の検索システムを用いて算出することができる。同一性の決定方法の詳細については、例えばAltschul et al, Nuc. Acids. Res. 25, 3389-3402, 1977及びAltschul et al., J. Mol. Biol. 215, 403-410, 1990を参照されたい。 As used herein, the term "amino acid identity" refers to the sequence number when two amino acid sequences are aligned and gaps are introduced as necessary to maximize the degree of amino acid matching between the two amino acids. The ratio (%) of the same amino acid residue between two amino acid sequences to the total amino acid residue of the galectin 9 protein containing the amino acid sequence represented by 1. Amino acid identity can be calculated using a protein search system using BLAST or FASTA. For details on how to determine identity, see, for example, Altschul et al, Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215, 403-410, 1990. I want to.
　「ガレクチン9遺伝子」は、前記ガレクチン9タンパク質をコードする遺伝子である。ガレクチン9遺伝子の具体例として、配列番号1で示されるアミノ酸配列を含むヒトガレクチン9タンパク質をコードするヒトガレクチン9遺伝子が挙げられる。より具体的には、ガレクチン9遺伝子は、配列番号2で示される塩基配列を含む、又はからなる遺伝子である。 The "galectin 9 gene" is a gene encoding the galectin 9 protein. Specific examples of the galectin 9 gene include a human galectin 9 gene encoding a human galectin 9 protein containing the amino acid sequence shown in SEQ ID NO: 1. More specifically, the galectin 9 gene is a gene containing or consisting of the nucleotide sequence shown in SEQ ID NO: 2.
　また、ガレクチン9遺伝子には、配列番号2で示されるガレクチン9遺伝子がコードするガレクチン9タンパク質と機能的に同等の活性を有するガレクチン9バリアントや他生物種のガレクチン9オルソログをコードするガレクチン9遺伝子も包含される。具体的には、配列番号2で示される塩基配列において1若しくは数個の塩基が欠失、置換又は付加された塩基配列、あるいは配列番号2で示される塩基配列に対して80%以上、90％以上、95％以上、97％以上、98％以上又は99％以上の塩基同一性を有するガレクチン9遺伝子が包含される。さらに、配列番号2で示される塩基配列に対して相補的な塩基配列の一部を含む核酸断片と高ストリンジェントな条件下でハイブリダイズする塩基配列を含み、かつガレクチン9タンパク質と機能的に同等の活性を有するタンパク質をコードする遺伝子が包含される。 In addition, the galectin 9 gene includes a galectin 9 variant having an activity functionally equivalent to that of the galectin 9 protein encoded by the galectin 9 gene shown in SEQ ID NO: 2 and a galectin 9 gene encoding a galectin 9 ortholog of another species. Included. Specifically, 80% or more and 90% of the base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 2 or the base sequence shown in SEQ ID NO: 2 As described above, galectin 9 genes having 95% or more, 97% or more, 98% or more, or 99% or more base identity are included. Furthermore, it contains a base sequence that hybridizes under highly stringent conditions with a nucleic acid fragment containing a part of the base sequence complementary to the base sequence shown in SEQ ID NO: 2, and is functionally equivalent to the galectin 9 protein. Contains genes encoding proteins with the activity of.
　本明細書において「塩基同一性」とは、2つの塩基配列を整列(アラインメント)し、必要に応じてギャップを導入して、両者の塩基一致度が最も高くなるようにしたときに、配列番号2で示される塩基配列を含むガレクチン9遺伝子の全塩基に対する2つの塩基配列間で同一塩基の割合(％)をいう。 As used herein, the term "base identity" refers to a sequence number when two base sequences are aligned and a gap is introduced as necessary so that the degree of base matching between the two is the highest. The ratio (%) of the same base between two base sequences to all the bases of the Galectin 9 gene including the base sequence shown by 2.
　本明細書において「高ストリンジェントな条件下でハイブリダイズする」とは、低塩濃度及び/又は高温の条件下でハイブリダイゼーションと洗浄を行うことをいう。例えば、6×SSC、5×Denhardt試薬、0.5％SDS、100μg/mL変性断片化サケ精子DNA中で65℃～68℃にてプローブと共にインキュベートし、その後、2×SSC、0.1％SDSの洗浄液中で室温から開始して、洗浄液中の塩濃度を0.1×SSCまで下げ、かつ温度を68℃まで上げて、バックグラウンドシグナルが検出されなくなるまで洗浄することが例示される。高ストリンジェントなハイブリダイゼーションの条件については、Green, M.R. and Sambrook, J., 2012, Molecular Cloning： A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New Yorkに記載されているので参考にすることができる。 As used herein, "hybridizing under high stringent conditions" means performing hybridization and washing under low salt concentration and / or high temperature conditions. For example, incubate with probe in 6 × SSC, 5 × Denhardt reagent, 0.5% SDS, 100 μg / mL denatured fragmented salmon sperm DNA at 65 ° C-68 ° C, then in a wash solution of 2 × SSC, 0.1% SDS. It is exemplified by starting from room temperature at room temperature, lowering the salt concentration in the cleaning solution to 0.1 × SSC, and raising the temperature to 68 ° C. until no background signal is detected. The conditions for high stringent hybridization are described in Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. It can be used as a reference.
　このようなガレクチン9遺伝子の塩基配列情報は、公共のデータベース(GenBank、EMBL、DDBJ)より検索可能である。例えば、配列番号2で示されるガレクチン9遺伝子の既知塩基配列情報に基づいて、塩基同一性の高い遺伝子をデータベースから検索し、入手することができる。 The nucleotide sequence information of such galectin 9 gene can be searched from public databases (GenBank, EMBL, DDBJ). For example, a gene having high base identity can be searched and obtained from a database based on the known base sequence information of the galectin 9 gene shown in SEQ ID NO: 2.
　「ガレクチン9遺伝子の転写産物」とは、ガレクチン9 mRNAを意味する。mRNAは、mRNA前駆体(pre-mRNA)及び成熟mRNA(mature mRNA)を問わない。通常、mRNA前駆体は、核内において直ちにスプライシングされて、成熟mRNA成熟体となることから、本発明のバイオマーカーとなるガレクチン9遺伝子の転写産物は、ガレクチン9成熟mRNAであってよい。 "Transcript of galectin 9 gene" means galectin 9 mRNA. The mRNA may be an mRNA precursor (pre-mRNA) or a mature mRNA (mature mRNA). Normally, the mRNA precursor is immediately spliced in the nucleus to become a mature mRNA, so that the transcript of the galectin 9 gene, which is a biomarker of the present invention, may be a galectin 9 mature mRNA.
　本明細書において「ペプチド断片」とは、ガレクチン9タンパク質を構成するアミノ酸配列の一部を含む、又はからなるペプチド断片であって、その断片を構成するアミノ酸配列からガレクチン9タンパク質の断片であることを同定することができるものをいう。例えば、「ペプチド断片」は、ガレクチン9タンパク質の全長アミノ酸配列のうちの10個以上、20個以上、30個以上、40個以上、又は50個以上の連続するアミノ酸酸残基であってよく、また、200個以下、150個以下、120個以下、100個以下、又は80個以下の連続するアミノ酸残基からなるペプチドであってよい。例えば、「ペプチド断片」は、10個～200個、30個～120個、50個～80個の連続するアミノ酸残基からなるペプチドであってよい。 As used herein, the term "peptide fragment" is a peptide fragment containing or consisting of a part of the amino acid sequence constituting the galectin 9 protein, and the fragment of the galectin 9 protein from the amino acid sequence constituting the fragment. Can be identified. For example, the "peptide fragment" may be 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more consecutive amino acid acid residues in the full-length amino acid sequence of the galectin 9 protein. Further, it may be a peptide consisting of 200 or less, 150 or less, 120 or less, 100 or less, or 80 or less consecutive amino acid residues. For example, a "peptide fragment" may be a peptide consisting of 10 to 200, 30 to 120, and 50 to 80 consecutive amino acid residues.
