WO2024055378A1 - 用于检测阿尔兹海默病自身抗体的蛋白抗原组合及其应用 - Google Patents

用于检测阿尔兹海默病自身抗体的蛋白抗原组合及其应用 Download PDF

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WO2024055378A1
WO2024055378A1 PCT/CN2022/124874 CN2022124874W WO2024055378A1 WO 2024055378 A1 WO2024055378 A1 WO 2024055378A1 CN 2022124874 W CN2022124874 W CN 2022124874W WO 2024055378 A1 WO2024055378 A1 WO 2024055378A1
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antigen polypeptide
modification
fusion
cells
adap
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PCT/CN2022/124874
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English (en)
French (fr)
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于欣
王华丽
张楠
谷冬梅
韦卓飞
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北京湃德智健科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the present invention relates to the technical field of immunology, and specifically to antigen polypeptides for detecting ADAP autoantibodies and their applications.
  • AD Alzheimer's disease
  • a ⁇ amyloid- ⁇
  • Microglia are homologous to peripheral macrophages and retain strong immune response functions. Studies have shown that autoantibodies to some endogenous protein molecules can activate the phagocytic function of microglia.
  • Some well-known foreign pharmaceutical companies have carried out clinical research on vaccines and therapeutic monoclonal antibodies related to AD (such as Roche's A ⁇ monoclonal antibody crenezumab, etc.). However, because the focus is on A ⁇ itself, the target selection is narrow, and the actual effect is not satisfactory. As expected.
  • the present invention involves 10 Alzheimer’s Disease Associated Protein (ADAP) autoantibodies (SORL1, TMEM163, SPRED2, CLU, PTK2B, TSPAN14, FERMT2, ADAM10, TREM2, VKORC1):
  • ADAP Alzheimer’s Disease Associated Protein
  • Sortilin is a protein encoded by the SORT1 gene on chromosome 1 in humans. This protein is a type I membrane glycoprotein in the vacuolar protein sorting 10 protein (Vps10) family of sorting receptors. SORL1 is ubiquitously expressed in many tissues but is most abundant in the central nervous system. At the cellular level, SORL1 plays a role in protein transport between the Golgi apparatus, endosomes, lysosomes, and the plasma membrane.
  • Vps10 vacuolar protein sorting 10 protein
  • SORL1 This molecular function enables SORL1 to participate in a variety of biological processes, including transporting GLUT4 to the plasma membrane of adipose and skeletal muscle cells in response to insulin; mediating the interaction between proNGF and the p75NTR:sortilin complex by acting as a coreceptor; Sends cell death signal; finely regulates brain-derived neurotrophic factor (BDNF) necessary for neuronal survival.
  • BDNF brain-derived neurotrophic factor
  • SORL1 is the first member of the Vps10 family that has been proven to be a late-onset AD risk gene by GWAS, and its expression level is reduced in the brains of patients with late-onset AD and mild cognitive impairment.
  • SORL1 is the cargo receptor of reverse transcriptase.
  • APP protein can transport APP protein from the endoplasmic reticulum system and the highly active environment of BACE protein to the reverse Golgi apparatus for re-modification. It can also directly regulate the oligomerization of APP to regulate its proteolysis. process. Reduction in SORL1 expression level or activity can promote the accumulation of A ⁇ .
  • TMEM163 a zinc ion transmembrane transporter, is a GWAS risk gene for sporadic AD.
  • TMEM163 is responsible for the internal and external outflow of zinc ions at neuronal synapses, including participating in the release of synaptic vesicles.
  • SPRED2 belongs to the Sprouty/SPRED family, a member of the budding protein family, and participates in the activation of the MAPK kinase cascade process induced by growth factors. Genetically, SPRED2 has been shown to be a risk gene for sporadic AD.
  • the present invention uses the self-designed SPRED2 epitope polypeptide to detect the levels of autoantibodies in the serum and plasma of AD patients and develop response reagents to predict the risk of AD and the progression of the disease, providing a reliable reference for clinical treatment.
  • CLU is the second largest lipoprotein in the brain after apolipoprotein APOE and is a GWAS risk gene for sporadic AD.
  • CLU may be involved in the transport of ⁇ -amyloid protein between the brain and plasma, and helps regulate the clearance of ⁇ -amyloid protein in the brain in the pathogenesis of AD.
