WO2022186486A1 - Utilisation de la chaîne alpha-4 de la tropomyosine et de la chaîne bêta de la glycoprotéine ib plaquettaire pour le diagnostic de la maladie d'alzheimer - Google Patents

Utilisation de la chaîne alpha-4 de la tropomyosine et de la chaîne bêta de la glycoprotéine ib plaquettaire pour le diagnostic de la maladie d'alzheimer Download PDF

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WO2022186486A1
WO2022186486A1 PCT/KR2022/001558 KR2022001558W WO2022186486A1 WO 2022186486 A1 WO2022186486 A1 WO 2022186486A1 KR 2022001558 W KR2022001558 W KR 2022001558W WO 2022186486 A1 WO2022186486 A1 WO 2022186486A1
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disease
alzheimer
tpm4
gp1bb
expression level
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English (en)
Korean (ko)
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최영기
정유현
강성민
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주식회사 피플바이오
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates to the use of tropomyosin alpha-4 chain (TPM4) and platelet glycoprotein Ib beta chain (GP1BB) for the diagnosis of Alzheimer's disease. More specifically, the present invention relates to a composition for diagnosing Alzheimer's disease, a diagnostic kit, and a method for providing information necessary for diagnosing Alzheimer's disease using the same.
  • TPM4 tropomyosin alpha-4 chain
  • GP1BB platelet glycoprotein Ib beta chain
  • AD Alzheimer's disease
  • NFTs neurofibrillary tangles
  • NINCDS-ADRDA a diagnostic standard widely used for clinical research of Alzheimer's disease, biological biomarkers are recommended to differentiate Alzheimer's disease from other types of dementia.
  • the newly revised NINCDS-ADRDA in 2008 includes biological biomarkers as supportive features of diagnostic criteria.
  • Alzheimer's disease drugs currently approved by the FDA only show a positive effect on symptom improvement, but do not treat the underlying disease.
  • biomarkers are being used for the development of new treatments (disease modifying drugs) in countries around the world and research is being conducted, it is questionable whether biomarkers or diagnostic methods that are accurate, low-cost and capable of improving the efficiency of drug application are being used.
  • the new drug development process has limitations such as astronomical cost and dropout of many subjects because the clinical research period is long and requires a large number of subjects. Therefore, it is expected that identifying and using new biological biomarkers that reflect the pathology of Alzheimer's disease will reduce drug development costs and enable accurate target selection.
  • the present inventors found that the biomarkers in plasma, TPM4, GP1BB, or a combination thereof, are expressed at a low level in the plasma of the Alzheimer's patient group compared to normal people, and whether the Alzheimer's disease patient is present by measuring the expression level of these biomarkers through ROC analysis It was confirmed that it can be accurately diagnosed early, and the present invention was completed.
  • an object of the present invention is to provide an agent for detecting tropomyosin alpha-4 chain (TPM4), platelet glycoprotein Ib beta chain (GP1BB), or a combination thereof. It is to provide a composition for diagnosis of Alzheimer's disease, comprising.
  • Another object of the present invention is to provide a kit for diagnosing Alzheimer's disease, comprising the composition for diagnosing Alzheimer's disease.
  • Another object of the present invention is the biomarker in the blood tropomyosin alpha-4 chain (TPM4), platelet glycoprotein Ib beta chain (GP1BB), or a combination thereof
  • TPM4 blood tropomyosin alpha-4 chain
  • GP1BB platelet glycoprotein Ib beta chain
  • biomarker refers to a marker that can be detected in a sample, eg, a marker necessary for diagnosis and/or prognosis.
  • a biomarker can serve as a marker of a particular subtype of disease or disorder (eg, Alzheimer's disease) characterized by particular molecular, pathological, histological and/or clinical features.
  • the biomarker is a gene.
  • Biomarkers include polynucleotides (eg, DNA and/or RNA), polynucleotide copy number alterations (eg, DNA copy number), polypeptides, polypeptides and polynucleotide modifications (eg, post-translational modifications), carbohydrates and/or glycolipid-based molecular markers.
  • an “amount” or “level” of a biomarker that is associated with an increased clinical benefit to an individual is a detectable level in a biological sample. It can be determined by methods known to the person skilled in the art and also disclosed by the present invention. The expression, level or amount of the biomarker being evaluated can be used to determine the presence or absence of a disease, the severity of the disease, and/or response to treatment.
