WO2022070798A1 - Marqueurs combinés pour différencier des états pathologiques de la maladie d'alzheimer et procédé de différenciation d'états pathologiques de la maladie d'alzheimer les utilisant - Google Patents

Marqueurs combinés pour différencier des états pathologiques de la maladie d'alzheimer et procédé de différenciation d'états pathologiques de la maladie d'alzheimer les utilisant Download PDF

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WO2022070798A1
WO2022070798A1 PCT/JP2021/032787 JP2021032787W WO2022070798A1 WO 2022070798 A1 WO2022070798 A1 WO 2022070798A1 JP 2021032787 W JP2021032787 W JP 2021032787W WO 2022070798 A1 WO2022070798 A1 WO 2022070798A1
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protein
sugar chain
mci
man
fragment
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康弘 橋本
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康弘 橋本
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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
    • 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
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention presents Alzheimer's disease and a combination biomarker for early identification of cognitive disorders that may progress to Alzheimer's disease from other central nervous system diseases, a kit for detecting them, and an Alzheimer's disease using the combination biomarker. Regarding the method of distinguishing the pathological condition.
  • AD Alzheimer's disease
  • a cognitive dysfunction accompanied by memory impairment and thinking disorder.
  • Behavioral disorders or personality changes. It is the most common dementia disease and accounts for about 60-80% of all dementia patients. It generally develops in the elderly aged 65 and over, but some also develop in the age of 64 and under, and is called juvenile Alzheimer's disease.
  • AD hydrophobic peptide amyloid ⁇ protein
  • APP amyloid precursor protein
  • PSEN1 presenilin 1
  • Non-Patent Document 1 Currently, based on the pathogenic mechanism of AD, many AD therapeutic agents that inhibit the pathogenic process are being developed. However, most of them have not yet been shown to be sufficiently effective in clinical trials. It is hypothesized that this is not because the investigational drug is ineffective, but because the study group is not appropriate.
  • the target group for which clinical trials have been conducted in the past was late-stage patients with advanced brain lesions due to AD and massive neuronal cell death. However, even if treatment is started at this stage, recovery of nerve function cannot be expected. That is, it is considered that an effective therapeutic effect could not be obtained because the administration time of the therapeutic agent was too late (Non-Patent Document 1).
  • Non-Patent Document 2 a monoclonal antibody against soluble A ⁇ aggregates was approved by the FDA for the first time in the world as a treatment for AD.
  • this therapeutic agent improves amyloid pathology, it does not improve cognitive symptoms (Non-Patent Document 2). This means that the effect of this therapeutic agent is limited to suppressing the progression of AD pathology. Therefore, if the AD therapeutic agent can be administered at an appropriate timing, that is, at an early stage in which nerve cell death can be avoided, its effectiveness can be expected. For that purpose, it is necessary to distinguish the AD pathological condition with higher accuracy.
  • MCI mini-mental state examination
  • NC Normal Control: often abbreviated as "NC” in the present specification
  • a method using a causative factor of AD as a marker for AD diagnosis is known.
  • biomarkers include tau protein, overly phosphorylated tau (p-tau) protein, amyloid ⁇ 42 (A ⁇ 42) peptide, amyloid ⁇ 40 (A ⁇ 40) peptide or cytokines in inflammatory diseases (Non-Patent Document 3).
  • p-tau overly phosphorylated tau
  • a ⁇ 42 amyloid ⁇ 42
  • a ⁇ 40 amyloid ⁇ 40
  • cytokines in inflammatory diseases
  • Patent Document 1 a transferase sugar protein having a mannose non-reducing terminal sugar chain
  • tau protein is not an AD-specific biomarker because it also increases in dementia such as frontotemporal dementia and progressive supranuclear palsy.
  • the p-tau protein is an excellent marker for AD diagnosis, but the appearance of this protein means the death of nerve cells, and although it is appropriate for early diagnosis of AD in which nerve cell death should be avoided. I didn't.
  • the A ⁇ 42 peptide did not change until after the disease had progressed.
  • cytokines can be measured with high sensitivity, they have the drawback of poor disease specificity.
  • the terminal Man-Tf has high specificity, it cannot be said that it is sufficient in terms of sensitivity.
  • the present invention develops a biomarker capable of specifically and accurately distinguishing the presence or absence of AD pathological condition from before the onset of the disease to the late stage of the onset of the disease and the risk of the onset of the disease, and a kit for detecting the biomarker, and a kit for detecting them.
  • An object of the present invention is to provide a highly accurate method for differentiating the AD pathological condition used.
  • the present inventors have conducted intensive studies, and conducted in situ hybridization in the human cerebrum, mass analysis of mannose sugar chains bound to Tf in the brain, and immunohistochemical analysis of AD patients.
  • the hippocampus we found that both terminal Man-Tf and p-tau, which are markers for AD diagnosis, are co-localized in neurons. This result suggests that terminal Man-Tf secretion and tau phosphorylation may be correlated. Therefore, as a result of investigating the relationship between sensitivity and specificity by combining the amounts of terminal Man-Tf protein and p-tau protein or tau protein in the cerebrospinal fluid collected from AD patients and MCI patients, the product of both was obtained, respectively.
  • AD pathology As compared with the case of using it alone.
  • AD diagnostic markers are available for transferase glycoproteins (terminal GlcNAc-Tf proteins) having N-acetylglucosamine non-reducing terminal sugar chains and prostaglandin-H2 D-isomerase (PGDS). It was found that when combined, they can be excellent biomarkers for differentiating AD pathology compared to the case of each alone.
  • a combination marker for differentiating Alzheimer's disease pathological condition consisting of any of the following (a) to (c).
  • Man-Tf protein Transtransferase glycoprotein
  • PGDS prostaglandin D2 synthase
  • the AD index is calculated from the formula (amyloid ⁇ 40 peptide / amyloid ⁇ 42 peptide) ⁇ p-tau protein.
  • Tf transtransferase
  • a sugar chain having mannose at the non-reducing end of the Man-Tf protein is added to the asparagine residue at position 432
  • the GlcNAc- The differential combination marker according to (1), wherein the sugar chain having N-acetylglucosamine at the non-reducing end of the Tf protein is added to the asparagine residues at positions 432 and 630.
  • AD Alzheimer's disease
  • MCI mild cognitive impairment
  • NC healthy control
  • the differential combination marker according to (3) which further differentiates the NC into an MCI transitional healthy control (pre-MCI) and an MCI non-transitional healthy control (non-pre-MCI).
  • a peptide-binding molecule and a sugar chain bond that are a kit for differentiating Alzheimer's pathology and specifically bind to each of the combination markers for differentiating Alzheimer's pathology according to any one of (1) to (4).
  • the polypeptide containing the molecule and specifically bound to the peptide bond molecule is at least one selected from the group consisting of Tf protein, PGDS, and tau protein, and the sugar chain binding molecule specifically binds to the polypeptide.
  • the above-mentioned discrimination kit wherein the sugar chain is a sugar chain having mannose at the non-reducing end and / or a sugar chain having N-acetylglucosamine at the non-reducing end.
  • the method for differentiating Alzheimer's disease pathology according to any one of (1) to (4), which is present in a predetermined amount of body fluid obtained from a subject suspected of having dementia.
  • the amount of each marker in the combination marker for use was measured using a peptide bond molecule that specifically binds to the polypeptide of the marker and a sugar chain binding molecule that specifically binds to the sugar chain of the marker, and the measured value thereof.
  • the discrimination method including a step of determining which of NC and NC can be applied.
  • the discrimination method including a step of determining whether the subject can correspond to pre-MCI or non-pre-MCI from the product value.
  • a combination marker for differentiating Alzheimer's disease pathological condition which comprises any of the following (a) to (c).
  • PGDS prostaglandin D2 synthase
  • This specification includes the disclosure content of Japanese Patent Application No. 2020-163105, which is the basis of the priority of the present application.
  • the AD pathological condition of the subject that is, the presence or absence of AD or MCI and the possibility of being affected can be discriminated with high accuracy.
  • A is a terminal Sia-Tf glycoprotein having two sialic acid non-reducing terminal sugar chains
  • B is a terminal GlcNAc-Tf glycoprotein having two GlcNAc non-reducing terminal sugar chains
  • C is a Man non-reducing terminal sugar chain.
  • GlcNAc show a terminal Man-Tf glycoprotein having one non-reducing terminal sugar chain each.