  • An imbalance in the production and clearance of beta-amyloid is a core factor in the development of Alzheimer's disease.
  • PTK2B is a cytoplasmic protein tyrosine kinase involved in the calcium-induced ion channel regulation and activation map kinase signaling pathways. PTK2B serves as an important signaling intermediate between neuropeptide-activated receptors or neurotransmitters that represent increased calcium flux, and downstream signals that regulate neuronal activity. It undergoes rapid tyrosine phosphorylation and activation in response to increases in intracellular calcium concentration, nicotinic acetylcholine receptor activation, membrane depolarization, or protein kinase C activation. Genetically, PTK2B is a GWAS risk gene for sporadic AD.
  • Phosphorylated PTK2B can co-localize with phosphorylated Tau in the brains of AD patients and transgenic Tau mice, participate in the phosphorylation and aggregation of Tau, and is an early marker and in vivo regulator of Tau toxicity.
  • TSPAN14 is a four-spanning protein on the cell membrane surface and a GWAS risk gene for sporadic AD. In terms of biological functions, TSPAN14 can affect the activity of signaling pathway kinases on the inner side of the cell membrane, including the activation and regulation of the Notch signaling pathway. In terms of distribution, TSPAN14 is highly expressed in the central nervous system and peripheral immune cells, and has the function of regulating neuroimmune mechanisms.
  • FERMT2 is a focal adhesion protein on the cell membrane surface and is a GWAS risk gene for sporadic AD.
  • FERMT2 is involved in regulating the binding activity of cytoskeletal tubulin, affecting the binding activity of phosphatidylinositol, and the activity of transforming growth factor ⁇ receptor.
  • FERMT2 participates in important events such as cell differentiation and local cell synthesis by affecting signaling pathways involving multiple cell surface receptors. Abnormal functional regulation of FERMT2 plays a key role in the development of AD.
  • the ADAM10 gene encodes a protein molecule containing disintegrin and metalloproteinase domains and is a GWAS risk gene for sporadic AD. Multiple functional mutations of the ADAM10 gene have also been found in familial AD. In neurons, ADAM10 is one of the most important enzymes with ⁇ -secretase activity for the proteolytic processing of amyloid precursor proteins. ADAM10 and ADAM17 together cleave the extracellular domain of the triggering receptor expressed on myeloid cells 2 (TREM2) to produce soluble TREM2 (sTREM2), which is one of the key molecules for the clearance of A ⁇ 42 in the brain. Abnormal functional regulation of ADAM10 plays a key role in the development of AD.
  • TREM2 myeloid cells 2
  • sTREM2 soluble TREM2
  • TREM2 is a stress-expressed receptor on the surface of myeloid cells and a GWAS risk gene for sporadic AD. Multiple functional mutations of the TREM2 gene have also been found in familial AD. In terms of biological function, this gene encodes a membrane protein that forms a receptor signaling complex with TYRO protein tyrosine kinase-binding protein, plays a role in immune responses, and may be involved in chronic disease by triggering the production of constitutive inflammatory cytokines. inflammation. In the brain, TREM2 is one of the key receptors by which microglia monitor and respond to neurodegenerative signals. In animal experiments, it was found that TREM2 antibodies can inhibit the hyperinflammatory state of microglia and restore their normal phagocytic function. Abnormal functional regulation of TREM2 plays a key role in the development of AD.
  • VKORC1 encodes a vitamin K oxidoreductase complex subunit and is a GWAS risk gene for sporadic and familial AD.
  • the VKORC1 protein is responsible for reducing inactive vitamin K 2,3-epoxide to active vitamin K in the endoplasmic reticulum membrane.
  • Vitamin K is a necessary cofactor for the carboxylation of glutamate residues by vitamin K-dependent gamma-carboxylase in thrombin.
  • the present invention uses self-designed antigenic epitope polypeptides to detect the levels of autoantibodies in the serum and plasma of AD patients and develop response reagents to predict the risk of AD and the progression of the disease, providing a reliable reference for clinical treatment.
  • the binding of antigens and antibodies actually only occurs between the antigenic determinant and the antigen-binding site of the antibody, and the two are completely complementary in spatial structure and configuration. Therefore, the antigenic determinant can represent the binding state and affinity characteristics of the entire protein to the antibody.