　本明細書において「核酸断片」とは、ガレクチン9 mRNAを構成する塩基配列の一部を含む、又はからなる核酸断片であって、その断片を構成する塩基配列からガレクチン9 mRNAの断片であることを同定することができるものをいう。例えば、「核酸断片」は、ガレクチン9 mRNAの全長塩基配列のうちの10個以上、20個以上、30個以上、40個以上、又は50個以上の連続する塩基からなる核酸であってよく、また600個以下、450個以下、300個以下、200個以下、又は100個以下の連続する塩基からなる核酸であってよい。例えば、「核酸断片」は、ガレクチン9 mRNAの全長塩基配列のうちの30個～600個、50個～300個、又は70個～100個の連続する塩基からなる核酸であってよい。 In the present specification, the "nucleic acid fragment" is a nucleic acid fragment containing or consisting of a part of the base sequence constituting galectin 9 mRNA, and is a fragment of galectin 9 mRNA from the base sequence constituting the fragment. Can be identified. For example, the "nucleic acid fragment" may be a nucleic acid consisting of 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more consecutive bases in the full-length base sequence of galectin 9 mRNA. Further, the nucleic acid may be composed of 600 or less, 450 or less, 300 or less, 200 or less, or 100 or less consecutive bases. For example, the "nucleic acid fragment" may be a nucleic acid consisting of 30 to 600, 50 to 300, or 70 to 100 consecutive bases in the full-length base sequence of galectin 9 mRNA.
(バイオマーカーを使用するNPSLEの検出)
　一態様において、本発明は、NPSLEを検出するための、本明細書に記載のバイオマーカーの使用に関する。 (Detection of NPSLE using biomarkers)
In one aspect, the invention relates to the use of the biomarkers described herein to detect NPSLE.
　一態様において、本発明は、被検動物がNPSLEに罹患しているか否か、又はNPSLE罹患リスクの有無を判定する（又は判定を補助する）方法に関する。本方法は、被検動物由来の試料において、本明細書に記載のバイオマーカーを検出又は定量する工程を含む。検出又は定量工程は、インビトロで行うことができる。また、本発明の方法は、前記工程で得られた検出又は定量結果に基づいて、該被検動物がNPSLEに罹患しているか否か、又はNPSLE罹患リスクの有無を判定する工程をさらに含んでもよい。 In one aspect, the present invention relates to a method for determining (or assisting) whether or not a test animal has NPSLE or is at risk of developing NPSLE. The method comprises the step of detecting or quantifying the biomarkers described herein in a sample derived from a test animal. The detection or quantification step can be performed in vitro. In addition, the method of the present invention further includes a step of determining whether or not the test animal has NPSLE or whether or not there is a risk of suffering from NPSLE based on the detection or quantification result obtained in the above step. Good.
　上記NPSLEの検出、NPSLEの罹患有無又は罹患リスクの判定は、NPSLE罹患患者と健常者の鑑別、及びNPSLEと他の疾患との鑑別を包含する。NPSLEと鑑別され得る他の疾患の例として、非NPSLEのSLE、Bechet病等の炎症性疾患、及びANCA関連血管炎等の膠原病、並びに脳炎、ステロイド誘発性精神障害(corticosteroid-induced psychiatric disorders(CIPDs))、視神経脊髄炎(NMO)、進行性多巣性白質脳症(PML)、及び逆性後頭葉白質脳症（PRES）の少なくとも一つが挙げられる。上記NPSLEの検出、NPSLEの罹患有無の判定は、SLEがNPSLEであるか否かを判別するものであってもよい。 The above-mentioned detection of NPSLE, determination of the presence or absence of NPSLE or determination of the risk of morbidity includes differentiation of NPSLE-affected patients from healthy subjects, and differentiation of NPSLE from other diseases. Examples of other diseases that can be differentiated from NPSLE include non-NPSLE inflammatory diseases such as SLE and Bechet's disease, and collagen diseases such as ANCA-related vasculitis, as well as encephalitis and corticosteroid-induced psychotric disorders (corticosteroid-induced psychiatric disorders). CIPDs))), neuromyelitis optica (NMO), progressive multifocal leukoencephalopathy (PML), and reverse posterior reversible leukoencephalopathy (PRES). The detection of NPSLE and the determination of the presence or absence of NPSLE may be used to determine whether or not SLE is NPSLE.
　上記の検出、罹患有無又は罹患リスクの判定の対象とするNPSLEは、上記の中枢神経系（精神症状及び神経症状）又は末梢神経系の症状のいずれかを有するNPSLEであってもよい。例えば、無菌性髄膜炎を有するNPSLEを検出、又は罹患有無若しくは罹患リスクの判定の対象としてもよい。 The NPSLE to be detected, whether or not it is affected or the risk of being affected may be an NPSLE having either the above-mentioned central nervous system (psychiatric and neurological symptoms) or peripheral nervous system symptoms. For example, NPSLE having aseptic meningitis may be detected, or the presence or absence of morbidity or the risk of morbidity may be determined.
　以下に各工程について詳細に説明する。
(1)検出又は定量工程
　本発明における被験動物は、例えば哺乳動物、例えばヒト及びアカゲザル等の霊長類、ラット、マウス、及びドブネズミ等の実験動物、ブタ、ウシ、ウマ、ヒツジ、及びヤギ等の家畜動物、並びにイヌ及びネコ等の愛玩動物が挙げられ、好ましくはヒトである。被験動物は、上記他の疾患、例えばSLEに罹患している、又は罹患している疑いのある被験体であってよい。 Each step will be described in detail below.
(1) Detection or quantification step The test animals in the present invention include, for example, mammals, for example, primates such as humans and red-tailed monkeys, experimental animals such as rats, mice, and rats, pigs, cows, horses, sheep, goats, etc. Domestic animals and pet animals such as dogs and cats are mentioned, and humans are preferable. The test animal may be a subject who has or is suspected of having the other diseases mentioned above, such as SLE.
　本発明において使用する試料は、特に限定されないが、例えば、体液、細胞、及び生検サンプル等の組織が挙げられる。体液の例として、髄液、血液（血清、血漿及び間質液を含む）、リンパ液、各組織若しくは細胞の抽出液、胸水、痰、涙液、鼻汁、唾液、尿等が挙げられ、例えば内部標準物質との補正が可能な髄液又は血液（特に血清）である。当該体液、細胞又は組織は、そのままバイオマーカーの測定に用いてもよいが、測定のために適宜前処理してもよい。例えば、免疫組織化学染色法でバイオマーカーを検出する場合は、被験動物由来の試料からパラフィン包埋切片を調製してもよい。また、例えば、ウエスタンブロット法又はRT-PCR法によってバイオマーカーを検出する場合は、被験動物由来の試料からタンパク質抽出液又はmRNA抽出液を調製してもよい。 The sample used in the present invention is not particularly limited, and examples thereof include tissues such as body fluids, cells, and biopsy samples. Examples of body fluids include cerebrospinal fluid, blood (including serum, plasma and interstitial fluid), lymph, extracts of tissues or cells, pleural fluid, sputum, tears, nasal juice, saliva, urine, etc. Cerebrospinal fluid or blood (particularly plasma) that can be corrected with standard substances. The body fluid, cells or tissue may be used as it is for the measurement of the biomarker, or may be appropriately pretreated for the measurement. For example, when biomarkers are detected by immunohistochemical staining, paraffin-embedded sections may be prepared from samples derived from test animals. Further, for example, when biomarkers are detected by Western blotting or RT-PCR, a protein extract or mRNA extract may be prepared from a sample derived from a test animal.
　本方法で測定されるバイオマーカーは、ガレクチン9タンパク質若しくはそのペプチド断片又はガレクチン9遺伝子の転写産物若しくはその核酸断片のいずれであってもよい。検出又は定量は、バイオマーカーの発現の有無、又は発現量若しくは発現濃度の大小等を測定することを包含する。本発明において、「測定」という用語には、半定量が包含される。 The biomarker measured by this method may be either a galectin 9 protein or a peptide fragment thereof, a transcript of the galectin 9 gene or a nucleic acid fragment thereof. Detection or quantification includes measuring the presence or absence of expression of a biomarker, or the magnitude of expression level or expression concentration. In the present invention, the term "measurement" includes semi-quantitative.
　測定されるバイオマーカーがガレクチン9タンパク質又はそのペプチド断片の場合、その測定方法は、公知のタンパク質定量方法であればよく、特に限定はしないが、例えば、免疫学的検出法が挙げられる。 When the biomarker to be measured is a galectin 9 protein or a peptide fragment thereof, the measurement method may be any known protein quantification method, and is not particularly limited, and examples thereof include an immunological detection method.