  • expression level generally refers to the amount of a biomarker in a biological sample. “Expression” generally refers to the process by which genetic information (eg, gene-coding and/or epigenetics) is converted into structures that exist and function within a cell. Thus, as used herein, “expression” may refer to transcription into a polynucleotide, translation into a polypeptide, or even modification of a polynucleotide and/or a polypeptide (eg, post-translational modification of a polypeptide).
  • polypeptides Transcribed polynucleotides, translated polypeptides, or polynucleotide and/or polypeptide modifications (e.g., post-translational modifications of a polypeptide), whether they are derived from transcripts produced by alternative splicing or degraded transcripts or from post-translational processing of the polypeptide, for example, by proteolysis, all are considered to be expressed.
  • “Expressed gene” includes those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (eg, tRNA and rRNA).
  • “Expressed protein” includes translation of mRNA into a polypeptide and post-translational processing.
  • Elevated expression refers to a control, such as a sample obtained from an individual or individuals (normal) not suffering from a disease or disorder (eg, Alzheimer's disease), or Refers to increased expression or increased level of a biomarker in an individual compared to an internal control (eg, a housekeeping biomarker) in a sample.
  • a control such as a sample obtained from an individual or individuals (normal) not suffering from a disease or disorder (eg, Alzheimer's disease), or Refers to increased expression or increased level of a biomarker in an individual compared to an internal control (eg, a housekeeping biomarker) in a sample.
  • “Decreased expression”, “reduced expression level” or “reduced level” refers to a control, such as a sample obtained from an individual or individuals (normal) not suffering from a disease or disorder (eg, Alzheimer's disease), or Refers to reduced expression or reduced level of a biomarker in an individual compared to an internal control (eg, a housekeeping biomarker) in a sample. In some embodiments, reduced expression means little or no expression.
  • the term “housekeeping biomarker” refers to a biomarker or group of biomarkers (eg, polynucleotides and/or polypeptides) that are typically similarly present in all cell types.
  • the housekeeping biomarker is a “housekeeping gene”.
  • a “housekeeping gene” herein refers to a gene or group of genes that encodes a protein whose activity is essential for the maintenance of cellular function and is typically present similarly in all cell types.
  • polynucleotide when used in the singular or plural generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides as defined herein include, but are not limited to, single- and double-stranded DNA, DNA comprising single- and double-stranded regions, single- and double-stranded RNA, and single- and double-stranded DNA.
  • - RNA comprising a region, single-stranded or more typically double-stranded, or hybrid molecule comprising DNA and RNA, which may comprise single- and double-stranded regions.
  • polynucleotide refers to a triple-stranded region comprising RNA or DNA, or both RNA and DNA. Strands in these regions may be from the same molecule or from different molecules. The regions may all include more than one molecule, but more typically include regions of only some molecules. One of the molecules of the triple-helix region is often an oligonucleotide.
  • polynucleotide specifically includes cDNA. The term includes DNA (including cDNA) and RNA containing one or more modified bases. Thus, DNA or RNA having a backbone that has been modified for stability or other reasons is a "polynucleotide" as the term is intended herein.
  • polynucleotide as defined herein is DNA or RNA comprising an unconventional base such as inosine or a modified base such as a tritiated base.
  • polynucleotide refers to all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as DNA and RNA characteristic of viruses and cells (including simple and complex cells). includes chemical forms.
  • oligonucleotide refers to a relatively short polynucleotide including, but not limited to, single-stranded deoxyribonucleotides, single- or double-stranded ribonucleotides, RNA:DNA hybrids, and double-stranded DNA. . Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using commercially available automated oligonucleotide synthesizers. However, oligonucleotides can be prepared by a variety of other methods, including in vitro recombinant DNA-mediated techniques, and by expression of DNA in cells and organisms.
  • diagnosis is used herein to refer to the identification or classification of a molecular or pathological condition, disease or condition (eg, Alzheimer's disease).
  • diagnosis may refer to being determined by a physician to be present in a subject with Alzheimer's disease.