  • Traditional enzyme-linked immunoassay (ELISA) detection of autoantibodies mainly uses recombinant proteins as antigens, which requires a series of tedious processes such as vector construction, transfection, expression, screening, and purification, and the production cost is expensive.
  • the complete protein has a complex spatial structure and the antigen epitope is not easily exposed, so the specificity of antigen-antibody binding is not strong, resulting in a low signal detection rate.
  • an antigen polypeptide for detecting Alzheimer's disease marker ADAP autoantibodies comprising at least one of the amino acid sequences shown in SEQ ID NO. 1-10:
  • SORL1 CEVWTQRLHGGSAPLPQDRGFLVVQGDPR;
  • PTK2B SGVSEPLSRVKLGTLRRPEGPAEPM
  • TSPAN14 GVPFSCCVPDPAQKVVNTQCGYDVRIQ
  • ADAM10 FSDEFKVETSNKVLDYDTSHIYTGH
  • TREM2 PLRLLILLFVTELSGAHNTTVFQG;
  • VKORC1 KAARARDRDYRALCDVGTAISCSRV;
  • fragments, variants, fusions or derivatives thereof, or the fusions of the fragments, variants or derivatives thereof retain the activity of specifically binding to ADAP autoantibodies of SEQ ID NO. 1-10.
  • the variants include those obtained by adding and/or replacing one or more amino acids to the antigen polypeptide.
  • the variant contains at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the amino acid sequence shown in any one of SEQ ID NO. 1-10 %, 96%, 97%, 98% or 99% homology to the amino acid sequence, purity >95%, pH >7.0.
  • amino acid includes the standard 20 genetically encoded amino acids and their corresponding "D" form stereoisomers (as compared to the natural "L” form), omega-amino acids, other naturally occurring amino acids, unconventional Amino acids (for example, ⁇ , ⁇ -disubstituted amino acids, N-hydrocarbyl amino acids, etc.) and chemically derivatized amino acids.
  • amino acid refers to both L-alanine and D-alanine, unless expressly stated otherwise.
  • amino acid refers to both L-alanine and D-alanine, unless expressly stated otherwise.
  • Other non-conventional amino acids may also be suitable components of the polypeptides of the invention, as long as the polypeptide retains the desired functional properties.
  • each encoded amino acid residue is (where appropriate) represented by a one-letter name corresponding to the conventional amino acid common name.
  • “Variants” of a polypeptide include insertions, deletions, and substitutions, which are either conservative or non-conservative.
  • a conservative substitution refers to the substitution of an amino acid within the same general class (eg, acidic amino acid, basic amino acid, non-polar amino acid, polar amino acid, or aromatic amino acid) with another amino acid within the same class.
  • conservative and non-conservative amino acid substitutions is well known in the art.
  • variants of polypeptides that exhibit activity that specifically binds to ADAP autoantibodies are included.
  • the derivatives include modification products of the antigen polypeptide, wherein the modifications include but are not limited to amination modification, methylation modification, amidation modification, hydroxylation modification, carboxylation modification, carbonylation modification, alkyl modification, etc.
  • PEGylated proteins can exhibit reduced renal clearance and proteolysis, reduced toxicity, reduced immunogenicity, and increased solubility.
  • PEG molecules can vary, and PEG variants that have been used for protein PEGylation include PEG and monomethoxy-PEG. Additionally, they can be either linear or branched.
  • PEG can be coupled at naturally occurring disulfide bonds, as described in WO 2005/007197. Disulfide bonds can be stabilized via the addition of chemical bridges that do not damage the polypeptide structure. This allows the conjugation thiol selectivity of the two sulfurs constituting the disulfide bond to be exploited to create a bridge for site-specific attachment of PEG. Thus, the need to engineer residues into the peptide to attach to the target molecule is circumvented.
  • the antigen polypeptide includes the TREM2 antigen polypeptide shown in SEQ ID NO. 9.
  • the antigen polypeptide includes the VKORC1 antigen polypeptide shown in SEQ ID NO. 10.