  • It is an immunohistochemical staining diagram showing the localization of Tf protein and phosphorylated tau (p-tau) protein in the hippocampus.
  • A indicates the hippocampus of a healthy body
  • B indicates the hippocampus of an AD patient.
  • merge is a diagram in which a staining diagram of Tf protein and p-tau protein is synthesized. It is a dot graph which shows the amount of Man-Tf protein in the cerebrospinal fluid in various dementia patients.
  • the black triangles indicate NC patients who have transitioned from NC to MCI by follow-up
  • the black squares indicate MCI patients who have transitioned from MCI to AD by follow-up. It is a dot graph in each AD pathological condition when two markers are combined.
  • A is a dot graph for comparison when only Man-Tf protein is used as a marker
  • B is a dot graph when a combination marker of Man-Tf protein and p-tau protein is used
  • C is Man-Tf protein.
  • the dot graph when the combination marker of tau protein is used is shown.
  • the broken line in the figure shows the cutoff value of each group when iNPH is used as a disease control.
  • a first aspect of the present invention is a combination marker for differentiating Alzheimer's disease (often referred to herein as "combination marker for AD pathology" or simply “combination marker”).
  • the combination marker of the present invention comprises a combination of two or more kinds of proteins or peptide fragments thereof, and can be used in the AD pathological condition differentiation method of the third aspect described later for a subject suspected of having dementia.
  • Alzheimer's disease state (often referred to as “AD condition” in the present specification) is a tauopathy whose pathogenic mechanism is abnormal accumulation of phosphorylated tau protein in nerve cells.
  • AD condition is a tauopathy whose pathogenic mechanism is abnormal accumulation of phosphorylated tau protein in nerve cells.
  • AD condition is a tauopathy whose pathogenic mechanism is abnormal accumulation of phosphorylated tau protein in nerve cells.
  • MCI mild cognitive impairment
  • AD Alzheimer's disease
  • MCI Mild cognitive impairment
  • NC normal control
  • patient with suspected dementia refers to all patients who have undergone a forgetfulness outpatient clinic.
  • Patients with suspected dementia include AD, MCI, progressive supranuclear palsy (abbreviated as “PSP” in this specification), and frontotemporal dementia; FTD ”), dementia with Lewy bodies (abbreviated as“ DLB ”in the present specification), Parkinson's disease (abbreviated as“ PD ”in the present specification) and NC. It falls under either category and is usually divided into either group according to the diagnosis of a doctor according to clinical diagnostic criteria.
  • the diagnostic criteria used in this case are well known to those skilled in the art, including the above-mentioned Dementia Score (MMSE) (see, for example, Dementia Disease Clinical Practice Guidelines 2017, Igaku-Shoin).
  • NC cognitive impairment transition type NC
  • pre-MCI refers to a pathological condition among NCs that is likely to eventually transition to MCI as the condition progresses without therapeutic intervention.
  • NC has been diagnosed as normal for dementia because it does not correspond to any dementia in the clinical diagnostic criteria for dementia.
  • the combination marker for AD pathological condition identification of the present invention has revealed that there may be a population in which a part of NC may be transferred to MCI in the future. Therefore, in the present specification, NC is considered to include two groups of pre-MCI and non-pre-MCI.
  • Non-pre-MCI is almost synonymous with conventional NC, and the possibility of transition to MCI in the future is low, and it may be normal for dementia. High NC.
  • diagnosis refers to the differentiation or possibility of morbidity of a disease, or the determination of the pathophysiology of a disease.
  • diagnosis act is the exclusive business of a doctor, a veterinarian, or a dentist, but the diagnosis in the present specification includes an auxiliary act of assisting the diagnosis by a doctor or the like without going through the act of the doctor or the like. ..
  • the term "affected () differentiation” refers to determining whether or not a person has a specific disease. It also includes distinguishing from other diseases with similar lesions and conditions.
  • the term "possibility of morbidity” refers to the probability of shifting from the current state to a specific disease in the future and suffering from that specific disease. As used herein, it means the probability of contracting AD when a specific disease is AD, and in particular, the probability of transition from MCI to AD.
  • the term "subject” refers to an object of application in each aspect of the present invention. Specifically, it refers to a subject to be subjected to the differentiation method of this embodiment for the purpose of differentiating the morbidity of AD pathology.
  • it is an individual animal, but it also includes its tissues and cells. Specific examples thereof include mammals, preferably humans, dogs, cats, horses and the like. It is preferably human.
  • the individual may be either an individual suffering from some kind of disease, such as a person undergoing a medical examination, an individual having a possibility of suffering from some kind of disease, or a healthy body.
  • the “disease-potential individual” is, for example, an individual having a possibility of transition from MCI to AD in the future or a pre-MCI individual having a possibility of transition from NC to MCI in the future.
  • the "healthy body” means an individual in a healthy state.
  • the term “healthy state” means at least a state not suffering from AD pathology or the like, preferably a healthy state without any disease or disorder.
  • the "disease control" described in Examples described later is an individual suffering from idiopathic normal pressure hydrocephalus (idiopathic Normal Pressure Hydrocephalus; often abbreviated as "iNPH” in the present specification). iNPH is clearly different from AD pathology. Therefore, the disease control corresponds to a healthy body herein.
  • a healthy body is mainly used as a control body as a reference for comparison with a subject. Therefore, in the present specification, the subject and the healthy body are the same species. Further, it is preferable that the conditions such as subspecies (including race), gender, age (including monthly and weekly age), height, and weight are the same.
  • body fluid refers to a liquid sample collected from a subject and a control body.
  • cerebrospinal fluid cerebrospinal fluid
  • blood including serum, plasma and interstitial fluid
  • urine urine
  • lymph digestive fluid
  • ascites pleural effusion
  • perineural fluid extracts of tissues or cells.
  • cerebrospinal fluid or blood more preferably cerebrospinal fluid.
  • Cerebrospinal fluid refers to a colorless and transparent extracellular fluid that exists only around the central nervous system (CNS: Central Nerve System) of the brain and spinal cord. It is separated from the blood by the hard membrane and by the blood-cerebrospinal and blood-cerebrospinal fluid barriers. Most of the cerebrospinal fluid exudes from the parenchyma of the brain and between brain cells and cerebrospinal fluid. Recent studies have shown that there are virtually no barriers (Wang C, et al., 2012, Cerebrospinal Fluid: Physiology, biomarker and methodology. In: V.S, Dolezal, T., editor.
  • Cerebrospinal Fluid Functions, Composition and Disorders. New York: Nova Science Publishers; pp.1-37.).
  • proteins derived from the central nervous system in this cerebrospinal fluid and they are associated with central nervous system diseases. It is known that the expression increases or decreases. For example, in AD pathology, it is suggested that the amount of cerebrospinal fluid increases and the waste products of the brain including A ⁇ are eliminated (Lliff, et al). ., Sci. Transl. Med., 2010, 4).
  • glycoprotein refers to a protein to which one or more sugar chains are added.
  • sugar chains are added as one of post-translational modifications, and are said to be present in more than 50% of proteins in vivo.
  • Sugar chains play an important role in imparting various functions to proteins, such as being involved in protein stabilization, protection, bioactivity, antigen-antibody reaction, viral infection, and pharmacokinetics.
  • the "peptide fragment (including the sugar chain)" is a peptide consisting of a part of the glycoprotein and containing an amino acid residue to which the target sugar chain is added.
  • a peptide consisting of a part of terminal Man-Tf and containing an asparagine residue at position 432 to which a mannose non-reducing terminal sugar chain is added can be mentioned.
  • the number of amino acids in the peptide fragment is not particularly limited. Since it is a part of glycoprotein, the lower limit may be a length that can include the epitope, for example, 8 amino acids or more, preferably 10 or 15 amino acids or more, more preferably 20 or 30 amino acids or more, and the upper limit is glycoprotein. It suffices to have amino acids less than the total length of.
  • the term "marker” refers to an index molecule for differentiating the onset or possibility of morbidity of a disease.
  • the marker corresponds to a biomarker targeting a molecule derived from a living body, particularly a polypeptide (including a glycoprotein and a fragment thereof). The marker is detected in the body tissue of the subject, but is preferably detected in body fluids herein.
  • the term “combination marker” refers to a marker that can achieve a predetermined purpose by combining a plurality of the markers.
  • predetermined purpose refers to the differentiation of Alzheimer's disease.
  • significant means statistically significant.
  • Statistically significant means that there is a significant difference between the measured and control values of the subject when statistically processed.