  • Diagnosis also refers to, for example, one or a combination of histopathological criteria, imaging criteria, or molecular characteristics (eg, biomarkers (eg, a particular gene or protein encoded by said gene)). It can refer to the classification of a particular type by subtype) characterized by the expression of
  • sample refers to a subject of interest containing a cellular and/or other molecular entity that is characterized and/or identified based on, for example, physical, biochemical, chemical and/or physiological characteristics. and/or a composition obtained from or derived from an individual.
  • disease sample and variant expressions thereof mean any sample obtained from a subject of interest expected or known to contain the cellular and/or molecular entity being characterized.
  • Samples may be primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, fluid, whole blood, blood-derived cells, urine, cerebrospinal fluid. , saliva, sputum, tears, sweat, mucus, and tissue culture media, tissue extracts such as blood (whole blood, serum, plasma), homogenized tissue, cell extracts, and combinations thereof.
  • reference sample As used herein, “reference sample (sample),” “reference cell,” “reference tissue,” “reference blood,” “reference plasma,” “control sample,” “control cell,” or “control tissue,” etc. are for comparative purposes. Refers to a sample, cell, tissue, standard or level used for In one embodiment, a reference sample (sample), reference cell, reference tissue, reference blood, reference plasma, control sample, control cell or control tissue is a healthy and/or non-diseased body part (e.g., eg, from tissue, blood or cells). In another embodiment, the reference sample is obtained from untreated tissues and/or cells of the body of the same subject or individual.
  • a reference sample (sample), reference cell, reference tissue, control sample, control cell or control tissue is from a part of the body (eg, tissue, blood or cells) of a healthy individual other than the subject or individual. obtain In another embodiment, a reference sample (sample), reference cell, reference tissue, control sample, control cell or control tissue is obtained from untreated tissue, blood and/or cells of the body of a healthy individual other than the subject or individual.
  • the term “individual”, “subject”, or “subject” is a mammal. Mammals include domestic animals (eg, cattle, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, mice and rats). including, but not limited to. In certain embodiments, the individual or subject is a human.
  • the present invention detects tropomyosin alpha-4 chain (TPM4), platelet glycoprotein Ib beta chain (GP1BB), or a combination thereof It provides a composition for diagnosis of Alzheimer's disease, comprising an agent for:
  • the tropomyosin alpha-4 chain (TPM4) is a protein encoded by the TPM4 gene, and is also called TM30p1 or tropomyosin-4 (Tropomyosin-4).
  • the tropomyosin alpha-4 chain binds to actin filaments and calcium in muscle and non-muscle cells, and is associated with the troponin complex to regulate calcium-dependent regulation of striated muscle contraction in vertebrates. is known It is known that the tropomyosin alpha-4 chain is increased in brain tissue or tear samples of humans with Alzheimer's disease or in brain tissue of mice.
  • platelet glycoprotein Ib beta chain is a protein encoded by the GP1BB gene, also called CD42b-beta, or CD42c, and is also referred to as GP-Ib beta, GPIb beta, or GPIbB. marked
  • the platelet glycoprotein Ib beta chain is a surface membrane protein of platelets and is involved in the formation of a blood clot in platelets by binding to von Willebrand factor, which is bound to endothelial cells. As with the tropomyosin alpha-4 chain, no association is known between the platelet glycoprotein Ib beta chain and the pathogenesis of Alzheimer's disease.
  • detecting the tropomyosin alpha-4 chain (TPM4), platelet glycoprotein Ib beta chain (GP1BB), or a combination thereof The agent for the present invention is an antibody or antigen-binding fragment thereof, a target binding protein, or an aptamer that specifically binds to TPM4 and GP1BB, which are the biomarkers of the present invention.
  • the composition further comprises an agent for detecting amyloid beta oligomer.
  • the agent comprises an antibody or antigen-binding fragment thereof, a target binding protein, or an aptamer that specifically binds to the amyloid beta oligomer, which is the biomarker of the present invention.
  • the antibody specifically binding to the amyloid beta oligomer is 6E10, FF51, and WO2, but is not limited thereto.
  • antibody refers to an antibody that specifically binds to a specific antigen, and includes not only a complete antibody form but also an antigen binding fragment of an antibody molecule.
  • the antibody includes an antibody selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a single domain antibody, and a single chain variable fragment.