  • fusion comprising the antigen polypeptide or a fragment, variant or derivative thereof, or a fusion of the fragment, variant or derivative thereof, or a fluorescent label, his-tag, CPP, connecting peptide.
  • a "fusion" of a polypeptide includes an amino acid sequence corresponding to a reference sequence (e.g., SEQ ID NOs. 1-10, or fragments or variants thereof) fused to any other polypeptide.
  • the polypeptide can be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A to facilitate purification of the polypeptide. Examples of such fusions are well known to those skilled in the art.
  • the polypeptide may be fused to an oligohistidine tag such as His6 or an epitope recognized by an antibody such as the well-known Myc tag epitope.
  • fusions containing hydrophobic oligopeptide terminal tags can be used. Also included within the scope of the present invention are fusions with any variant or derivative of the polypeptide.
  • Fusions may contain additional moieties that confer desired characteristics to the polypeptide of the invention; for example, such moieties may be used to detect or isolate the polypeptide, or to promote cellular uptake of the polypeptide.
  • the moiety may be, for example, a biotin moiety, a streptavidin moiety, a radioactive moiety, a fluorescent moiety, such as a small fluorophore or a green fluorescent protein (GFP) fluorophore, as is well known to those skilled in the art.
  • GFP green fluorescent protein
  • the module may be an immunogenic tag, such as a Myc tag, as is known to those skilled in the art, or may be a lipophilic molecule or polypeptide domain capable of promoting cellular uptake of the polypeptide, as is known to those skilled in the art.
  • composition comprising the antigen polypeptide or a fragment, variant, fusion or derivative thereof, or a fusion of the fragment, variant or derivative thereof, or comprising Fluorescent tag, his-tag, CPP, linker peptide.
  • the composition contains the antigen polypeptide shown in SEQ ID NO.1-10 or SEQ ID NO.1-8 or its fragment, variant, fusion or derivative,
  • the composition contains the antigen polypeptide shown in SEQ ID NO.9 or its fragment, variant, fusion or derivative, or at least one of the antigen polypeptide shown in SEQ ID NO.1-8 and SEQ ID NO.10.
  • the composition contains the antigen polypeptide shown in SEQ ID NO. 10 or its fragment, variant, fusion or derivative, or at least one antigen polypeptide or its fragment shown in SEQ ID NO. 1-9 , variants, fusions or combinations of derivatives.
  • composition which includes the antigen polypeptide or the fusion or the composition.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable buffer, carrier or excipient.
  • Such pharmaceutically acceptable buffers, carriers or excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th Edition, edited by A.R Gennaro, Mack Publishing Company (1990) and the handbook of Pharmaceutical Excipients, 3rd Edition , edited by A. Kibbe, Pharmaceutical Press (2000).
  • buffer is intended to mean an aqueous solution containing an acid-base mixture for the purpose of stabilizing the pH.
  • buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate , borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazole lactic acid, PIPES, SSC , SSPE, POPSO, TAPS, TABS, TAPSO and TES.
  • Carriers according to the present invention include antimicrobial agents, isotonic agents, antioxidants, local anesthetics, suspending agents, dispersing agents, emulsifiers, chelating agents, thickeners or solubilizers.
  • Excipients may be one or more of the following: carbohydrates, polymers, lipids and minerals.
  • carbohydrates include lactose, sucrose, mannitol, and cyclodextrin, which are added to the composition, for example to facilitate lyophilization.
  • polymers are starch, cellulose ethers, cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, ethylcellulose, hydrolyzed to varying degrees , methylcellulose, propylcellulose, alginates, carageenans, hyaluronic acid and its derivatives, polyacrylic acid, polysulphonate, polyethylene glycol/ Polyethylene oxide, polyethylene oxide/polypropylene oxide copolymer, polyvinyl alcohol/polyvinyl acetate, poly(lactic acid), poly(glycolic acid) or their copolymers with various compositions, and polyvinylpyrrolidone ( They all have different molecular weights), which are added to the composition, for example to control viscosity, achieve bioadhesion, or protect the active ingredient from chemical and proteolytic degradation.
  • lipids are fatty acids, phospholipids, mono-, di- and triglycerides, ceramides, sphingolipids and glycolipids (all with different acyl chain lengths and degrees of saturation), lecithin (egg lecithin), soy lecithin, hydrogenated Lecithin and soy lecithin, which are added to the composition for similar reasons as polymers.