  • the significant difference between the measured value of the terminal Man-Tf amount of the subject and the healthy body group when statistically processed is applicable.
  • the risk factor (significance level) of the obtained value is small, specifically, when it is smaller than 5% (p ⁇ 0.05), when it is smaller than 1% (p ⁇ 0.01), and when it is smaller than 0.1% (p). ⁇ 0.001) can be mentioned.
  • the "p (value)” shown here indicates the probability that the test statistic will happen to be the value in the distribution based on the null hypothesis in the statistical test.
  • test method for statistical processing a known test method capable of determining the presence or absence of superiority may be appropriately used, and is not particularly limited. For example, Student's t-test method, covariate ANOVA, etc. can be used.
  • Composition The "Combination marker for differentiating Alzheimer's disease (combination marker for AD pathological condition)" of the present invention is composed of a combination of markers consisting of two or more types of protein or peptide fragments, and is intended for subjects suspected of having dementia. It is a biomarker capable of differentiating the susceptibility of AD or MCI in AD pathology.
  • the combination markers of the present invention are composed of two or more markers.
  • the combination markers of the present invention are a transferrin glycoprotein or a fragment thereof containing a predetermined sugar chain, a prostaglandin D2 synthase or a fragment thereof, a tau-related protein or a fragment thereof, and an AD index.
  • each marker will be described.
  • Transferrin glycoprotein (Tf glycoprotein)
  • transferrin glycoprotein is a transferrin protein to which an N-linked sugar chain has been added.
  • the transferrin (Tf) protein (often referred to herein as the "Tf protein") is a carrier with a molecular weight of approximately 80 KDa that reversibly binds to two iron (Fe) ions and is responsible for its in vivo transport. It is a protein.
  • Specific examples of the Tf protein include a human Tf protein composed of 698 amino acid residues and consisting of the amino acid sequence shown in SEQ ID NO: 1.
  • Tf glycoproteins there are three types of isoforms in which the protein part is common to Tf glycoproteins and only the structure of the added N-linked sugar chain is different. Specifically, it is a terminal Man-Tf protein, a terminal GlcNAc-Tf protein, and a terminal Sia-Tf protein. Of these, the Tf glycoproteins that can constitute the combination marker of the present invention are the terminal Man-Tf protein and the terminal GlcNAc-Tf protein. Each Tf glycoprotein will be specifically described below.
  • Terminal Man-Tf protein is an N-linked sugar chain having mannose (often referred to as "Man” in the present specification) at the non-reducing end, that is, the Man non-reducing end.
  • a Tf glycoprotein having a structure in which a Man non-reducing terminal sugar chain is added to one of the two sugar chains and a GlcNAc non-reducing terminal sugar chain is added to the other can be mentioned. ..
  • the terminal Man-Tf protein is considered to be biosynthesized in the brain because it is not detected in the cerebrospinal fluid in anencephaly in which most of the cerebrum is congenitally deleted.
  • the amount of terminal Man-Tf increases in AD pathology. That is, the amount of terminal Man-Tf in body fluid, mainly in cerebrospinal fluid, is increased as compared with a healthy body, and therefore, terminal Man-Tf alone can be a marker for differentiating AD pathological condition reflecting AD pathological condition.
  • iNPH patients cannot be used as a differential marker for iNPH because there is no significant difference in the amount of terminal Man-Tf in body fluids, mainly in cerebrospinal fluid, as compared with healthy bodies.
  • AD can be easily distinguished from DLB or FTD by measuring the amount of terminal Man-Tf in body fluid.
  • a peptide fragment of a terminal Man-Tf protein containing a Man non-reducing terminal sugar chain can also be a constituent marker of the combination marker of the present invention.
  • Terminal GlcNAc-Tf protein is an N-linked sugar chain having N-acetylglucosamine (often referred to as "GlcNAc" in the present specification) at the non-reducing end, that is, GlcNAc. It is a Tf glycoprotein containing only a non-reducing terminal sugar chain, and is a constituent marker of the combination marker of the present invention. For example, as shown in B of FIG. 1, a Tf glycoprotein to which two GlcNAc non-reducing terminal sugar chains are added can be mentioned.
  • the asparagine (Asn: N) residue at position 432 (denoted by "N432") and the asparagine residue at position 630 in the amino acid sequence shown in SEQ ID NO: 1
  • This is a Tf glycoprotein in which a GlcNAc non-reducing terminal sugar chain is added to the side chain of the group (denoted by "N630").
  • the terminal GlcNAc-Tf protein is produced in cells of the central nervous system such as the brain and / or spinal cord and is considered to be a brain-type glycoprotein found mainly in the cerebrospinal fluid. These brain-type glycoproteins are also known as biomarkers that reflect the production of cerebrospinal fluid (Murakami, et al., Proc. Jpn. Acad., Ser.B, 2019, 95, 198-210. ).
  • the terminal GlcNAc-Tf protein is known to be a marker for iNPH differentiation (Japanese Patent Laid-Open No. 2010-121980).
  • the amount of terminal GlcNAc-Tf protein in body fluids, mainly in cerebrospinal fluid was significantly reduced as compared with healthy bodies.
  • a peptide fragment of a terminal GlcNAc-Tf protein containing a GlcNAc non-reducing terminal sugar chain can also be a constituent marker of the combination marker of the present invention.
  • Terminal Sia-Tf protein is an N-linked glycoprotein having ⁇ 2,6 sialic acid at the non-reducing end, that is, a Tf glycoprotein containing only the Sia non-reducing terminal sugar chain. .. Although it is an isoform of Tf glycoprotein, it is not a constituent marker of the combination marker of the present invention.
  • Terminal Sia-Tf is present in both serum and cerebrospinal fluid, but is also referred to as "serum Tf (glycoprotein)" because it is present in a particularly large amount in serum.
  • Terminal Sia-Tf is considered to be a Tf glycoprotein biosynthesized in the liver due to its similarity in sugar chain structure to other glycoproteins.
  • the terminal GlcNAc-Tf protein is not detected in the cerebrospinal fluid in anencephaly, so it is considered to be biosynthesized in the brain.
  • Prostaglandin D2 synthase is an enzyme that catalyzes the reaction of prostaglandin endoperoxides to prostaglandin D2 (PGD 2 ) and is the present invention.
  • the PGDS protein include a human PGDS protein composed of 190 amino acid residues and composed of the amino acid sequence shown in SEQ ID NO: 2. It is known that PGDS has a relatively high content in cerebrospinal fluid, but a concentration of about 1% thereof can also be detected in blood.
  • PGDS is also known as a biomarker that reflects the production of cerebrospinal fluid (Murakami, et al., Proc. Jpn. Acad., Ser.B, 2019, 95, 198-210.).
  • tau-related protein refers to tau protein and phosphorylated tau protein (often referred to as "p-tau protein” in the present specification).
  • Tau protein is a microtubule-associated protein involved in axonal transport and is present in nerve cells and glial cells of the central and peripheral nervous systems.
  • tau protein is excessively phosphorylated to p-tau protein. It is thought that this p-tau protein inhibits the stability of microtubules and causes intracellular cell death by forming intracellular aggregates. Since both tau protein and p-tau protein are intracellular proteins, their detection in body fluid usually means neuronal cell death. It is known that tau-related proteins can be markers for dementia such as PSP and FTD in addition to AD and MCI, and are also used as AD markers in clinical practice.
  • constituent marker of the combination marker of the present invention As a specific example of a certain tau protein, there is a human tau protein composed of 441 amino acid residues and composed of the amino acid sequence shown in SEQ ID NO: 3.
  • the human tau protein in addition to the amino acid sequence shown in SEQ ID NO: 3, there are 6 types of isoforms consisting of 352 to 412 amino acid residues which are splicing variants thereof.
  • These isoforms can also be constituent markers of the combination markers of the present invention. Phosphorylation occurs at various locations in p-tau proteins, and usually in AD, excessive phosphorylation occurs, so that phosphorylation can occur at multiple amino acid residues.
  • Detection of p-tau can be performed, for example, by phosphorylation site-specific detection, site-non-specific phosphorylation detection, or a combination thereof.
  • AD index is a value usually calculated by (A ⁇ 40 / A ⁇ 42) ⁇ p-tau, and is a marker that reflects both metabolic changes of A ⁇ peptide and p-tau protein in AD. Further, for the calculation of the AD index, for example, (A ⁇ 42 / A ⁇ 40) ⁇ p-tau may be used, but in the present specification, the value calculated by any of the formulas is included.