　「免疫学的検出法」は、抗原である標的分子と特異的に結合する抗体又は抗体断片を用いて、標的分子の量を測定する方法である。 The "immunological detection method" is a method of measuring the amount of a target molecule using an antibody or antibody fragment that specifically binds to the target molecule that is an antigen.
　抗体は、哺乳動物及び鳥を含めたいずれの動物由来とすることができる。例えば、マウス、ラット、モルモット、ウサギ、ヤギ、ロバ、ヒツジ、ラクダ、ウマ、ニワトリ又はヒト等が挙げられる。 The antibody can be derived from any animal, including mammals and birds. For example, mice, rats, guinea pigs, rabbits, goats, donkeys, sheep, camels, horses, chickens or humans can be mentioned.
　免疫学的検出法で使用する抗体は、特に限定されないが、モノクローナル抗体又はポリクローナル抗体を使用してよい。 The antibody used in the immunological detection method is not particularly limited, but a monoclonal antibody or a polyclonal antibody may be used.
　本明細書において「モノクローナル抗体」とは、単一免疫グロブリンのクローン群をいう。モノクローナル抗体を構成する各免疫グロブリンは、共通するフレームワーク領域及び共通する相補性決定領域を含み、同一抗原の同一エピトープを認識し、それに結合することができる。モノクローナル抗体は、単一細胞由来のハイブリドーマから得ることができる。 As used herein, the term "monoclonal antibody" refers to a group of clones of a single immunoglobulin. Each immunoglobulin constituting a monoclonal antibody contains a common framework region and a common complementarity determining region, and can recognize and bind to the same epitope of the same antigen. Monoclonal antibodies can be obtained from single cell hybridomas.
　本明細書において「ポリクローナル抗体」とは、同一抗原の異なるエピトープを認識し結合する複数種の免疫グロブリン群をいう。ポリクローナル抗体は、標的分子を抗原として動物に免疫後、その動物の血清から得ることができる。 As used herein, the term "polyclonal antibody" refers to a group of multiple immunoglobulins that recognize and bind to different epitopes of the same antigen. The polyclonal antibody can be obtained from the serum of an animal after immunizing the animal with the target molecule as an antigen.
　抗体がポリクローナル抗体又はモノクローナル抗体の場合、免疫グロブリン分子には、IgG、IgM、IgA、IgE、及びIgDの各クラスが知られているが、本発明の抗体は、いずれのクラスであってもよく、例えばIgGであってよい。 When the antibody is a polyclonal antibody or a monoclonal antibody, each class of IgG, IgM, IgA, IgE, and IgD is known as an immunoglobulin molecule, but the antibody of the present invention may be in any class. , For example, IgG.
　ガレクチン9タンパク質を認識し結合するポリクローナル抗体、又はモノクローナル抗体を産生するハイブリドーマを作製する方法は、ガレクチン9タンパク質又はその断片を抗原として当該分野で公知の抗体作製方法に準じて行えばよい。抗体はまた、製造業者から得てもよい。ガレクチン9タンパク質又はその断片の発現量は、市販のキットによって測定してもよく、そのようなキットとして、QuantikineTM ELISA Human Galectin-9 immunoassayが挙げられる。一実施形態において、ガレクチン9タンパク質又はその断片は、QuantikineTM ELISA Human Galectin-9 immunoassayに含まれる抗体によって認識されるエピトープを含む。 The method for producing a polyclonal antibody that recognizes and binds to galectin 9 protein or a hybridoma that produces a monoclonal antibody may be carried out according to an antibody production method known in the art using galectin 9 protein or a fragment thereof as an antigen. Antibodies may also be obtained from the manufacturer. The expression level of the galectin-9 protein or a fragment thereof may be measured by a commercially available kit, and such a kit includes Quantikine TM ELISA Human Galectin-9 immunoassay. In one embodiment, the galectin-9 protein or fragment thereof comprises an epitope recognized by an antibody contained in Quantikine TM ELISA Human Galectin-9 immunoassay.
　本明細書において「抗体断片」とは、ポリクローナル抗体又はモノクローナル抗体の部分断片であって、該抗体が有する抗原特異的結合活性と実質的に同等の活性を有するポリペプチド鎖又はその複合体をいう。例えば、抗原結合部位を少なくとも1つ包含する抗体部分、すなわち、少なくとも1組のVLとVHを有するポリペプチド鎖、又はその複合体が該当する。具体例としては、免疫グロブリンを様々なペプチダーゼで切断することによって生じる多数の十分に特徴付けられた抗体断片等が挙げられる。より具体的な例としては、Fab、F(ab')2、Fab'等が挙げられる。これらの抗体断片は、いずれも抗原結合部位を包含しており、抗原である標的分子と特異的に結合する能力を有している。 As used herein, the term "antibody fragment" refers to a polypeptide chain or a complex thereof that is a partial fragment of a polyclonal antibody or a monoclonal antibody and has an activity substantially equivalent to the antigen-specific binding activity of the antibody. .. For example, an antibody moiety that includes at least one antigen-binding site, that is, a polypeptide chain having at least one set of VL and VH, or a complex thereof. Specific examples include a large number of well-characterized antibody fragments produced by cleaving immunoglobulins with various peptidases. More specific examples include Fab, F (ab') 2 , Fab'and the like. All of these antibody fragments include an antigen-binding site and have the ability to specifically bind to a target molecule that is an antigen.
　免疫学的検出法としては、例えば、免疫組織化学染色法、酵素免疫測定法(ELISA法、EIA法を含む)、ウエスタンブロット法、放射免疫測定(RIA)法、免疫沈降法、又はフローサイトメトリー法が挙げられる。 Immunological detection methods include, for example, immunohistochemical staining, enzyme-linked immunosorbent assay (including ELISA and EIA), Western blotting, radioimmunoassay (RIA), immunoprecipitation, or flow cytometry. The law can be mentioned.
　「免疫組織化学染色法」は、公知の方法を採用することができる。例えば、被験動物由来の試料をホルマリン固定後、パラフィンに包埋して組織片に薄切し、スライドガラスに貼り付けたものを切片試料として使用してよい。切片試料は、場合により熱処理して抗原を賦活化し、その後、Dako EnVision+ System(Agilent社)などの市販の検出システムを用いて切片試料について免疫組織化学染色を行ってよい。 A known method can be adopted as the "immunohistochemical staining method". For example, a sample derived from a test animal may be fixed in formalin, embedded in paraffin, sliced into tissue pieces, and attached to a slide glass, which may be used as a section sample. The section sample may be heat-treated to activate the antigen, and then immunohistochemical staining may be performed on the section sample using a commercially available detection system such as DakoEnVision + System (Agilent).
　また、測定されるバイオマーカーがガレクチン9遺伝子転写産物又はその核酸断片の場合、その測定方法は、公知の核酸定量方法であればよく、特に限定はしないが、例えば、プライマーを用いる核酸増幅法又はプローブを用いるハイブリダイゼーション法が挙げられる。 When the biomarker to be measured is a galectin 9 gene transcript or a nucleic acid fragment thereof, the measurement method may be any known nucleic acid quantification method, and is not particularly limited, but for example, a nucleic acid amplification method using a primer or a nucleic acid amplification method using a primer. A hybridization method using a probe can be mentioned.
　「核酸増幅法」は、フォワード/リバースプライマーセット用いて、標的核酸の特定の領域を核酸ポリメラーゼによって増幅させる方法をいう。核酸増幅法としては、例えば、RT-PCR(逆転写ポリメラーゼ連鎖反応)法などのPCR(ポリメラーゼ連鎖反応)法が挙げられる。 "Nucleic acid amplification method" refers to a method in which a specific region of a target nucleic acid is amplified by a nucleic acid polymerase using a forward / reverse primer set. Examples of the nucleic acid amplification method include PCR (polymerase chain reaction) methods such as RT-PCR (reverse transcription polymerase chain reaction).