  • antigen binding fragment refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2 and Fv.
  • the antibody or antigen-binding fragment is specifically in the form of Fab, scFv, or complete antibody.
  • target binding polypeptide refers to a non-immunoglobulin polypeptide molecule that has binding affinity for a target antigen or hapten, such as an antibody, but is structurally unrelated to the antibody.
  • the target-binding polypeptide is also called an antibody-like molecule or antibody mimetics, and generally has a molecular weight of 3-20 kDa, unlike an antibody having a molecular weight of about 150 kDa.
  • the target-binding polypeptide is an affibody derived from the Z-domain of protein A, affilin derived from gamma-B crystallin or ubiquitin, and an affimer derived from cystatin.
  • target-binding polypeptide may be engineered to have binding affinity for any target antigen or hapten through various screening methods known in the art, such as phage display and ribosome display.
  • the present invention provides a kit for diagnosing Alzheimer's disease, comprising the composition for diagnosing Alzheimer's disease described above.
  • the kit includes an agent for detecting the aforementioned TPM4, GP1BB, or a combination thereof.
  • the kit further comprises an agent for detecting amyloid beta oligomer.
  • the TPM4, GP1BB, amyloid beta oligomer, or a combination thereof is TPM4, GP1BB, amyloid beta oligomer, or a combination thereof detected in a sample obtained from an individual using the kit and a reference sample. Comparing the expression levels of the combinations can be used to provide information necessary for the diagnosis of Alzheimer's disease.
  • the kit for diagnosing Alzheimer's disease of the present invention includes all of the composition for diagnosing Alzheimer's disease according to an aspect of the present invention, the contents of the composition common to the composition for diagnosing Alzheimer's disease are the same and overlap with each other. are omitted to avoid complexity of the description of the present specification within the scope of
  • the present invention provides a method for providing information necessary for the diagnosis of Alzheimer's disease, comprising the following steps:
  • the expression level of TPM4, GP1BB, or a combination thereof measured in a sample isolated from the subject is more than the expression level of TPM4, GP1BB, or a combination thereof measured in a reference sample. If it is low, it indicates that you are more likely to have Alzheimer's disease.
  • the expression level of the TPM4, GP1BB, or a combination thereof is expressed as a concentration (w/v) of the protein in plasma.
  • the protein concentration of the TPM4, GP1BB, or a combination thereof is expressed in ng/ml, but is not limited thereto.
  • the method further comprises measuring the expression level of amyloid beta oligomer in a sample isolated from the subject.
  • Alzheimer's disease patients are more likely indicates that
  • the level of the amyloid beta oligomer is a ratio value of the amyloid beta oligomerization tendency (OA ⁇ tendency) derived by Equation 1 below:
  • Amyloid beta oligomerization tendency OA ⁇ concentration of sample/average OA ⁇ concentration of control A&B
  • control A is a positive sample in which OA ⁇ is present
  • control B is a negative sample in which OA ⁇ is not present.
  • the method measures the expression level of TPM4, GP1BB or a combination thereof, and the expression level of amyloid beta oligomer measured in a sample isolated from an individual and a reference sample, respectively, and the following formula 2 Comparing the value (V2) derived by
  • V2 TPM4 expression level X GP1BB expression level.
  • the method comprises: i) the expression level of TPM4, GP1BB or a combination thereof measured in a sample isolated from a subject and a reference sample; and ii) measuring the expression level of amyloid beta oligomer, respectively, and comparing the value (V3, V4, or V5) derived by any one of the following formulas 3 to 5. Indicative of higher, the method:
  • V3 amyloid beta oligomerization propensity/TPM4 expression level
  • V4 amyloid beta oligomerization propensity/GP1BB expression level
  • V5 amyloid beta oligomerization tendency/(TPM4 expression level X GP1BB expression level)
  • amyloid beta oligomerization tendency is derived by the following formula 1:
  • Amyloid beta oligomerization tendency OA ⁇ concentration of sample/average OA ⁇ concentration of control A&B
  • control A is a positive sample in which OA ⁇ is present
  • control B is a negative sample in which OA ⁇ is not present.
  • the expression level of the TPM4, GP1BB, or a combination thereof is expressed as the concentration of the protein in plasma.