  • the AD index is considered to have the highest AD diagnostic ability by itself among the AD markers currently used clinically.
  • (1) Combination of Man-Tf / GlcNAc-Tf / PGDS and tau / p-tau As the first combination pattern, it has Man-Tf protein or a fragment thereof containing a sugar chain having Man, GlcNAc-Tf protein or GlcNAc.
  • fragment containing a sugar chain having Man refers to a peptide fragment containing an asparagine residue to which a Man non-reducing terminal sugar chain is added in the Man-Tf protein.
  • a Man-Tf protein fragment containing N432 and / or N630 to which a Man non-reducing terminal sugar chain has been added is added.
  • a fragment containing a sugar chain having GlcNAc refers to a peptide fragment containing an asparagine residue to which a Man non-reducing terminal sugar chain is added in the GlcNAc-Tf protein.
  • a Man-Tf protein fragment containing N432 and N630 added with a Man non-reducing terminal sugar chain is added in the GlcNAc-Tf protein.
  • a more specific combination of constituent markers is a Man-Tf protein or a fragment thereof containing a sugar chain having Man and a tau protein or a fragment thereof, a Man-Tf protein or a fragment thereof containing a sugar chain having Man and a p-tau protein.
  • a fragment containing a phosphorylation site thereof a fragment thereof containing a sugar chain having GlcNAc-Tf protein or GlcNAc and a tau protein or a fragment thereof, a fragment thereof containing a sugar chain having GlcNAc-Tf protein or GlcNAc and a p-tau protein or A fragment containing the phosphorylation site, and a PGDS protein or a fragment thereof and a tau protein or a fragment thereof, a PGDS protein or a fragment thereof and a p-tau protein or a fragment containing the phosphorylation site thereof.
  • a fragment containing a Man-Tf protein or a sugar chain having Man, a GlcNAc-Tf protein or a sugar chain having GlcNAc is used. Included is a combination of at least two proteins selected from a constitutive marker consisting of the fragment comprising and any one polypeptide selected from the group consisting of PGDS or fragments thereof.
  • a more specific combination of constituent markers is a combination of two constituent markers, a fragment thereof containing a Man-Tf protein or a sugar chain having Man, and a fragment thereof containing a GlcNAc-Tf protein or a sugar chain having GlcNAc, Man-Tf.
  • any of the above combination markers can distinguish AD and MCI from NC as AD pathology. It is also possible to distinguish between pre-MCI and non-pre-MCI in NC.
  • the third combination pattern includes a Man-Tf protein or a fragment thereof containing a sugar chain having Man, and a sugar chain containing a GlcNAc-Tf protein or GlcNAc.
  • the combination of the fragment and the AD index can be mentioned.
  • a second aspect of the present invention is an Alzheimer's disease pathological differentiation kit.
  • the kit of the present invention can detect the marker for AD pathological condition according to the first aspect that can be contained in the sample, thereby distinguishing the morbidity or possibility of morbidity of AD pathological condition of the subject.
  • the AD diagnostic kit of the present invention contains a peptide bond molecule and a sugar chain bond molecule that specifically bind to each of the combination markers for AD pathological condition discrimination according to the first aspect as essential constituents.
  • a peptide bond molecule and a sugar chain bond molecule that specifically bind to each of the combination markers for AD pathological condition discrimination according to the first aspect as essential constituents.
  • Peptide bond molecule refers to a molecule that specifically binds to a polypeptide or a peptide fragment thereof in a constituent marker of a combination marker for AD pathological condition differentiation.
  • constituent marker polypeptide that is, the target polypeptide
  • tau protein examples include Tf protein, PGDS, and tau protein.
  • the AD diagnostic kit of the present invention contains one or more peptide bond molecules that bind to these polypeptides.
  • the peptide bond molecule may be any of a peptide, nucleic acid, small molecule compound, or a combination thereof.
  • the peptide bond molecule composed of a peptide include an antibody or an active fragment thereof.
  • the antibody may be either a polyclonal antibody, a monoclonal antibody or a recombinant antibody. Monoclonal or recombinant antibodies are preferred to allow for more specific detection.
  • the globulin type of the antibody is not particularly limited and may be any of IgG, IgM, IgA, IgE, IgD and IgY, but IgG and IgM are preferable.
  • the species from which the antibody of this embodiment is derived is not particularly limited. It can be of any animal origin, including mammals and birds. Examples include mice, rats, guinea pigs, rabbits, goats, donkeys, sheep, camels, horses, chickens, or humans.
  • the term "recombinant antibody” refers to, for example, a chimeric antibody, a humanized antibody, and a synthetic antibody.
  • a “chimeric antibody” is an antibody in which the constant region of a light chain and a heavy chain (C region: Constant region) is replaced with the C region of a light chain and a heavy chain of another antibody.
  • C region Constant region
  • an antibody in which the C region of the light chain and the heavy chain is replaced with the C region of an appropriate human antibody is applicable. That is, in this case, the variable region (V region: Variable region) including the CDR is derived from the mouse antibody, and the C region is derived from the human antibody.
  • a “humanized antibody” is also referred to as a reshaped human antibody, and is a mosaic antibody in which the CDR in an antibody derived from a non-human animal to a target antigen is replaced with the CDR of a human antibody.
  • a recombinant antibody gene was prepared by substituting a DNA sequence encoding each CDR region (CDR1 to CDR3) of a mouse anti-human Man-Tf antibody with a DNA sequence encoding each corresponding CDR derived from an appropriate human antibody. , Antibodies obtained by expressing it.
  • Synthetic antibody refers to an antibody synthesized by using a chemical method or a recombinant DNA method.
  • a monomeric polypeptide molecule in which one or more VLs and one or more VHs of a specific antibody are artificially linked via a linker peptide having an appropriate length and sequence, or a multimeric polypeptide thereof.
  • linker peptide having an appropriate length and sequence
  • Specific examples of such polypeptides include single-chain Fv (scFv: single chain Fragment of variable region) (Pierce Catalog and Handbook, 1994-1995, Pierce Chemical Co., Rockford, IL) and diabody. ), Triabody, tetrabody and the like.
  • Single-stranded Fv is a synthetic antibody fragment having a structure in which the V regions on these two polypeptide chains are linked by a flexible linker of sufficient length and contained in one polypeptide chain. Within a single-stranded Fv, both V regions can self-assemble with each other to form one functional antigen-binding site. Single-stranded Fv can be obtained by incorporating the recombinant DNA encoding it into the phage genome using a known technique and expressing it. Diabody is a molecule with a structure based on a single-stranded Fv dimer structure (Holliger et al., 1993, Proc.
  • the triabodies and tetrabodies have trimer and tetramer structures based on a single-stranded Fv structure, similar to diabodies. They are trivalent and tetravalent antibody fragments, respectively, and may be multispecific antibodies.
  • the antibody preferably has a high affinity with a target polypeptide having a dissociation constant of 10 -8 M or less, preferably 10 -9 M or less, more preferably 10 -10 M or less.
  • the dissociation constant can be measured using a technique known in the art. For example, it may be measured using the speed evaluation kit software by the BIAcore system (GE Healthcare).
  • the polyclonal antibody of this embodiment used in this step is known from immune animals in the art after immunizing a suitable animal with a polypeptide serving as an antigen, that is, Tf protein, PGDS, and tau protein, or a peptide fragment thereof. It can be recovered by the method.
  • a monoclonal antibody can also be obtained by a known method which is a conventional technique in the art. For example, after immunizing a mouse with the antigen, antibody-producing cells are collected from the immune mouse. Hybridomas may be identified that fuse the antibody-producing cells to a myeloma cell line, thereby producing hybridoma cells and producing monoclonal antibodies that bind to the target polypeptide.
  • the “antibody fragment” is a peptide fragment having the antigen-binding activity of the above-mentioned antibody, and examples thereof include Fab, F (ab') 2, Fv and the like.
  • the antibody or antibody fragment thereof used in this step may be modified.
  • modification includes a label required for antibody detection or a functional modification required for antigen-specific binding activation. Labeling includes, for example, the aforementioned fluorescent substances, fluorescent proteins (eg, PE, APC, GFP), enzymes (eg, horseradish peroxidase, alkaline phosphatase, glucose oxidase), or labeling with biotin or (streptavidin) avidin. ..
  • modifications include glycosylation of antibodies performed to adjust the affinity for the target polypeptide.
  • FR Framework region
  • the "active fragment thereof” refers to a partial fragment of an antibody that retains antigen-binding property and immune response activity.