　「ハイブリダイゼーション法」は、検出すべき標的核酸の塩基配列の全部又は一部に相補的な塩基配列を有する核酸断片をプローブとして用い、その核酸と該プローブ間の塩基対合を利用して、標的核酸若しくはその断片を検出、定量する方法である。ハイブリダイゼーション法には、検出手段の異なるいくつかの方法が知られているが、例えば、ノザンハイブリダイゼーション法(ノザンブロットハイブリダイゼーション法)、in situハイブリダイゼーション法、又はマイクロアレイ法が挙げられる。 The "hybridization method" uses a nucleic acid fragment having a base sequence complementary to all or part of the base sequence of the target nucleic acid to be detected as a probe, and utilizes the base pairing between the nucleic acid and the probe. A method for detecting and quantifying a target nucleic acid or a fragment thereof. As the hybridization method, several methods with different detection means are known, and examples thereof include a Northern hybridization method (Northern blot hybridization method), an in situ hybridization method, and a microarray method.
　プライマー及びプローブ等の核酸鎖は、公知のバイオマーカー配列情報を基に当業者に公知の方法により適宜設計し、化学合成などの公知の作製方法により得ることができる。 Nucleic acid chains such as primers and probes can be appropriately designed by a method known to those skilled in the art based on known biomarker sequence information, and can be obtained by a known production method such as chemical synthesis.
　上述の各測定法は、いずれも当該分野に公知の技術である。したがって、具体的な測定方法については、公知の方法に準じて行えばよい。例えば、Green, M.R. and Sambrook, J., 2012(前述)に記載の方法を参考にすることができる。 Each of the above-mentioned measurement methods is a technique known in the art. Therefore, the specific measurement method may be performed according to a known method. For example, the method described in Green, M.R. and Sambrook, J., 2012 (described above) can be referred to.
(2)判定工程
　本工程では、前記検出又は定量工程で得られた結果に基づいて、該被検動物がNPSLEに罹患しているか否か、又はNPSLE罹患リスクの有無を判定する。 (2) Judgment step In this step, based on the result obtained in the detection or quantification step, it is determined whether or not the test animal has NPSLE or whether or not there is a risk of suffering from NPSLE.
　一実施形態において、本工程は、前記検出又は定量工程で得られた結果から、上記試料がバイオマーカーについて陽性であるか又は陰性であるか判定することを含む。上記試料がバイオマーカーについて陰性である場合、被験動物はNPSLEに罹患していないか、又はその罹患リスクは低いと判定し得る。一方、上記試料がバイオマーカーについて陽性である場合、被験動物はNPSLEに罹患しているか、又はその罹患リスクは高いと判定し得る。 In one embodiment, the step comprises determining whether the sample is positive or negative for the biomarker from the results obtained in the detection or quantification step. If the sample is negative for biomarkers, it can be determined that the test animal does not have NPSLE or is at low risk of developing it. On the other hand, if the sample is positive for biomarkers, it can be determined that the test animal has NPSLE or is at high risk of developing it.
　免疫組織化学染色法を用いる場合、例えば、一つ又は複数の細胞又は細胞クラスターが染色された場合、陽性と判定し、染色された細胞が存在しない場合、陰性と判定することができる。あるいは、細胞総数に対して染色された細胞数が一定割合(例えば10％、15％又は20％)を超える場合、陽性と判定し、細胞総数に対して染色された細胞数が前記一定割合以下の場合、陰性と判定してもよい。 When the immunohistochemical staining method is used, for example, when one or more cells or cell clusters are stained, it can be determined as positive, and when the stained cells do not exist, it can be determined as negative. Alternatively, if the number of stained cells exceeds a certain ratio (for example, 10%, 15% or 20%) with respect to the total number of cells, it is determined to be positive, and the number of stained cells with respect to the total number of cells is equal to or less than the certain ratio. In the case of, it may be determined as negative.
　一実施形態において、判定工程は、前記検出又は定量工程で得られた試料におけるバイオマーカーの発現レベルが、(例えば所定の閾値より)高いか低いかを判定することを含む。試料におけるバイオマーカーの発現レベルが、所定の閾値より低い(例えば、統計学的に有意に低い)場合、NPSLEに罹患していないか、又は(例えば、所定の閾値より高い発現レベルを有する集団に対して)その罹患リスクは低いと判定し得る。一方、試料におけるバイオマーカーの発現レベルが所定の閾値より高い(例えば、統計学的に有意に高い)場合、NPSLEに罹患しているか、又は(例えば、所定の閾値より低い発現レベルを有する集団に対して)その罹患リスクは高いと判定し得る。 In one embodiment, the determination step comprises determining whether the expression level of the biomarker in the sample obtained in the detection or quantification step is high or low (eg, below a predetermined threshold). If the expression level of the biomarker in the sample is below a predetermined threshold (eg, statistically significantly lower), then the population is not affected by NPSLE or (eg, has an expression level higher than the predetermined threshold). On the other hand, it can be judged that the risk of morbidity is low. On the other hand, if the expression level of the biomarker in the sample is higher than a predetermined threshold (eg, statistically significantly higher), then the population is either suffering from NPSLE or (eg, having an expression level lower than the predetermined threshold). On the other hand, the risk of morbidity can be determined to be high.
　所定の閾値は、対照試料(対照細胞又は組織、例えば対照大腸細胞又は大腸組織)において測定した対照量であってもよい。対照試料は、健常個体(例えば健常人)、非NPSLEのSLE患者、又は他の疾患に罹患した患者に由来してもよい。本発明において「健常個体」とは、被験個体と同じ生物種の、NPSLEに罹患していない健康な個体をいい、NPSLEに罹患したことがあるが、NPSLEが治療された個体を包含する。 The predetermined threshold value may be a control amount measured in a control sample (control cell or tissue, for example, control colon cell or colon tissue). The control sample may be derived from a healthy individual (eg, a healthy person), a non-NPSLE SLE patient, or a patient suffering from another disease. In the present invention, the "healthy individual" refers to a healthy individual of the same species as the test individual, which is not affected by NPSLE, and includes an individual who has been affected by NPSLE but has been treated with NPSLE.
　例えば、これらの対照試料における発現量、又は複数の個体における発現量の中央値、平均値、上限レベル、下限レベル、一定範囲の値を所定の閾値として用いることができる。また、上記の値若しくは範囲に適当な係数（例えば、0.3以上、0.5以上、0.8以上、0.9以上、又は3以下、2以下、1.5以下、又は1.1以下）をかけたものを閾値として用いてもよい。閾値は、予測の精度等に応じて適宜設定することができ、例えば、ROC(receiver operating characteristic curve：受信者動作特性曲線)解析により定めることができる。 For example, the median value, average value, upper limit level, lower limit level, and a certain range of values of the expression level in these control samples or the expression level in a plurality of individuals can be used as a predetermined threshold value. Also, the above value or range multiplied by an appropriate coefficient (for example, 0.3 or more, 0.5 or more, 0.8 or more, 0.9 or more, or 3 or less, 2 or less, 1.5 or less, or 1.1 or less) may be used as the threshold value. Good. The threshold value can be appropriately set according to the accuracy of prediction and the like, and can be determined by, for example, ROC (receiver operating characteristic curve) analysis.
　本明細書において「統計学的に有意」とは、得られた値の危険率(有意水準)が小さい場合、具体的には、p＜0.05(5％未満)、p＜0.01(1％未満)又はp＜0.001(0.1％未満)の場合を指す。統計学的検定方法は、有意性の有無を判断可能な公知の検定方法を適宜使用すればよく、特に限定しない。例えば、スチューデントt検定法、多重比較検定法、ログランク検定法を用いることができる。 In the present specification, "statistically significant" means that when the risk rate (significance level) of the obtained value is small, specifically, p <0.05 (less than 5%) and p <0.01 (less than 1%). ) Or p <0.001 (less than 0.1%). As the statistical test method, a known test method capable of determining the presence or absence of significance may be appropriately used, and is not particularly limited. For example, the Student's t-test method, the multiple comparison test method, and the log rank test method can be used.