  • the protein concentration of the TPM4, GP1BB, or a combination thereof is expressed in ng/ml, but is not limited thereto.
  • the value derived by Equations 1 to 5 may determine whether the patient is Alzheimer's disease based on the cut-off values shown in Table 3 below, but is not limited thereto.
  • the method may further comprise the following steps:
  • the Alzheimer's disease therapeutic agent may be, for example, aducanumab (Aducanumab, Biogen, USA), but if it is a therapeutic agent with proven Alzheimer's disease therapeutic efficacy, it can be used without limitation as the Alzheimer's disease therapeutic agent of the present invention. have.
  • the present invention provides a method for treating Alzheimer's disease, comprising the steps of:
  • TPM4 tropomyosin alpha-4 chain
  • GP1BB platelet glycoprotein Ib beta chain
  • the Alzheimer's disease treatment method of the present invention includes all of the methods for providing information necessary for diagnosing Alzheimer's disease according to an aspect of the present invention, the configuration is common to the above-described method for providing information necessary for diagnosing Alzheimer's disease. All of the contents are equally applied, and are omitted in order to avoid the complexity of the description of the present specification within the scope of overlapping contents.
  • TPM4, GP1BB, or a combination thereof are expressed at a low level in the plasma of the Alzheimer's patient group compared to normal individuals, it is possible to determine whether or not Alzheimer's disease patients are present by measuring the expression level of these biomarkers. It can be diagnosed early and accurately.
  • FIG. 1 is a diagram showing the overall sequence of plasma proteome profiling of the present invention using TMT-labeled LC-MS/MS.
  • FIG. 2 is a diagram showing the results of analysis of the concentration of TPM4 and ROC curve in the plasma of the normal group and the AD patient group matched by age.
  • 3 is a diagram showing the results of analysis of the concentration and ROC curve of GP1BB in the plasma of the normal group and the AD patient group matched by age.
  • FIG. 4 is a diagram showing the multiplication value of TPM4 concentration and GP1BB concentration in the plasma of the normal group and the AD patient group matched by age, and the ROC curve analysis result accordingly.
  • FIG. 5 is a diagram showing the detection value of amyloid beta oligomer in the plasma of the normal group and the AD patient group matched by age, and the ROC curve analysis result accordingly.
  • FIG. 6 is a diagram showing a value obtained by dividing the detection value of amyloid beta oligomer in the plasma of the normal group and the AD patient group matched by age by the concentration of TPM4, and the ROC curve analysis results accordingly.
  • FIG. 7 is a diagram showing a value obtained by dividing the detection value of amyloid beta oligomer in the plasma of the normal group and the AD patient group matched by age by the concentration of GP1BB, and the ROC curve analysis results accordingly.
  • FIG. 8 is a diagram showing a value obtained by dividing the detection value of amyloid beta oligomer in the plasma of the normal group and the AD patient group matched by age by the concentration of TPM4 and the concentration of GP1BB, and the ROC curve analysis results accordingly.
  • AD dementia Alzheimer's disease dementia
  • EOAD Early onset Alzheimer's disease
  • LOAD late onset Alzheimer's disease
  • 1160 protein groups were identified by performing proteomics analysis through TMT (Tandem Mass Tag)-labeling mass spectrometry experiments with samples of 10 patients in each group. Biomarker candidate proteins that were different for each group were selected. Among the identified biomarker candidate proteins, the blood biomarkers of the present invention were selected from the results of quantification through ELISA in blood in the order of increasing or decreasing proteins in Alzheimer's disease patients compared to the normal group.
  • pooled plasma samples were removed using a Pierce TM Top 2 abundant protein depletion spin column (Thermo Fisher Scientific, Rockford, IL) or a High Select Top 14 abundant protein depletion mini spin column (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, 10 mL of pooled plasma samples were added directly to the resin slurry in depletion spin columns, followed by incubation for 10 minutes and centrifugation at 1,000 ⁇ g for 2 minutes. The removed samples were digested using an S-Trap spin column (Protifi, Huntington, NY). The sample was reduced by DTT and then IAA (alkylated by iodoacetamide).
  • TMT6plex reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, each prepared TMT reagent was added to the peptide sample, the mixture was incubated for 1 hour, then quenched by adding 8 mL of 5% hydroxylamine and incubated at room temperature for 15 minutes.