  • Specific antibodies in the AD diagnostic kit of the present invention include, for example, anti-Tf antibody, anti-PGDS antibody, anti-tau antibody, anti-p-tau antibody, anti-A ⁇ 40 antibody, and anti-A ⁇ 42 antibody.
  • nucleic acid aptamers examples include nucleic acid aptamers.
  • a "nucleic acid aptamer” is an aptamer composed of nucleic acids, which is strong with a target substance due to the secondary structure of a single-stranded nucleic acid molecule via hydrogen bonds and the three-dimensional structure formed based on the tertiary structure.
  • a ligand molecule that has the ability to specifically bind.
  • the nucleic acid aptamer used in the present specification can be produced by a method known in the art.
  • an in vitro sorting method using a SELEX (systematic evolution of ligands by exponential enrichment) method can be mentioned.
  • the SELEX method is, for example, in the case of separating RNA aptamers, "selecting an RNA molecule bound to a target molecule from an RNA pool composed of a large number of RNA molecules having a random sequence region and primer binding regions at both ends thereof. After amplifying the recovered RNA molecule by RT-PCR reaction, transcription is performed using the obtained cDNA molecule as a template to obtain an amplification product of the selected RNA molecule, which is used as the RNA pool for the next round.
  • the base sequence lengths of the random sequence region and the primer binding region are not particularly limited. Generally, the random sequence region is preferably in the range of 20 to 80 bases, and the primer binding region is preferably in the range of 15 to 40 bases, respectively.
  • a molecule similar to the target molecule and an RNA pool or an RNA pool are mixed in advance, and a pool consisting of an RNA molecule or a DNA molecule that does not bind to a molecule similar to the target molecule is formed. It may be used.
  • the SELEX method is a known method, and the specific method may be, for example, according to Pan et al. (Proc. Natl. Acad. Sci. 1995, U.S.A. 92: 11509-11513).
  • the target molecule can be used as a sugar chain mannose
  • the nucleic acid molecule finally obtained by carrying out the above method can be used as a nucleic acid aptamer for mannose.
  • RNA aptamers and DNA aptamers are generally known, but the nucleic acids constituting the nucleic acid aptamers in the present specification are not particularly limited. For example, it includes a DNA aptamer, an RNA aptamer, an aptamer composed of a combination of DNA and RNA, and the like. Generally, RNA aptamers are frequently used, but DNA aptamers are superior in terms of stability, production cost in chemical synthesis, and the number of steps in aptamer production.
  • Nucleic acid aptamers used in this step include fluorescent substances (eg, FITC, Texas, Cy3, Cy5, Cy7, Cyanine3, Cyanine5, Cyanine7, FAM, HEX, VIC, fluoresamine and so on, as long as they do not inhibit the ability to bind to the target molecule. It can also be labeled with its derivatives, such as rhodamine and its derivatives), radioactive isotopes (eg, 32P, 33P, 35S), or labeling substances such as biotin or (streptavidin) avidin.
  • fluorescent substances eg, FITC, Texas, Cy3, Cy5, Cy7, Cyanine3, Cyanine5, Cyanine7, FAM, HEX, VIC, fluoresamine and so on, as long as they do not inhibit the ability to bind to the target molecule. It can also be labeled with its derivatives, such as rhodamine and its derivatives), radioactive isotopes (eg, 32P,
  • nucleic acid aptamer in the AD diagnostic kit of the present invention examples include Tf protein-binding RNA aptamer, PGD-binding RNA aptamer, tau protein-binding RNA aptamer, and p-tau protein-binding RNA aptamer.
  • the peptide bond molecule in the AD diagnostic kit of the present invention may be immobilized on a carrier or labeled with a fluorescent dye, a luminescent substance, or the like, if necessary.
  • the “sugar chain-binding molecule” refers to a molecule that specifically binds to the sugar chain of a glycoprotein in a constituent marker of a combination marker for AD pathological condition differentiation.
  • the constituent marker polypeptide that is, the target glycoprotein
  • the constituent marker polypeptide include Man-Tf protein and GlcNAc-Tf protein, which are Tf glycoproteins.
  • the AD diagnostic kit of the present invention contains one or more sugar chain-binding molecules that bind to the sugar chains of these Tf glycoproteins.
  • the sugar chain binding molecule may be any of a peptide, a nucleic acid, a small molecule compound, or a combination thereof.
  • sugar chain-binding molecule composed of a peptide examples include a lectin, an antibody, or an active fragment thereof.
  • Lectin refers to a protein or glycoprotein that binds to a sugar chain other than an immune response.
  • the lectins that can be included in the Alzheimer's disease pathological identification kit of the present invention include sugar chains containing Man or GlcNAc, preferably sugar chains containing terminal Man or terminal GlcNA, and more preferably Man non-reducing terminal sugar chains or GlcNA non-reducing terminals. Examples include lectins that bind to sugar chains.
  • Man non-reducing end sugar chain-binding lectin When the non-reducing end of the sugar chain is Man, a Man non-reducing end sugar chain-binding lectin can be mentioned.
  • Specific examples of Man non-reducing terminal sugar chain-binding lectins include 45 types of Man-binding lectins described in http://jcggdb.jp/rcmg/glycodb/LectinSearch including UDA lectins and BC2L-A lectins. Can be mentioned.
  • GlcNA non-reducing end sugar chain-binding lectin When the non-reducing end of the sugar chain is GlcNAc, GlcNA non-reducing end sugar chain-binding lectin can be mentioned.
  • Specific examples of the GlcNA non-reducing terminal sugar chain-binding lectin include, for example, the agglutinin GSL-II lectin derived from the mushroom family Griffonia simplicifolia, the agglutinin ABA lectin derived from Agaricus bisporus, and the agglutinin derived from the child body of Psathyrella velutina.
  • the agglutinin PVL lectin is known.
  • lectins there may be a difference in apparent sugar chain binding specificity between the case where the target molecule is a glycoprotein and the case where the target molecule is only a sugar chain.
  • UDA lectins derived from urtica UDA lectins derived from urtica (Urtica dioica) are classified as GlcNAc-binding lectins when the target molecule is a sugar chain, but when the target molecule is a glycoprotein, they do not bind to the terminal GlcNAc-Tf protein. It becomes specifically bound to the Man non-reduced end of the terminal Man-Tf protein.
  • the lectin constituting the kit of the present invention is Man of the terminal Man-Tf protein or GlcNac of the terminal GlcNAc-Tf protein, preferably terminal Man of the terminal Man-Tf protein or terminal GlcNAc of the terminal GlcNAc-Tf protein, more preferably.
  • the lectin constituting the kit of the present invention is Man of the terminal Man-Tf protein or GlcNac of the terminal GlcNAc-Tf protein, preferably terminal Man of the terminal Man-Tf protein or terminal GlcNAc of the terminal GlcNAc-Tf protein, more preferably.
  • lectin a commercially available lectin may be used.
  • biotinylated UDA lectin Cat No. BA-8005-1; EY
  • BC2L-A lectin derived from bacteria Backholderia cenocepacia lectin-A; Wako Pure Chemical Industries, Ltd.
  • the composition of the antibody or its active fragment is specifically described in "(1) Peptide bond molecule, (i) Peptide bond molecule composed of peptide" described above, and the same applies to this invention. Then, the explanation is omitted.
  • Specific examples of the antibody that binds to the sugar chain include a sugar chain containing Man, preferably a sugar chain containing a terminal Man, and more preferably an antibody that recognizes and binds to a sugar chain containing a non-reducing terminal Man.
  • Examples thereof include an antibody that recognizes and binds to a sugar chain containing GlcNAc, preferably a sugar chain containing a terminal GlcNAc, and more preferably a sugar chain containing a non-reducing terminal GlcNAc.
  • sugar chain-binding molecule composed of nucleic acid examples include nucleic acid aptamers.
  • the composition of the nucleic acid aptamer and the method for producing the same are specifically described in the above-mentioned "(1) Peptide bond molecule, (ii) Peptide bond molecule composed of nucleic acid", and the same applies to the present invention. Then, the explanation is omitted.
  • the nucleic acid-binding molecule in the AD diagnostic kit of the present invention may be immobilized on a carrier or labeled with a fluorescent dye, a luminescent substance, or the like, if necessary.
  • AD therapeutic agents especially monoclonal antibodies against A ⁇
  • these therapeutic agents may be most effective when administered to patients in the early stage of onset to suppress the progression of symptoms at an early stage. For this reason, it is most desirable to administer to pre-MCI patients with dementia.