　本明細書において、NPSLEの「罹患リスクが高い」とは、現在NPSLEに罹患していないものの、ある一定期間中（例えば、1週間以内、数週間以内、1カ月以内、2カ月以内、3カ月以内、6カ月以内、1年以内、2年以内、又は5年以内）にNPSLEに罹患する可能性が高いことを意図する。一方、NPSLEの「罹患リスクが低い」とは、現在NPSLEに罹患しておらず、かつある一定期間中（例えば、1週間以内、数週間以内、1カ月以内、2カ月以内、3カ月以内、6カ月以内、1年以内、2年以内、又は5年以内）にNPSLEに罹患する可能性が低いことを意図する。 As used herein, the term "high risk of morbidity" for NPSLE refers to a period of time (eg, within a week, within a few weeks, within a month, within 2 months, within 3 months), although not currently suffering from NPSLE. Within, within 6 months, within 1 year, within 2 years, or within 5 years), it is intended to be more likely to have NPSLE. On the other hand, "low risk of morbidity" of NPSLE means that the patient is not currently suffering from NPSLE and has a certain period of time (for example, within 1 week, within several weeks, within 1 month, within 2 months, within 3 months, It is intended that the likelihood of contracting NPSLE within 6 months, 1 year, 2 years, or 5 years) is low.
　本発明の被検動物がNPSLEに罹患しているか否か、又はNPSLE罹患リスクの有無を判定する（又は判定を補助する）方法は、さらに精度を高めるために、別のNPSLE診断法と組み合わせてもよい。そのような別のNPSLE診断法としては、画像検査(CT、MRI、SPECT、及びPET等、例えば頭部MRIにおける異常所見の有無)、脳波検査(脳波異常の有無)、髄液検査(髄液細胞数、髄液タンパク質及び髄液糖量等の髄液一般検査、髄液IL-6等のサイトカインの測定)、及び自己抗体(抗カルジオリピン抗体、ループスアンチコグラント、抗ribosomal P抗体、抗Ro/SS-A抗体、抗Sm抗体、抗ds-DNA抗体等)の検出等が挙げられる。このような診断法の診断基準については当業者に公知であり、例えば、山下裕之著、2017、日本内科学会雑誌106巻8号、1555頁～1563頁、西村勝治ら著、2011、総合病院精神医学、23(1)、42頁～51頁等を参照されたい。 The method of determining (or assisting) whether or not the test animal of the present invention has NPSLE or is at risk of developing NPSLE is combined with another NPSLE diagnostic method in order to further improve the accuracy. May be good. Other such NPSLE diagnostic methods include imaging tests (CT, MRI, SPECT, PET, etc., for example, presence or absence of abnormal findings in head MRI), electroencephalography (presence or absence of electroencephalogram abnormalities), and spinal fluid test (medullary fluid). General spinal fluid tests such as cell number, spinal protein and sugar content, measurement of cytokines such as spinal fluid IL-6), and autoantibodies (anti-cardiolipin antibody, lupus anticogrant, anti-ribosomal P antibody, anti-Ro / SS-A antibody, anti-Sm antibody, anti-ds-DNA antibody, etc.) detection and the like. Diagnostic criteria for such diagnostic methods are known to those skilled in the art, for example, Hiroyuki Yamashita, 2017, Journal of the Japanese Society of Internal Medicine 106, No. 8, pp. 1555 to 1563, Masaru Nishimura et al., 2011, General Hospital Psychiatry. See Medicine, 23 (1), pp. 42-51, etc.
(治療及び/又は予防方法)
　本発明によれば、NPSLEの罹患の有無又は罹患リスクを判定することができ、その結果に基づき、治療方針(例えば、併用薬の使用の有無、ステロイド剤（及び使用する場合には併用薬）の種類、投与量、投与間隔、及び投与期間)を決定することができる。 (Treatment and / or prevention method)
According to the present invention, the presence or absence or risk of morbidity of NPSLE can be determined, and based on the result, the treatment policy (for example, the presence or absence of concomitant drug use, steroid drug (and concomitant drug if used)). Type, dose, dosing interval, and dosing period) can be determined.
　本発明により、被験体がNPSLEに罹患している又はその罹患リスクが高いと判定された場合、NPSLEを治療及び／又は予防するために、被験動物にステロイド剤を投与してもよい。したがって、本発明はまた、本発明の方法によりNPSLEに罹患している又はその罹患リスクが高いと判定された被験動物にステロイド剤を投与することを含む、NPSLEの治療及び／又は予防方法を提供する。 If the present invention determines that a subject has or is at high risk of developing NPSLE, steroids may be administered to the test animal to treat and / or prevent NPSLE. Therefore, the present invention also provides a method for treating and / or preventing NPSLE, which comprises administering a steroid agent to a test animal which is determined to have NPSLE or is at high risk of developing NPSLE by the method of the present invention. To do.
　ステロイド剤としては、限定されないが、コルチコステロイド、例えばプレドニゾロン、メチルプレドニゾロン、ベタメタゾン、デキサメタゾン、トリアムシノロン及びヒドロコルチゾン並びにそれらの溶媒和物などが挙げられる。ステロイド剤は、単独で又は組みあわせて使用できる。ステロイド剤は、注射、静脈内投与、経口投与などの経路で投与され得る。 Examples of steroids include, but are not limited to, corticosteroids such as prednisolone, methylprednisolone, betamethasone, dexamethasone, triamcinolone and hydrocortisone, and their solvents. Steroids can be used alone or in combination. Steroids can be administered by routes such as injection, intravenous administration, or oral administration.
　ステロイド剤の投与量は、限定しないが、NPSLEでは高用量のステロイド（例えば、1.0mg/体重kg/日）を使用することが多く、症状を見ながら2週間おきに10％前後減量していくことが多い。投与量は、例えば0.005～100mg/体重kg/日、0.05～10mg/体重kg/日、0.1～5.0mg/体重kg/日、0.25～2.0mg/体重kg/日、又は0.5～1.0mg/体重kg/日であってよい。投与回数は、限定するものではないが、1日3回、1日2回、1日1回、2日に1回、3日に1回、1週間に1回、2週間に1回、1カ月に1回等であってよい。また、投与期間は、限定するものではないが、1日、2日、3日、1週間、2週間、1カ月、半年、一年、又はそれ以上であってよい。数日間、高用量の投与を行うステロイド・パルス療法を行ってもよい。ステロイド・パルス療法における投与量は、メチルプレドニゾロン換算で250mg～1000mg/日、点滴静注であってよい。ステロイド・パルス療法の期間は、限定するものではないが、1日、2日、3日、又はそれ以上であってよい。 The dose of steroids is not limited, but NPSLE often uses high doses of steroids (for example, 1.0 mg / kg body weight / day), and the dose is reduced by about 10% every two weeks while observing the symptoms. Often. The dosage is, for example, 0.005 to 100 mg / kg body weight / day, 0.05 to 10 mg / kg body weight / day, 0.1 to 5.0 mg / kg body weight / day, 0.25 to 2.0 mg / kg body weight / day, or 0.5 to 1.0 mg / body weight. It may be kg / day. The number of administrations is not limited, but is 3 times a day, 2 times a day, once a day, once every 2 days, once every 3 days, once a week, once every two weeks, It may be once a month or the like. The administration period may be, but is not limited to, 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, 6 months, 1 year, or more. Steroidal pulse therapy with high doses may be given for several days. The dose in steroid pulse therapy may be 250 mg to 1000 mg / day in terms of methylprednisolone, and may be intravenous drip infusion. The duration of steroid pulse therapy may be, but is not limited to, 1 day, 2 days, 3 days, or longer.
　ステロイド剤は他の薬剤と共に併用してもよく、他の薬剤の例として、免疫抑制剤が挙げられる。免疫抑制剤の例としては、限定されないが、アザチオプリン、シクロホスファミド、ミコフェノール酸モフェチル及びミゾリビン等が挙げられる。 Steroids may be used in combination with other drugs, and examples of other drugs include immunosuppressants. Examples of immunosuppressants include, but are not limited to, azathioprine, cyclophosphamide, mycophenolate mofetil, mizoribine and the like.