  • Each set of 6 TMT labeled peptide samples was pooled and dried in a vacuum concentrator.
  • Peptide fractionation was performed by gradient for 115 minutes at a flow rate of 500 mL/min (0% solvent B (10 mM TEAB in 90% acetonitrile) for 10 minutes, 0-5% solvent B for 10 minutes). , 5-35% solvent B 60 min, 35-70% solvent B 15 min, 70% solvent B 10 min, 70-0% solvent B 10 min). A total of 96 fractions were collected every minute from 15 min to 110 min and pooled every 24 non-consecutive peptide fractions (i.e., #1-#25-#49-#73, #2-#26-#50-#74, ..., #24-#48-#72-#96).
  • TMT-labeled 24 peptide fractions were analyzed with a Q Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific) coupled with an Ultimate 3000 RSLCnano system (Thermo Scientific). Peptides were loaded onto a trap column (100 ⁇ m x 2 cm) packed with Acclaim PepMap100 C18 resin, and then in solvent B (0.1% formic acid in ACN) with a gradient of 7 to 36% at a flow rate of 0.3 mL/min for 200 min.
  • solvent B 0.1% formic acid in ACN
  • the eluted peptides separated by the analytical column (EASY-Spray column, 75 ⁇ m x 50 cm, Thermo Fisher Scientific) were sprayed into a nano-ESI source with an electron spray voltage of 2.1 kV.
  • the Q Exactive Orbitrap mass analyzer was operated with the top 10 data-dependent methods. Full MS scans were acquired in the m/z 350-2000 range with a mass resolution of 140,000 (at m/z 200).
  • the AGC target value was 3.00E+06.
  • Orbitrap with a mass resolution of 35,000 at m/z 200 fragmented in a higher-energy collisional dissociation (HCD) collision cell, in which the 10 most intense peaks, with charge states of 2 or greater, have a normalized collision energy of 32 Tandem mass spectra were acquired on a mass spectrometer.
  • HCD collisional dissociation
  • Database search parameters included precursor ion mass tolerance of 10 ppm, fragment ion mass tolerance of 0.08 Da, carbamidomethyl cysteine (+57.021 Da/C), and TMT. Fixed modifications to the tag (+229.163 Da/K and N-terminus) and variable modifications for oxidation (+15.995 Da/M) were included. We obtained less than 1% FDR at the peptide level and filtered with high peptide confidence.
  • ELISA Enzyme-Linked Immunoprecipitation Assay
  • ELISA is a method of quantitatively measuring the strength and amount of an antigen-antibody reaction by labeling an antibody or antigen with an enzyme, measuring the activity of the enzyme, and measuring the amount of a specific protein in a biological sample.
  • GP1BB ELISA KIT MyBioSource, catalog#: MBS945024
  • TPM4 ELISA KIT MyBioSource, catalog#: MBS944560
  • the blood amyloid beta oligomerization tendency was measured by detecting the aggregation type of the aggregation type-forming polypeptide using the MDS-OA ⁇ test (cat. # - MD1002PE, PeopleBio).
  • the antibody amyloid beta specific binding antibodies 6E10 (Biolegend) and WO2-HRP (Absolute) having amino acids 3-8 and 4-10 of the human A ⁇ peptide sequence, respectively, as epitopes were used.
  • 30 ⁇ g of 6E10 antibody (anti-A ⁇ protein, Biolegend) was diluted in 10 ml of coating buffer (Sigma-Aldrich), and 100 ⁇ l of the plate (Nunc) was dispensed into each well, reacted in a refrigerator at 4° C.
  • the sample was used after vortexing after thawing the frozen plasma sample at room temperature.
  • a total of 285 ⁇ l was prepared by mixing 25 ⁇ l of plasma with 235 ⁇ l of assay buffer and 25 ⁇ l of recombinant A ⁇ (1 ng/10 ⁇ l) inducing aggregation. Samples prepared by spiking the aggregation-type formation-inducing recombinant A ⁇ were incubated for 1 hour in an incubator at 37°C.
  • the present inventors designed an experiment as follows to identify plasma proteins involved in the diagnosis of normal group and Alzheimer's disease (AD) patients and changes in A ⁇ oligomerization.