  • the combination marker of the present invention is not only an excellent marker for differentiating AD pathology, but also enables differentiation of pre-MCI patients, and is expected to contribute to early diagnosis and treatment of AD in the future.
  • a third aspect of the present invention is a method for differentiating AD pathological conditions.
  • the amount of the combination marker for differentiating AD pathology of the first aspect present in the body fluid derived from a subject suspected of having dementia is measured, and the subject is AD or MCI based on the measured value.
  • NC can be determined.
  • the AD pathological condition discrimination method of the present invention includes a measurement step, a product value calculation step, and a determination step as essential steps. Hereinafter, each step will be specifically described.
  • Measurement step the amount of each marker in the combination marker for AD pathological condition discrimination according to the first aspect present in a predetermined amount of body fluid obtained from a subject suspected of having dementia is measured. , Is the process of obtaining the measured value.
  • the body fluid is preferably cerebrospinal fluid or blood.
  • the method for collecting cerebrospinal fluid and blood may be any known method and is not particularly limited. For example, if it is cerebrospinal fluid, it may be collected by lumbar puncture. Lumbar puncture is relatively less invasive because pain can be reduced to less than blood sampling by using a commercially available local anesthetic in advance, and side effects can be reduced by using a traumatic needle. This is a suitable method for collecting cerebrospinal fluid. If it is blood, it may be collected according to a known blood collection method. As a general rule, the body fluids of the subject and the control body should be the same kind of body fluids as if one is cerebrospinal fluid and the other is cerebrospinal fluid.
  • Predetermined amount means an amount predetermined by capacity or weight. Although the predetermined amount is not particularly limited, it is necessary that the marker for AD pathological condition discrimination according to the first aspect contained at least in the body fluid of the subject, preferably in the cerebrospinal fluid, is a measurable amount.
  • the amount of cerebrospinal fluid may be 5 ⁇ L to 1 mL, or the amount of cerebrospinal fluid protein may be 5 ⁇ g to 200 ⁇ g.
  • the "measured value” is a value indicating the amount of each marker in the combination marker for AD pathological condition discrimination measured in this step.
  • the measured value may be an absolute value such as volume or weight, or may be a relative value such as concentration, ionic strength, absorbance or fluorescence intensity.
  • the amount of the marker is determined by using a peptide-binding molecule that specifically binds to the polypeptide of each constituent marker of the combination marker for AD pathological condition differentiation and a sugar chain-binding molecule that specifically binds to the sugar chain of the constituent marker. Just measure. Since the configurations of the peptide bond molecule and the sugar chain bond molecule are described in detail in the second aspect, specific description thereof will be omitted here.
  • the measuring method may be any known protein or glycoprotein quantification method using a peptide bond molecule and a sugar chain binding molecule, and is not particularly limited.
  • an immunological detection method using an antibody a lectin detection method using a lectin, a mass spectrometry method or a combination method thereof can be mentioned.
  • Immunological detection methods include, for example, enzyme immunoassay (including ELISA and EIA methods), fluorescent immunoassay, radioimmunoassay (RIA), luminescence immunoassay, and surface plasmon resonance (SPR). , Crystal transducer microbalance (QCM) method, immunoturbidimetric method, latex agglutination immunoassay, latex turbidimetric method, hemagglutination reaction, particle agglutination reaction method, gold colloid method, capillary electrophoresis method, western blot method or immunity Histochemical method (immunostaining method) can be mentioned.
  • enzyme immunoassay including ELISA and EIA methods
  • fluorescent immunoassay including radioimmunoassay (RIA), luminescence immunoassay, and surface plasmon resonance (SPR).
  • QCM Crystal transducer microbalance
  • immunoturbidimetric method immunoturbidimetric method
  • Examples of the lectin detection method include a lectin blotting method.
  • High-speed liquid chromatograph mass spectrometry LC-MS
  • high-speed liquid chromatograph tandem mass spectrometry LC-MS / MS
  • gas chromatograph mass spectrometry GC-MS
  • gas chromatograph tandem mass spectrometry include mass spectrometry.
  • Analytical methods GC-MS / MS
  • capillary electromass spectrometry CE-MS
  • ICP mass spectrometry ICP mass spectrometry
  • the combination method for example, a sandwich ELISA method using a lectin and an antibody and an automated method using a similar principle, preferably a high-throughput lectin inhibition-automatic lattice aggregation method can be used.
  • the constituent marker of the combination marker for AD pathological condition identification is glycoprotein
  • the combination method of detecting each of the sugar chain and the protein as a target is preferable because the glycoprotein can be quantified with high accuracy.
  • a body fluid for example, spinal fluid
  • a carrier such as a plate on which an anti-Tf antibody is adsorbed.
  • a method of adsorbing the terminal Man-Tf protein and then detecting the terminal Man-Tf protein using the UDA lectin, which is a Man-binding molecule, as a probe can be mentioned.
  • a method of adsorbing the terminal Man-Tf protein through a body fluid (for example, cerebrospinal fluid) on a carrier such as a plate on which UDA lectin is adsorbed and then detecting the terminal Man-Tf protein with an anti-Tf antibody.
  • the “product value calculation step” is a step of obtaining a product value obtained by multiplying the measured values of the respective markers obtained in the measurement step.
  • the "judgment step” determines whether the subject can correspond to AD, MCI, or NC from the product value based on the preset AD cutoff value and MCI cutoff value. It is a process.
  • the "cutoff value” refers to a value that can determine the presence or absence of the risk of illness or the pathological condition based on the value.
  • the cutoff value indicates a sufficiently high value for both sensitivity and specificity.
  • it is derived from the ROC curves drawn based on a direct comparison between the control group and the disease group to be compared using a known method, but is not limited to this. It does not have to be a direct comparison between the control group and the disease group to be compared, and the cutoff value may be set without using the ROC curve.
  • ROC curve Receiveiver Operating Characteristic curve
  • TPF True Position Fraction
  • FPF False Position Fraction
  • the "AD cutoff value” is a cutoff that serves as a reference for determining whether or not a subject subject to the discrimination method of the present invention is likely to be affected by AD, based on the product value obtained in the product value calculation step. The value. If the product value is higher than the AD cutoff value, the subject is determined to be more likely to have AD. If the product value is lower than the AD cutoff value, the subject is determined to be less likely to have AD and more likely to be either MCI or NC.
  • the specific value of the AD cutoff value differs depending on the combination of constituent markers in the combination marker for AD discrimination.
  • the AD cutoff values for each combination are shown in Tables 1 to 4. For example, when the combination marker for AD discrimination is a combination of Man-Tf protein and p-tau protein, the AD cutoff value is 97.8 from Table 1.
  • the "MCI cutoff value” is a cutoff that serves as a reference for determining whether or not a subject subject to the discrimination method of the present invention is likely to be affected by MCI, based on the product value obtained in the product value calculation step. The value. If the product value is higher than the MCI cutoff value, the subject is determined to be more likely to have MCI. If the product value is lower than the AD cutoff value, it is determined that the subject is likely to be NC.
  • the specific value of the MCI cutoff value differs depending on the combination of constituent markers in the combination marker for AD pathological condition discrimination.
  • the MCI cutoff values for each combination are shown in Tables 1-4.
  • the MCI cutoff value is 110 from Table 1.
  • the AD pathological condition differentiation method of this embodiment can be carried out in place of or in combination with a known AD pathological condition discrimination method.
  • Known methods for differentiating AD pathology include, for example, measurement of dementia score, amyloid PET (positron emission tomography) using [ 11 C] Pittsburgh compound (PiB), and fluoro-2-. PET using deoxy-D-glucose (FDG) and the like can be mentioned.
  • a fourth aspect of the present invention is a method for differentiating a healthy control with MCI transfer (pre-MCI differentiating method).
  • the pre-MCI discrimination method of the present invention can determine whether the subject is NC or non-MCI or non-pre-MCI.
  • the pre-MCI discrimination method of the present invention includes a measurement step, a product value calculation step, a determination step, and a re-judgment step.
  • the measurement step, the product value calculation step, and the determination step are the same as the AD pathological condition discrimination method of the third aspect. That is, the pre-MCI differentiation method of the present invention is a differentiation method that follows the AD pathological condition differentiation method of the third aspect.
  • the re-determination step which is a feature of the present invention, will be described.