　免疫抑制剤の投与量は、限定しないが、例えば0.005～5000mg/体重kg/日、0.05～1000mg/体重kg/日、0.1～100mg/体重kg/日、又は1.0～10mg/体重kg/日であってよい。投与回数は、限定するものではないが、1日3回、1日2回、1日1回、2日に1回、3日に1回、1週間に1回、2週間に1回、1カ月に1回等であってよい。また、投与期間は、限定するものではないが、1日、2日、3日、1週間、2週間、1カ月、半年、一年、又はそれ以上であってよい。例えば、アザチオプリンは、0.5～2.5mg/体重kg/日を内服1日1回で連日投与することができ、シクロホスファミドは、500～750mg/体重kg/日、静注で2～3週間おきに計7～10回投与することができ、ミコフェノール酸モフェチルは1000mg～2000mg/体重kg/日、静注又は内服で、1日2回、連日投与することができ、ミゾリビンは、150～300mg/体重kg/日、内服で、1日1回～1日3回、連日投与することができる。 The dose of the immunosuppressant is not limited, but is, for example, 0.005 to 5000 mg / kg body weight / day, 0.05 to 1000 mg / kg body weight / day, 0.1 to 100 mg / kg body weight / day, or 1.0 to 10 mg / kg body weight / day. It may be there. The number of administrations is not limited, but is 3 times a day, 2 times a day, once a day, once every 2 days, once every 3 days, once a week, once every two weeks, It may be once a month or the like. The administration period may be, but is not limited to, 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, 6 months, 1 year, or more. For example, azathioprine can be administered at 0.5 to 2.5 mg / kg body weight / day once daily, and cyclophosphamide can be administered at 500 to 750 mg / kg body weight / day for 2 to 3 weeks by intravenous injection. It can be administered 7 to 10 times in total every other time, and mycophenolate mofetil can be administered 1000 mg to 2000 mg / kg body weight / day, intravenously or orally twice daily, and mizoribine can be administered 150 to 150 times. It can be administered at 300 mg / kg body weight / day orally once to three times a day every day.
　免疫抑制剤を投与する場合、ステロイド剤と、免疫抑制剤は同時に投与されてもよいし、異なる時点で投与されてもよい。同時に投与される場合、ステロイド剤、及び免疫抑制剤を含む組成物として同時に投与されてもよいし、ステロイド剤と、免疫抑制剤を別々に投与してもよい。ステロイド剤と免疫抑制剤が異なる時点で投与される場合、投与の順番は限定されず、ステロイド剤を投与した後に、免疫抑制剤を投与してもよいし、ステロイド剤を投与する前に、免疫抑制剤を投与してもよい。 When an immunosuppressant is administered, the steroid and the immunosuppressant may be administered at the same time or at different time points. When administered at the same time, it may be administered simultaneously as a composition containing a steroid agent and an immunosuppressive agent, or the steroid agent and the immunosuppressive agent may be administered separately. When the steroid and the immunosuppressant are administered at different times, the order of administration is not limited, and the immunosuppressant may be administered after the steroid is administered, or the immunosuppressant may be administered before the steroid is administered. Inhibitors may be administered.
(バイオマーカーを使用するNPSLEに対する治療効果の判定)
　一態様において、本発明は、(例えば、治療方法又は薬剤等の)NPSLEに対する治療効果を判定するための、本明細書に記載のバイオマーカーの使用に関する。 (Determination of therapeutic effect on NPSLE using biomarkers)
In one aspect, the invention relates to the use of the biomarkers described herein to determine a therapeutic effect on NPSLE (eg, a therapeutic method or agent, etc.).
　一態様において、本発明は、(例えば、治療方法、又は薬剤の)NPSLEに対する治療効果を判定する(又は判定を補助する)方法に関する。本方法は、被検動物由来の試料において、本明細書に記載のバイオマーカーを検出又は定量する工程を含む。本検出又は定量工程は、NPSLEに罹患しているか否か、又はNPSLE罹患リスクの有無を判定する方法における当該工程について記載した通りである。 In one aspect, the invention relates to a method of determining (or assisting) a therapeutic effect on NPSLE (eg, a therapeutic method or drug). The method comprises the step of detecting or quantifying the biomarkers described herein in a sample derived from a test animal. This detection or quantification step is as described for the step in the method of determining whether or not the patient has NPSLE or the risk of suffering from NPSLE.
　また、本方法は、前記工程で得られた検出又は定量結果に基づいて、NPSLEに対する治療効果があるか否かを判定する工程をさらに含んでもよい。本判定工程は、NPSLEに対する治療効果があるか否かを判定することを包含する。本判定工程についても、NPSLEに罹患しているか否か、又はNPSLE罹患リスクの有無を判定する方法における当該工程について記載した通りである。例えば、判定工程は、前記検出又は定量工程で得られた結果から、上記試料がバイオマーカーについて陽性であるか又は陰性であるか判定することを含む。上記試料がバイオマーカーについて陰性である場合、治療効果がある又は高いと判定し得る。一方、上記試料がバイオマーカーについて陽性である場合、治療効果がない又は低いと判定し得る。陽性、陰性の判定基準については記載した通りである。 Further, the present method may further include a step of determining whether or not there is a therapeutic effect on NPSLE based on the detection or quantification result obtained in the above step. This determination step includes determining whether or not there is a therapeutic effect on NPSLE. This determination step is also as described in the method for determining whether or not the patient has NPSLE or whether or not the patient is at risk of developing NPSLE. For example, the determination step includes determining whether the sample is positive or negative for the biomarker from the results obtained in the detection or quantification step. If the sample is negative for the biomarker, it can be determined to be therapeutically effective or high. On the other hand, if the sample is positive for the biomarker, it can be determined that the therapeutic effect is ineffective or low. The criteria for determining positive and negative are as described.
　また、判定工程は、前記検出又は定量工程で得られた試料におけるバイオマーカーの発現レベルが、(例えば所定の閾値より)高いか低いかを判定することを含む。試料におけるバイオマーカーの発現レベルが、所定の閾値より低い(例えば、統計学的に有意に低い)場合、(例えば、所定の閾値より高い発現レベルを有する集団に対して)治療効果がある又は高いと判定し得る。一方、試料におけるバイオマーカーの発現レベルが所定の閾値より高い(例えば、統計学的に有意に高い)場合、(例えば、所定の閾値より低い発現レベルを有する集団に対して)治療効果がない又は低いと判定し得る。所定の閾値についても、既に記載した通りである。ただし、本態様における閾値は、治療前の被験動物における発現レベルを含んでもよい。 The determination step also includes determining whether the expression level of the biomarker in the sample obtained in the detection or quantification step is higher or lower (for example, than a predetermined threshold value). When the expression level of the biomarker in the sample is lower than a predetermined threshold (for example, statistically significantly lower), the therapeutic effect is effective or high (for example, for a population having an expression level higher than the predetermined threshold). Can be determined. On the other hand, if the expression level of the biomarker in the sample is higher than a predetermined threshold (for example, statistically significantly higher), there is no therapeutic effect (for example, for a population having an expression level lower than the predetermined threshold). It can be judged to be low. The predetermined threshold value is also as described above. However, the threshold in this embodiment may include the expression level in the test animal before treatment.
　また、本方法は、前記検出又は定量工程の前、最中、及び／又は後に、NPSLEを治療する工程を含んでもよい。治療工程は、被験動物にステロイド剤(及び任意に免疫抑制剤)を投与することを含んでもよい。ステロイド剤、及び免疫抑制剤の種類、投与量、及び投与期間等については、上記治療及び/又は予防方法において記載した通りである。 The method may also include the step of treating NPSLE before, during, and / or after the detection or quantification step. The treatment step may include administering to the test animal a steroid (and optionally an immunosuppressant). The types, doses, administration periods, etc. of steroids and immunosuppressants are as described in the above-mentioned treatment and / or prevention methods.
　上記判定工程の結果に応じて、治療工程を変更してもよい。例えば、判定工程において、治療効果がないと判定された場合、上記薬剤の投与量を増加及び／又は投与する薬剤（例えば、免疫抑制剤）を追加してもよい。 The treatment process may be changed according to the result of the above determination process. For example, if it is determined in the determination step that there is no therapeutic effect, a drug (for example, an immunosuppressant) for increasing and / or administering the drug may be added.
(キット)
　一態様において、本発明は、本明細書に記載のバイオマーカーの量を測定するための試薬を含む、NPSLEを検出するためのキットも提供する。 (kit)
In one aspect, the invention also provides a kit for detecting NPSLE, which comprises a reagent for measuring the amount of biomarker described herein.
　バイオマーカーの量を測定するための試薬としては、例えば、上述のような抗体若しくは抗体断片、又はプローブ若しくはプライマーが挙げられる。キットは、公知の免疫組織化学染色、ELISA、ウエスタンブロット、又はRT-PCR用の試薬等、例えば、標識試薬、緩衝液、発色基質、二次抗体、ブロッキング剤、並びに試験に必要な器具、コントロール及び説明書等の少なくとも一つをさらに含んでもよい。 Examples of the reagent for measuring the amount of biomarker include the above-mentioned antibody or antibody fragment, or probe or primer. The kit includes known immunohistochemical staining, ELISA, Western blot, or reagents for RT-PCR, such as labeling reagents, buffers, chromogenic substrates, secondary antibodies, blocking agents, and instruments and controls required for testing. And at least one of the instructions and the like may be further included.