  • the normal group and AD patients were recruited based on amyloid PET scan and clinical course judged to be AD by the judgment of a doctor who has specialized in dementia at a tertiary hospital.
  • 10 normal patients with negative amyloid PET scan results and normal clinical trials and 10 AD patients with positive amyloid PET scan results and clinical AD were selected (Table 1).
  • Plasma samples from the normal group and AD patient group were collected and pooled, respectively, and proteomic analysis was performed according to the procedure shown in FIG. 1 .
  • pooled plasma samples were depleted (removing albumin and IgG, or 14 abundant proteins).
  • i)/ii) non-depletion sample iii)/iv) albumin and IgG depletion sample
  • v)/vi) 14 abundant protein depletion sample a total of 6 samples, respectively.
  • a pooled plasma sample was obtained. Then, based on these samples, tandem mass tags (TMT)-labeled LC-MS/MS analysis was performed.
  • TMT tandem mass tags
  • TPM4 tropomyosin alpha-4 chain
  • GP1BB platelet glycoprotein Ib beta chain
  • Validation cohort consisted of 32 normal group and 30 AD patient group. Normal group and AD patient group were selected based on the same criteria as the previously pooled plasma samples, and each pooled 10 people were included in each group (Table). 2).
  • TPM4 is a protein involved in actin-binding in muscle cells and non-muscle cells, and is known to play an important role in muscle contraction. According to previous studies, the expression level of TPM4 is increased in human brain or tear samples with AD pathology, or in mouse brain tissue. However, as a result of this study, it was confirmed that TPM4 was quantitatively and significantly decreased in the AD patient group compared to the normal group (Fig. 2, a). In addition, as a result of analyzing the ROC curve using TPM4, an area under the curve (AUC) value of 0.818 was derived (FIG. 2, b).
  • AUC area under the curve
  • GP1BB is a cell membrane protein with high expression in blood and brain, and is known to have biological functions of blood coagulation, cell adhesion, and hemostasis. Studies on the potential of GP1BB as a diagnostic biomarker are scarce, and in particular, the potential for use in AD diagnosis is not suggested. As a result of the ELISA assay, the concentration of GP1BB was significantly reduced in the AD patient group compared to the normal group (FIG. 3, a). In addition, an AUC value of 0.635 was obtained through ROC curve analysis using the quantitative value of GP1BB in each group (FIG. 3, b).
  • TPM4 and GP1BB of the present invention selected from proteomics can be usefully used as novel plasma biomarkers for diagnosing the normal group and the AD patient group.
  • Example 3 AUC analysis by the combination of TPM4 and GP1BB
  • the present inventors searched for a way to improve the AUC value of AD diagnosis by combining two candidate substances.
  • concentrations of the two proteins obtained from the ELISA analysis results performed in each individual were multiplied (TPM4 X GP1BB), and the results were analyzed with a dot plot and an ROC curve.
  • TPM4 X GP1BB concentrations of the two proteins obtained from the ELISA analysis results performed in each individual were multiplied (TPM4 X GP1BB), and the results were analyzed with a dot plot and an ROC curve.
  • the AD patient group was significantly reduced compared to the normal group (Fig. 4, a).
  • the AUC value of the combined diagnosis of TPM4 and GP1BB was 0.830, which was improved compared to the diagnosis result of TPM4 alone (0.818) or GP1BB alone (0.635) ( FIGS. 4 and b ).
  • the above results indicate that TPM4 and GP1BB alone can be used as biomarkers for AD diagnosis, but furthermore, better accuracy can be improved by appropriately combining the two biomarkers.
  • Example 3 AUC analysis by TPM4, GP1BB, or a combination thereof and a combination of OA ⁇ propensity
  • the existing multimer detection system reflects the oligomerization tendency of amyloid beta, OA ⁇ tendency of plasma, and is known to increase in AD patients.
  • MDS multimer detection system
  • the OA ⁇ trend (represented by the OA ⁇ ratio value) was calculated as follows.
  • OA ⁇ tendency OA ⁇ concentration of sample/average OA ⁇ concentration of control A&B
  • control A is a positive sample in which OA ⁇ is present
  • control B is a negative sample in which OA ⁇ is not present
  • the oligomerization tendency of the control A or control B is greater than 0.9 and less than 2 (0.9 ⁇ OA ⁇ tendency of control A or B ⁇ 2)
  • the present inventors attempted an appropriate combination of a newly discovered biomarker with MDS in order to further improve the AUC value of AD diagnosis.