  • lecithin egg lecithin
  • soy lecithin soy lecithin
  • hydrogenated Lecithin and soy lecithin which are added to the composition for similar reasons as polymers.
  • minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduced liquid accumulation or favorable pigment properties.
  • compositions may also contain one or more monosaccharides or disaccharides, such as xylitol, sorbitol, mannitol, lactitol, isomalt, maltitol or xyloside, and/or Monoacylglycerols, such as monolaurin.
  • monosaccharides or disaccharides such as xylitol, sorbitol, mannitol, lactitol, isomalt, maltitol or xyloside
  • Monoacylglycerols such as monolaurin.
  • the characteristics of the carrier depend on the route of administration.
  • One route of application is topical application.
  • one preferred carrier is an emulsified emulsion containing the active peptide, but other common carriers such as certain petrolatum/mineral-based and plant-based ointments, and polymeric gels may be used , liquid crystal phase and microemulsion.
  • compositions of the invention may also be in the form of liposomes in which the polypeptide is combined with an amphipathic agent such as a lipid present in aggregated form as micelles, insoluble monolayers and liquid crystals, in addition to other pharmaceutically acceptable carriers.
  • an amphipathic agent such as a lipid present in aggregated form as micelles, insoluble monolayers and liquid crystals, in addition to other pharmaceutically acceptable carriers.
  • Lipids suitable for liposome formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponins, bile acids, and the like.
  • compositions of the present invention may also be in the form of biodegradable microspheres.
  • Aliphatic polyesters such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly(carprolactone) have been widely used in the generation of microspheres. (PCL), and polyanhydrides as biodegradable polymers.
  • PLA poly(lactic acid)
  • PGA poly(glycolic acid)
  • PCL polyanhydrides
  • the preparation of such microspheres can be found in US 5,851,451 and EP 213 303.
  • compositions of the present invention may also be in the form of polymer gels, wherein polymers such as starch, cellulose ethers, cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, hydrolyzed to varying degrees are used.
  • polymers such as starch, cellulose ethers, cellulose, carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, hydrolyzed to varying degrees are used.
  • Cellulose ethylhydroxyethylcellulose, ethylcellulose, methylcellulose, propylcellulose, alginate, chitosan, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, Polyvinylimidazole, polysulfonate, polyethylene glycol/polyoxyethylene, polyoxyethylene/polypropylene oxide copolymer, polyvinyl alcohol/polyvinyl acetate, and polyvinylpyrrolidone are used to thicken solutions containing peptides.
  • the polymer may also contain gelatin or collagen.
  • polypeptides of the invention can be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum and/or various buffers. Dissolve in liquid.
  • compositions of the present invention may be administered topically or systemically.
  • Routes of administration include topical, ocular, nasal, pulmonary, buccal, parenteral (intravenous, subcutaneous, and intramuscular), oral, vaginal, and rectal. Administration of self-implants is also possible.
  • Suitable preparation forms are, for example, granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, microemulsions (which are defined as optically distinct emulsions consisting of water, oil and surfactants).
  • liquid crystal phases which are defined as systems characterized by long-range order but short-range disorder (examples include lamellar, hexagonal and cubic phases, or water or oil continuous), or their dispersed counterparts, gels, ointments, dispersions, suspensions, creams, aerosols, injectable solutions in the form of droplets or ampoules, but also preparations with extended release of the active compound in which In the formulation, excipients, diluents, or carriers are typically used, as described above.
  • the pharmaceutical composition may also be provided in a bandage, cast, or suture, or the like.
  • compositions of the present invention must be sterile and stable under the conditions of manufacture and storage.
  • the preferred methods of preparation are vacuum drying and freeze-drying, which produce the active ingredient from previously sterile-filtered solutions of the active ingredient and other desired ingredients. ingredients and other desired ingredients.
  • the compositions of the invention may be in solution and appropriate pharmaceutically acceptable excipients may be added and/or mixed prior to or upon delivery to provide an injectable unit dosage form.
  • pharmaceutically acceptable excipients used in the present invention are suitable for high drug concentrations, can maintain appropriate flowability, and, if necessary, can delay absorption.