　以下、実施例を用いて本発明をさらに具体的に説明する。但し、本発明の技術的範囲はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples. However, the technical scope of the present invention is not limited to these examples.
＜実施例1:異なる患者群におけるガレクチン9濃度の測定＞
（材料と方法）
　サンプルとして、2004年6月から2019年1月の間に福島県立医科大学リウマチ膠原病内科を受診した、膠原病リウマチ性疾患患者の内、腰椎穿刺が施行され、-80℃にて髄液が凍結保存されていた全身性エリテマトーデス（SLE）26例、他疾患6例の髄液検体を用いた。髄液検体中のガレクチン9濃度を、QuantikineTM ELISA Human Galectin-9 immunoassay(R & D systems)を用いて、製造業者のプロトコルに従って、酵素免疫測定(ELISA)法で測定した。 <Example 1: Measurement of galectin 9 concentration in different patient groups>
(Materials and methods)
As a sample, among patients with rheumatoid arthritis who visited the Department of Internal Medicine for Rheumatoid Arthritis, Fukushima Prefectural Medical University between June 2004 and January 2019, lupus puncture was performed and the spinal fluid was discharged at -80 ° C. Twenty-six cases of systemic lupus erythematosus (SLE) and 6 cases of other diseases that had been cryopreserved were used. Galectin 9 concentrations in cerebrospinal fluid samples were measured by enzyme-linked immunosorbent assay (ELISA) using Quantikine TM ELISA Human Galectin-9 immunoassay (R & D systems) according to the manufacturer's protocol.
　上記26例のSLE患者は、髄液採取後に神経精神ループス(NPSLE)と診断した患者18例、及びNPSLEの関与は否定的と判断した患者8例（対照2）からなる。NPSLEは、SLEの活動性の血清学的評価、髄液検査、画像診断（MRI、SPECT）、臨床経過をふまえ、鑑別診断を行い、総合的に診断した（具体的には、1997年ACRの診断基準でSLEの確定診断がついており、頭痛・発熱などの症状に加え、SPECTで脳血流の低下を認め、MRIで脳実質に高吸収領域を認め、髄液のIg index及びIL-6の増加を認めた症例を、NPSLEと診断した。また、脳症を来たす他の疾患は鑑別診断で除外した。なお、現時点でNPSLEの確立した診断基準は存在しない）。コントロール群は、上記他疾患の6例（対照1、内訳としてBechet病5例、ANCA関連血管炎1例）からなる。各群間における髄液中ガレクチン9濃度につき比較検証を行った。 The above 26 SLE patients consist of 18 patients diagnosed with neuropsychiatric lupus (NPSLE) after CSF collection and 8 patients (control 2) who were judged to be negatively involved in NPSLE. NPSLE made a comprehensive diagnosis based on the serological evaluation of SLE activity, spinal fluid examination, diagnostic imaging (MRI, SPECT), and clinical course, and made a comprehensive diagnosis (specifically, 1997 ACR). A definitive diagnosis of SLE is made according to the diagnostic criteria, and in addition to symptoms such as headache and fever, SPECT shows a decrease in cerebral blood flow, MRI shows a highly absorbed region in the brain parenchyma, and the Ig index and IL-6 of the spinal fluid. Cases with an increase in the number of patients were diagnosed with NPSLE, and other diseases causing encephalopathy were excluded by differential diagnosis. There are no established diagnostic criteria for NPSLE at this time). The control group consists of 6 cases of the above other diseases (control 1, 5 cases of Behcet's disease, 1 case of ANCA-related vasculitis). A comparative verification was performed on the concentration of galectin 9 in the cerebrospinal fluid between each group.
（結果）
　結果を図1に示す。図1に示される通り、NPSLE群では、他の疾患群（対照1）、及び非NPSLEのSLE群（対照2）よりも検体中のガレクチン9濃度が有意に高かった。
　これは、ガレクチン9がNPSLEの検出用マーカーとして用い得ることを示唆している。 (result)
The results are shown in Figure 1. As shown in FIG. 1, the concentration of galectin 9 in the sample was significantly higher in the NPSLE group than in the other disease groups (control 1) and the non-NPSLE SLE group (control 2).
This suggests that galectin 9 can be used as a marker for detecting NPSLE.
＜実施例2：NPSLE治療前後のガレクチン9濃度の測定＞
（材料と方法）
　発症当時19歳の男性（主訴は、発熱、頭痛、及び皮疹）患者において、NPSLE治療前後のガレクチン9濃度を測定した。治療の経過は以下の通りである。 <Example 2: Measurement of galectin 9 concentration before and after NPSLE treatment>
(Materials and methods)
Galectin 9 levels were measured before and after NPSLE treatment in a 19-year-old male patient (mainly fever, headache, and rash) at the time of onset. The course of treatment is as follows.
　患者は、2004年顔面及び上肢の紅斑、並びに38℃台の発熱が出現し、同年6月福島県立医科大学附属病院皮膚科を受診した。血液検査にて炎症反応亢進や補体価の低下は認めず、抗核抗体陽性（Speckled, Granular型）、抗U1RNP抗体陽性であった。抗Sm抗体、抗DNA抗体は陰性であった。また、腎症を示唆する腎機能障害や尿所見は認めなかった。右前腕から皮膚生検を行ったところ、皮膚エリテマトーデスに矛盾しない所見を認めた。2004年6月、約38～39℃の発熱と強い頭痛が認められた。プレドニゾロン20mg/体重kg/日より内服を開始し、2004年6月7日、腰椎穿刺を施行した（治療前髄液ガレクチン9測定）。髄液検査からは髄圧上昇、リンパ球優位の細胞数の増多は認めるも、ウイルス及び細菌感染症は否定的であり無菌性髄膜炎の所見であった。脳SPECT検査にて脳全般に血流低下を認めた。 The patient developed erythema on the face and upper limbs and fever in the 38 ° C range in 2004, and visited the dermatology department of Fukushima Medical University Hospital in June of the same year. Blood tests showed no increase in inflammatory response or decrease in complement titer, and were positive for antinuclear antibody (Speckled, Granular type) and anti-U1RNP antibody. Anti-Sm antibody and anti-DNA antibody were negative. In addition, no renal dysfunction or urinary findings suggestive of nephropathy were observed. A skin biopsy from the right forearm revealed findings consistent with cutaneous lupus erythematosus. In June 2004, a fever of about 38-39 ° C and a strong headache were observed. Oral administration of prednisolone 20 mg / kg body weight / day was started, and lumbar puncture was performed on June 7, 2004 (measurement of pretreatment cerebrospinal fluid galectin 9). Cerebrospinal fluid examination revealed an increase in meningeal pressure and an increase in the number of lymphocyte-dominant cells, but viral and bacterial infections were negative, indicating aseptic meningitis. Brain SPECT examination revealed decreased blood flow throughout the brain.
　一連の経過よりNPSLEと診断され、プレドニゾロン60mg/体重kg/日に増量された。治療開始後、頭痛及び発熱といった症状の改善を認め、7月2日からはプレドニゾロン50mg/day、7月16日からはプレドニゾロン45mg/dayに漸減後も再燃は認めず、7月28日に再度施行した腰椎穿刺（治療後髄液ガレクチン9測定）では髄液中細胞数は基準値内にまで減少した。
　治療前、及び治療後の髄液検体中のガレクチン9濃度を、実施例1と同様に測定した。 NPSLE was diagnosed after a series of courses, and the dose of prednisolone was increased to 60 mg / kg body weight / day. After the start of treatment, symptoms such as headache and fever improved, and prednisolone 50 mg / day from July 2 and prednisolone 45 mg / day from July 16 did not relapse even after tapering, and again on July 28. In the lumbar puncture performed (measurement of cerebrospinal fluid galectin 9 after treatment), the number of cells in the cerebrospinal fluid decreased to within the standard value.
The galectin 9 concentration in the cerebrospinal fluid sample before and after the treatment was measured in the same manner as in Example 1.