  • OA ⁇ tendency and TPM4 were combined. Since the OA ⁇ trend was high in the AD patient group compared to the normal group and TPM4 was quantified low, the OA ⁇ trend was corrected by dividing the OA ⁇ tendency by the TPM4 concentration. As a result, it was confirmed that the 'OA ⁇ tendency / TPM4 concentration' value was significantly increased in the AD patient group (Fig. 6, a), and through the ROC curve analysis, the AUC value was 0.883 for MDS alone (0.817) or TPM4 alone (0.818) It was confirmed that there was more improvement (FIGS. 6, b)
  • TPM4 and GP1BB can be used alone as plasma biomarkers for AD diagnosis, and that the combination between TPM4 and GP1BB, TPM4, GP1BB and MDS improve the accuracy of AD diagnosis through appropriate combination It was confirmed that it can be an important factor that can make it happen.
  • AD senile plaque and neurofibrillary tangle
  • the MDS Multimer Detection System
  • MDS Multimer Detection System
  • OA ⁇ trend measured by MDS showed a statistically significant increase in the AD patient group compared to the normal group, confirming that it was correlated with both the cerebrospinal fluid biomarker and PIB PET. Therefore, it can be said that the difference in the OA ⁇ tendency between the AD patient group and the normal group measured by MDS is highly effective as a blood-based biomarker to help diagnose AD.
  • the present inventors tried to identify the plasma protein causing the difference between the two groups using amyloid PET scan and plasma samples obtained from the normal group and the AD patient group recruited on a clinical basis.
  • TMT-labeled LC-MS/MS was performed by pooling 10 plasma samples obtained from each group and classifying them according to whether plasma abundant protein depletion was present.
  • 1160 proteins obtained as a result proteins significantly higher or lower in the AD patient group than in the normal group were selected as biomarker candidates for AD diagnosis.
  • TPM4 and GP1BB were selected as final candidates for novel biomarkers capable of diagnosing AD.
  • the present invention identifies TPM4 and GP1BB as novel biomarkers for the diagnosis of AD patients through plasma proteome profiling of normal and AD patient groups obtained by amyloid PET scan and strict clinical criteria. It has priority in that it did.
  • the selected TPM4 and GP1BB are novel biomarkers capable of diagnosing AD even when used alone, as well as demonstrating that an appropriate combination of these can also increase the effectiveness of AD diagnosis.
  • the potential as an auxiliary biomarker to further improve the existing MDS technique was also suggested.
  • the present invention relates to the use of tropomyosin alpha-4 chain (TPM4) and platelet glycoprotein Ib beta chain (GP1BB) for the diagnosis of Alzheimer's disease. More specifically, the present invention relates to a composition for diagnosing Alzheimer's disease, a diagnostic kit, and a method for providing information necessary for diagnosing Alzheimer's disease using the same.
  • TPM4 tropomyosin alpha-4 chain
  • GP1BB platelet glycoprotein Ib beta chain

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Abstract

La présente invention concerne une composition et une trousse de diagnostic pour le diagnostic de la maladie d'Alzheimer, et une méthode destinée à fournir les informations nécessaires pour diagnostiquer la maladie d'Alzheimer à l'aide de la composition et de la trousse. TPM4, GP1BB, ou une combinaison associée, qui sont des biomarqueurs plasmatiques ayant récemment été développés par les inventeurs de la présente invention, sont exprimés à un faible niveau dans le plasma d'un groupe de patients atteints de la maladie d'Alzheimer par comparaison avec des individus normaux, et par conséquent, par l'intermédiaire d'une mesure des niveaux d'expression des biomarqueurs, il est possible de diagnostiquer de façon précoce et exacte si un patient est atteint ou non de la maladie d'Alzheimer.
PCT/KR2022/001558 2021-03-05 2022-02-08 Utilisation de la chaîne alpha-4 de la tropomyosine et de la chaîne bêta de la glycoprotéine ib plaquettaire pour le diagnostic de la maladie d'alzheimer WO2022186486A1 (fr)

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