  • the "re-judgment step” is a subject who is determined to correspond to NC in the determination step in the AD pathological condition discrimination method of the third aspect based on a preset pre-MCI cutoff value. This is a step of determining whether the subject can correspond to pre-MCI or non-pre-MCI from the product value obtained in the product value calculation step.
  • pre-MCI cut-off value is a cut that serves as a criterion for determining whether an NC has a high possibility of MCI transition or an NC that does not, when the subject is determined to be NC by the AD pathological condition discrimination method of the third aspect. Off value.
  • Subjects determined to be AD or MCI in the determination step in the AD pathological condition differentiation method of the third aspect are not subject to the pre-MCI differentiation method of the present invention, and only subjects determined to be NC are targeted. If the product value obtained in the product value calculation process is higher than the pre-MCI cutoff value, it is determined that the subject is pre-MCI and is currently NC but is likely to move to MCI in the future. On the other hand, if the product value is lower than the pre-MCI cutoff value, the subject is determined to be non-pre-MCI, that is, true NC, which is unlikely to move to MCI.
  • the pre-MCI differentiation method of this embodiment can be used in combination with a known AD pathological condition differentiation method or the like.
  • Example 1 Localization of Tf protein and p-tau protein in the hippocampus> (Purpose) To verify the localization of the Man-Tf protein, known as an AD marker, in the hippocampus. (Method) Since the hippocampus, which controls short-term memory, is the site where nerve cell death and tissue atrophy progress at the earliest stage, AD patients and healthy hippocampus for control are double-stained with anti-Tf protein antibody and anti-p-tau antibody. Was done. 5 micron sections were made from formalin-fixed hippocampus and immunostained on glass slides.
  • Anti-Tf protein antibody (A0061, Dako Ltd.) and anti-p-tau protein antibody (AT8, Cosmo Bio Co., Ltd.) are used as the primary antibody, and Alexa Fluor 488 or 594 labeled antibody (Thermo Fisher Scientific) is used as the secondary antibody. ) was used. It is clear from the results of mass spectrometry that the Man-Tf protein occupies 85% or more of the sugar chain isoform in the Tf protein of the cerebral cortex.
  • Tf protein secretion increases with the progression of AD pathology, and that Tf protein and p-tau protein are co-localized.
  • Example 2 Measurement of Man-Tf protein in cerebrospinal fluid> (Purpose) The amount of Man-Tf protein in the cerebrospinal fluid was measured, and the effect of Man-Tf protein as a marker on various dementias was verified.
  • Method 1 Diagnosis of each disease For 316 patients who visited the outpatient department for dementia, diagnosis was made by the existing diagnostic method, and 5 mL of cerebrospinal fluid was collected from each patient by lumbar puncture. 1-1. Diagnosis of AD pathology and other neurodegenerative diseases Patients suspected of having outpatient AD were classified into MCI and AD according to the following AD diagnostic criteria, and the group of patients who did not belong to either was designated as NC. At the same time, tauopathy other than AD, which accumulates tau protein, which is a dementia with neurodegenerative disease, and synucleinopathy, which accumulates ⁇ -synuclein, were also classified according to the following diagnostic criteria.
  • tauopathy and synucleopathy are often referred to as "other neurodegenerative diseases”.
  • Idiopathic normal pressure hydrocephalus (iNPH) showed dementia and ventricular enlargement like AD, but did not show neurodegenerative disease, so it was used as a disease control.
  • Diagnosis of AD According to the clinical diagnostic criteria by the National Institute on Aging-Alzheimer's association workgroup (NIA-AA).
  • NIA-AA National Institute on Aging-Alzheimer's association workgroup
  • MCI The clinical diagnostic criteria of MCI against the background of AD recommended by the NIA-AA Diagnostic Guideline Development Workgroup were followed.
  • Diagnosis of tauopathy Specific diseases of tauopathy include progressive supranuclear palsy (PSP) and frontotemporal dementia (FTD). For these diagnoses, the dementia disease clinical practice guideline 2017 (Igaku-Shoin electronic version ISBN 978-4-260-62858-7) was followed.
  • (4) Diagnosis of synucleinopathy Specific diseases of synucleinopathy include Lewy body dementia (DLB) and Parkinson's disease (PD).
  • DLB Lewy body dementia
  • PD Parkinson's disease
  • Man-Tf protein in cerebrospinal fluid In the measurement of Man-Tf protein, rBC2L-A lectin was used as a sugar chain binding molecule.
  • the capture reaction was carried out by immobilizing an anti-human Tf antibody on a plate. Specifically, anti-human Tf antibody (A0061, Dako Ltd) was diluted to 1 ⁇ g / mL with 100 mM carbonate buffer (pH 9.5), 100 ⁇ L was added to a microtiter plate and incubated overnight at 4 ° C. After washing once with TBS, TBS containing 10% N101 (Fuji Film Wako Pure Chemical Industries, Ltd.) was added as a blocking agent and incubated at room temperature for 1 hour to prepare an antibody-immobilized plate.
  • anti-human Tf antibody A0061, Dako Ltd
  • 100 mM carbonate buffer pH 9.5
  • Cerebrospinal fluid (5-10 ⁇ L) of the sample is placed at 55 ° C. for 60 minutes before in PBST (phosphate-buffered saline / 0.05% Tween-20) containing 0.6% 2-mercaptoethanol and 0.003% SDS at a final concentration. Processing was performed. The blocked antibody-immobilized plate was washed once with TBST, then the pretreated sample was diluted with TBST (TBST-CaCl 2 ) containing 10 mM CaCl 2 and incubated overnight at 4 ° C.
  • PBST phosphate-buffered saline / 0.05% Tween-20
  • TBST-CaCl 2 After washing 3 times with TBST-CaCl 2 , 100 ⁇ L of TBST-CaCl 2 containing biotinylated rBC2L-A (50 ng / mL) was added, and the mixture was incubated at room temperature for 2 hours. rBC2L-A was previously biotinylated using Ez-link NHS-biotin (# 21336, Pierce) according to the attached protocol. TBST-CaCl 2 was added and washed twice, 100 ⁇ L of TBST-CaCl 2 containing HRP-labeled streptavidin (50 ng / mL) was added, and the mixture was incubated at room temperature for 2 hours.
  • HRP-labeled streptavidin 50 ng / mL
  • T1147 Sigma-Aldrich
  • sialic acid ⁇ 2,6 galactose ⁇ 1,4GlcNAc ⁇ 1, (3/6) -mannose residue at the end of the sugar chain was used as a sialidase.
  • SPSS version 26
  • multiple comparisons were performed by Turkey-Kramer comparison.
  • a ⁇ 42 peptide Human / rat ⁇ amyloid (42) ELISA Kit, Wako, High Sensitive (cat No. 292-64501, Wako Pure Chemical Industries, Ltd.)
  • the AD index was calculated by (A ⁇ 40 / A ⁇ 42) ⁇ p-tau based on the measured amount of p-tau protein, A ⁇ 40 peptide, and A ⁇ 40 peptide.
  • the cutoff value of the combination marker was obtained based on the product value obtained by multiplying the measured values of each marker.
  • the cutoff values for AD and MCI were set from the ROC curve using the Yoden method.
  • the cutoff value obtained based on the comparison between "patients with suspected AD (NC + MC + AD)" and “disease control (iNPH)” was used in a pseudo manner.
  • the sensitivity and specificity were calculated from the cutoff value, and the AUC (area under the curve) representing the reliability was calculated using the receiver operating characteristic (ROC) curve.
  • the sensitivity, specificity, and AUC were calculated from the combination of Man-Tf protein and p-tau protein.
  • the MCI group has an MCI cutoff value (110a.u .: dashed line) based on the product value (p-tau ⁇ Man-Tf) of the measured values of Man-Tf protein and p-tau protein.
  • the sensitivity was 83.9%
  • the specificity was 90.4%
  • the AUC was 0.919.
  • an AUC above 0.9 is considered to be extremely accurate.
  • the sensitivity was as high as 93.9%, the specificity was as high as 88.5%, and the AUC was also extremely high as 0.957. Therefore, it was shown that the combination of Man-Tf protein and p-tau protein provides extremely high accuracy for the differentiation of MCI and AD.
  • the sensitivity, specificity, and AUC were calculated for the combination of Man-Tf protein and tau protein.
  • the MCI group was verified with the MCI cutoff value (559 a.u .: broken line), and as shown in Table 1, both the sensitivity was 88.9% and the specificity was 88.5%, and the AUC was 0.907. there were.