（結果）
　結果を図2に示す。図2は、NPSLEの治療により、検体中のガレクチン9濃度が顕著に低下することを示している。これは、ガレクチン9が、NPSLEの治療が成功したか否かを判定するために用い得ることを示唆している。 (result)
The result is shown in figure 2. FIG. 2 shows that treatment with NPSLE significantly reduces the concentration of galectin 9 in the sample. This suggests that galectin 9 can be used to determine if treatment for NPSLE was successful.
＜実施例3：無菌性髄膜炎を有するNPSLE患者を対象としたガレクチン9濃度の測定＞
（材料と方法）
　無菌性髄膜炎（aseptic meningitis; AM）は、NPSLEの神経症状の１つである。実施例1におけるNPSLE群の患者18例を、無菌性髄膜炎の有無に基づいて、AM有り群（7例）とAM無し群（11例）の2群に分類し、両群における髄液中ガレクチン9濃度を比較した。 <Example 3: Measurement of galectin 9 concentration in NPSLE patients with aseptic meningitis>
(Materials and methods)
Aseptic meningitis (AM) is one of the neurological symptoms of NPSLE. Eighteen patients in the NPSLE group in Example 1 were classified into two groups, an AM group (7 patients) and an AM-less group (11 patients), based on the presence or absence of aseptic meningitis, and the cerebrospinal fluid in both groups. Medium galectin 9 concentrations were compared.
（結果）
　結果を図3に示す。図3に示される通り、AM有り群（7例）では、AM無し群（11例）よりも髄液中ガレクチン9濃度が有意に高かった（スチューデントt検定、p＜0.01）。これは、ガレクチン9が無菌性髄膜炎を有するNPSLEの検出用マーカーとして用い得ることを示唆している。
　本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。 (result)
The results are shown in Figure 3. As shown in FIG. 3, the cerebrospinal fluid galectin 9 concentration was significantly higher in the group with AM (7 cases) than in the group without AM (11 cases) (Student's t-test, p <0.01). This suggests that galectin 9 can be used as a marker for detecting NPSLE with aseptic meningitis.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (15)

  1. 　ガレクチン9タンパク質若しくはそのペプチド断片、又はガレクチン9遺伝子の転写産物若しくはその核酸断片からなる、神経精神ループス（NPSLE）を検出するためのバイオマーカー。 A biomarker for detecting neuropsychiatric lupus (NPSLE), which consists of a galectin 9 protein or a peptide fragment thereof, or a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
  2. 　前記ガレクチン9タンパク質が、以下の(a)～(c)のいずれかのタンパク質：
    　(a)配列番号1で示されるアミノ酸配列を含むタンパク質
    　(b)配列番号1で示されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列を含むタンパク質、及び
    　(c)配列番号1で示されるアミノ酸配列に対して90％以上のアミノ酸同一性を有するアミノ酸配列を含むタンパク質、
    である、請求項１に記載のバイオマーカー。     The galectin 9 protein is one of the following proteins (a) to (c):
    (a) Protein containing the amino acid sequence shown in SEQ ID NO: 1 (b) Protein containing the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and (c) ) A protein containing an amino acid sequence having 90% or more amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 1.
    The biomarker according to claim 1.
  3. 　前記ガレクチン9遺伝子が、請求項２に示すタンパク質をコードする、請求項１又は２に記載のバイオマーカー。 The biomarker according to claim 1 or 2, wherein the galectin 9 gene encodes the protein shown in claim 2.
  4. 　NPSLEを検出するための、請求項１～３のいずれか一項に記載のバイオマーカーの使用。 Use of the biomarker according to any one of claims 1 to 3 for detecting NPSLE.
  5. 　全身性エリテマトーデス（SLE）が、NPSLEであるか否かを判別するための、請求項１～３のいずれか一項に記載のバイオマーカーの使用。 Use of the biomarker according to any one of claims 1 to 3 for determining whether systemic lupus erythematosus (SLE) is NPSLE.
  6. 　被検動物由来の試料において、請求項１～３のいずれか一項に記載のバイオマーカーを検出又は定量する工程を含む、該被検動物がNPSLEに罹患しているか否か、又はNPSLE罹患リスクの有無の判定を補助する方法。 Whether or not the test animal suffers from NPSLE, or the risk of suffering from NPSLE, which comprises the step of detecting or quantifying the biomarker according to any one of claims 1 to 3 in the sample derived from the test animal. A method to assist in determining the presence or absence of.
  7. 　被験動物がSLEに罹患している、請求項６に記載の方法。 The method according to claim 6, wherein the test animal has SLE.
  8. 　バイオマーカーを定量する工程を含む請求項６又は７に記載の方法であって、
    　定量されたバイオマーカーの量を閾値と比較する工程、
    　定量されたバイオマーカーの量が閾値以上である場合に、前記被検動物がNPSLEに罹患しているか、又は罹患リスクが高いと判定する工程、
    をさらに含む、前記方法。     The method of claim 6 or 7, comprising the step of quantifying the biomarker.
    The step of comparing the amount of quantified biomarker to the threshold,
    A step of determining that the test animal has NPSLE or is at high risk of morbidity when the amount of the quantified biomarker is equal to or higher than the threshold value.
    The method, further comprising.
  9. 　ガレクチン9タンパク質若しくはそのペプチド断片、又はガレクチン9遺伝子の転写産物若しくはその核酸断片からなる、NPSLEに対する治療効果の判定用バイオマーカー。 A biomarker for determining the therapeutic effect on NPSLE, which comprises a galectin 9 protein or a peptide fragment thereof, or a transcript of the galectin 9 gene or a nucleic acid fragment thereof.
  10. 　被検動物由来の試料において、請求項９に記載のバイオマーカーを検出又は定量する工程を含む、NPSLEに対する治療効果の判定を補助する方法。 A method for assisting in determining a therapeutic effect on NPSLE, which comprises a step of detecting or quantifying the biomarker according to claim 9 in a sample derived from a test animal.
  11. 　前記バイオマーカーを定量する工程を含む請求項１０に記載の方法であって、
    　定量されたバイオマーカーの量を閾値と比較する工程、
    　定量されたバイオマーカーの量が閾値以下である場合に、NPSLEに対する治療効果があると判定する工程、
    をさらに含む、前記方法。     The method of claim 10, comprising the step of quantifying the biomarker.
    The step of comparing the amount of quantified biomarker to the threshold,
    A step of determining that there is a therapeutic effect on NPSLE when the amount of quantified biomarker is below the threshold value.
    The method, further comprising.
  12. 　前記被験動物がヒトである、請求項６～８及び１０並びに１１のいずれか一項に記載の方法。 The method according to any one of claims 6 to 8 and 10 and 11, wherein the test animal is a human.
  13. 　前記試料が髄液である、請求項６～８及び１０～１２のいずれか一項に記載の方法。 The method according to any one of claims 6 to 8 and 10 to 12, wherein the sample is cerebrospinal fluid.
  14. 　請求項１～３のいずれか一項に記載のバイオマーカーの量を測定するための試薬を含む、NPSLEを検出するためのキット。 A kit for detecting NPSLE, which comprises a reagent for measuring the amount of the biomarker according to any one of claims 1 to 3.
  15. 　ガレクチン9タンパク質又はそのペプチド断片と特異的に結合する抗体又はその断片を含む、NPSLEを検出するためのキットであって、
    　前記ガレクチン9タンパク質が、以下の(a)～(c)のいずれかのタンパク質：
    　　(a)配列番号1で示されるアミノ酸配列を含むタンパク質
    　　(b)配列番号1で示されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列を含むタンパク質、及び
    　　(c)配列番号1で示されるアミノ酸配列に対して90％以上のアミノ酸同一性を有するアミノ酸配列を含むタンパク質
    である、前記キット。     A kit for detecting NPSLE, which comprises an antibody or fragment thereof that specifically binds to the galectin 9 protein or a peptide fragment thereof.
    The galectin 9 protein is one of the following proteins (a) to (c):
    (a) Protein containing the amino acid sequence shown in SEQ ID NO: 1 (b) Protein containing the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and (c) ) The kit, which is a protein containing an amino acid sequence having 90% or more amino acid identity with respect to the amino acid sequence shown in SEQ ID NO: 1.
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