  • the AD group as a result of verification with the AD cutoff value (615 a.u .: broken line), as shown in Table 1, the sensitivity is 100% and the specificity is 90.4%, both of which are extremely high, and the AUC is also extremely high at 0.962. showed that. Therefore, it was shown that the combination of Man-Tf protein and tau protein also provides extremely high accuracy for the differentiation of MCI and AD.
  • Example 4 Verification of AD discrimination accuracy using other combination markers> (Purpose) The accuracy of differentiating AD pathological conditions by a combination marker other than Example 3 will be verified.
  • the amount of GlcNAc-Tf protein and PGDS was measured by the following method.
  • the measurement of GlcNAc-Tf protein basically followed the method for detecting Man-Tf protein.
  • rPVL synthinant Psathyrella velutina lectin, Medical and Biological Laboratories Co., Ltd.
  • rPVL (1 ⁇ g / 0.1 mL) was added to the plate, and the mixture was incubated overnight at 4 ° C.
  • 50 mM TBS containing 10% Block Ace was added as a blocking agent, and blocking was performed at 4 ° C. for 4 hours.
  • the blocked biotin-immobilized plate was washed once with TBST, the pretreated sample was added, and the mixture was incubated overnight at 4 ° C. After washing twice with TBST, 100 ⁇ L of anti-Tf antibody (A0061, Dako Ltd.) solution (0.5 ⁇ g / mL) was added, and the mixture was incubated at room temperature for 2 hours. After washing twice with TBST, 100 ⁇ L of a 20,000-fold diluted Western wasabi peroxidase-labeled anti-rabbit IgG antibody (Promega, W4011) solution (0.1 ⁇ g / mL) was added, and the mixture was incubated at room temperature for 2 hours.
  • anti-Tf antibody A0061, Dako Ltd.
  • T1147 Sigma-Aldrich
  • sialic acid ⁇ 2,6 galactose ⁇ 1,4GlcNAc ⁇ 1, (3/6) -mannose residue at the end of the sugar chain was used as a sialidase and
  • a Tf sugar chain isomer having a ⁇ 1,4GlcNAc ⁇ 1, (3/6) -mannose residue at the end of the sugar chain prepared by sequential digestion with galactosidase was used.
  • a commercially available ELISA kit Human prostaglandin D synthase (Lipocalin-type) ELISA (Cat. No .: RD191113100R, BioVendor) was used to measure the amount of PGDS. The cutoff value was set, the sensitivity, specificity, and AUC were calculated according to Example 4.
  • GlcNAc-Tf protein and PGDS concentrations were measured in the cerebrospinal fluid and compared with the iNPH group. As shown in Table 3, in the case of GlcNAc-Tf protein alone, there was a significant increase in the NC, MCI and AD groups, but the sensitivity and specificity were not high. In the case of PGDS alone, a significant increase was observed only in the AD group, but the sensitivity and specificity were not high, and the AUC was less than 80%, so it was hard to say that it was a good marker.
  • Example 3 when the cutoff value based on the product value of the combination markers of the present embodiment is applied to other neurodegenerative disease groups (PSP + FTD + DLB + PD), the ratio of the cutoff value or more is any. It was as low as 26% to 37%, indicating that the increase in these product values is AD pathologically specific.
  • the serum PGDS concentration was measured in iNPH individuals and MCI individuals by the same method as in this example.
  • PGDS alone did not show a clear difference in the amount between iNPH and MCI. Therefore, the product of the measured value of p-tau used in the above analysis of the same individual and the PGDS concentration in serum was calculated and used as a pseudo combination marker. Then, when this product value was used, the marker value was higher in MCI than in iNPH, and a significant tendency was observed between the two. This suggests that the combination marker is useful even when the measurement results from samples other than cerebrospinal fluid are used.
  • Example 5 Verification of AD discrimination accuracy by different AD indexes> (Purpose) We will verify the accuracy of differentiating the AD pathology of the combination marker when the formula (A ⁇ 42 / A ⁇ 40) ⁇ p-tau is used for the AD index. (Method) The procedure was the same as in Example 3 except for the calculation of the AD index. As the AD index, the value calculated by the formula (A ⁇ 42 / A ⁇ 40) ⁇ p-tau was used in this example.
  • the sensitivity was 83.3%, the specificity was 79.3%, and the AUC was 0.826 in the MCI group.
  • the sensitivity was as high as 100%, but the specificity was as low as 65.5%. Therefore, it was suggested that the AD index alone does not have sufficient discrimination accuracy.
  • the sensitivity, specificity, and AUC were calculated from the combination of the Man-Tf protein and the above AD index.
  • the sensitivity was 100% and the specificity was 72.4% in the AD group, which could not be sufficiently differentiated by themselves, and the discrimination accuracy was improved.
  • AUC also showed 0.872.
  • the sensitivity was 88.9% and the AUC was 0.895, showing even higher values.
  • the combination marker of the present invention is a calculation formula of the AD index. Regardless, it was shown that it is a highly accurate differential marker for MCI and AD, and can be an excellent predictive differential marker before the onset of MCI. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

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Abstract

La présente invention aborde le problème de la mise au point de biomarqueurs, qui permettent de différencier spécifiquement et avec une sensibilité élevée l'occurrence ou l'absence de la maladie d'Alzheimer et son risque d'apparition et concerne donc un kit de détection des biomarqueurs et un procédé de différenciation des états pathologiques de la maladie d'Alzheimer les utilisant.　Les marqueurs combinés selon la présente invention permettent une différenciation très précise des états pathologiques de la maladie d'Alzheimer chez un sujet, à savoir, l'occurrence ou l'absence de la maladie d'Alzheimer ou d'un trouble cognitif léger et leur risque d'apparition.
PCT/JP2021/032787 2020-09-29 2021-09-07 Marqueurs combinés pour différencier des états pathologiques de la maladie d'alzheimer et procédé de différenciation d'états pathologiques de la maladie d'alzheimer les utilisant WO2022070798A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024122459A1 (fr) * 2022-12-05 2024-06-13 アルメッド株式会社 Méthode de détermination d'un stade de dysfonctionnement cognitif

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007524847A (ja) * 2003-11-07 2007-08-30 サイファージェン バイオシステムズ インコーポレイテッド アルツハイマー病のためのバイオマーカー
WO2008029886A1 (fr) * 2006-09-06 2008-03-13 National University Corporation Tottori University Trousse pour le diagnostic de la maladie d'Alzheimer, marqueur de diagnostic et procédé de détection de l'indicateur pour l'état pathologique
US20140357525A1 (en) * 2013-03-26 2014-12-04 Duke University Markers for alzheimer's disease and mild cognitive impairment and methods of using the same
JP2015004664A (ja) * 2013-06-18 2015-01-08 磁量生技股▲ふん▼有限公司 アルツハイマー病罹患リスクを検出する方法
WO2017195778A1 (fr) * 2016-05-10 2017-11-16 橋本　康弘 Marquer diagnostique de la démence et procédé d'identification de la survenance de la démence à l'aide dudit marqueur
WO2019022064A1 (fr) * 2017-07-25 2019-01-31 長谷川　亨 Procédé d'aide au diagnostic pour jugement de maladies neurodégénératives

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007524847A (ja) * 2003-11-07 2007-08-30 サイファージェン バイオシステムズ インコーポレイテッド アルツハイマー病のためのバイオマーカー
WO2008029886A1 (fr) * 2006-09-06 2008-03-13 National University Corporation Tottori University Trousse pour le diagnostic de la maladie d'Alzheimer, marqueur de diagnostic et procédé de détection de l'indicateur pour l'état pathologique
US20140357525A1 (en) * 2013-03-26 2014-12-04 Duke University Markers for alzheimer's disease and mild cognitive impairment and methods of using the same
JP2015004664A (ja) * 2013-06-18 2015-01-08 磁量生技股▲ふん▼有限公司 アルツハイマー病罹患リスクを検出する方法
WO2017195778A1 (fr) * 2016-05-10 2017-11-16 橋本　康弘 Marquer diagnostique de la démence et procédé d'identification de la survenance de la démence à l'aide dudit marqueur
WO2019022064A1 (fr) * 2017-07-25 2019-01-31 長谷川　亨 Procédé d'aide au diagnostic pour jugement de maladies neurodégénératives

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024122459A1 (fr) * 2022-12-05 2024-06-13 アルメッド株式会社 Méthode de détermination d'un stade de dysfonctionnement cognitif

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