  • the pharmaceutical composition is suitable for pulmonary or nasal administration.
  • compositions of the present invention may be administered intranasally or by inhalation, and are conveniently delivered as a dry powder inhaler or an aerosol spray by use of a suitable propellant, such as dichlorodichloride.
  • a suitable propellant such as dichlorodichloride.
  • Fluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3, 3-Heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gases are supplied from pressurized containers, pumps, sprays or atomizers.
  • the dosage unit may be determined by providing a valve to deliver the metered Dosage.
  • Pressurized containers, pumps, sprays or nebulizers may contain a solution or suspension of the active compound, for example using ethanol and a propellant mixture as solvent, which may additionally contain a lubricant, for example, sorbitan trioleate.
  • Inhalers Or capsules and cartridges (eg, made of gelatin) for use in insufflators may be formulated to contain a powder mixture containing a polypeptide of the invention and a suitable powder base such as lactose or starch.
  • the pharmaceutical composition will be administered to the patient in a pharmaceutically effective dose.
  • “Pharmaceutically effective dose” means a dose sufficient to produce the desired effect with respect to the condition for which it is administered. The precise dosage will depend on the activity of the compound, the mode of administration, the nature and severity of the condition, the age and weight of the patient, and different dosages may be required. Dosage administration can be effected by a single administration in the form of individual dosage units (otherwise, several smaller dosage units) and also by multiple administrations of subdivided doses at specific time intervals.
  • the polypeptides of the invention can be prepared with carriers that will protect them against rapid release (such as a controlled release formulation), including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation such as a controlled release formulation
  • Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used in the present invention.
  • the polypeptide may be coated with, or administered concurrently with, materials or compounds that prevent inactivation of the polypeptide.
  • the polypeptide can be administered with an appropriate carrier such as liposomes or diluents.
  • Oral dosage forms may be formulated as tablets, troches, dragees, aqueous or oily suspensions, dispersed powders or granules, emulsions, hard capsules, soft gel capsules, syrups or elixirs, pills, dragees, liquids, gels or ointment.
  • These preparations may contain pharmaceutical excipients, including but not limited to: granulating and disintegrating agents, binding agents, lubricants, preservatives, coloring agents, flavorings or sweeteners, vegetable oils or minerals Oils, humectants, and thickeners.
  • Preparations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile non-toxic injection or perfusion solutions or suspensions.
  • the solution or suspension may include agents such as 1,3-butanediol, Ringer's solution, Hank's solution, isotonic solution, etc. in doses and concentrations that are non-toxic to the recipient.
  • agents such as 1,3-butanediol, Ringer's solution, Hank's solution, isotonic solution, etc. in doses and concentrations that are non-toxic to the recipient.
  • nucleic acid molecules encoding the antigen polypeptides are also provided.
  • nucleic acid sequence encoding the polypeptide of the present invention can be obtained entirely through chemical synthesis.
  • the nucleic acid sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the sequence of the polypeptide of the invention by chemical synthesis.
  • nucleic acid encoding a polypeptide includes nucleic acids comprising a sequence encoding a polypeptide of the invention, particularly a polypeptide having the amino acid sequence set forth in SEQ ID NOs. 1-10.
  • the term also includes nucleic acids that comprise a single contiguous region or multiple discontinuous regions encoding the polypeptide (e.g., due to integrating phage, integrating insert sequence, integrating vector sequence, integrating transposon sequence or due to RNA Polynucleotides interrupted by editing or reconstruction of genomic DNA) as well as additional regions that may also include coding and/or non-coding sequences.
  • a recombinant vector containing the nucleic acid molecule is also provided.
  • vector refers to a non-chromosomal nucleic acid containing an intact replicon such that when placed within a permissive cell, the vector can be replicated, such as by a transformation process.
  • the vector can replicate in one cell type (e.g., bacteria) but has a limited ability to replicate in another cell type (e.g., mammalian cells).
  • Vectors can be viral or non-viral.
  • Exemplary non-viral vectors for delivering nucleic acids include naked DNA; and DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and DNA complexes containing cationic polymers Particles of condensed DNA (such as heterogeneous polylysine, fixed-length oligopeptides, and polyethylenimine) are also contained in liposomes in some cases.
  • condensed DNA such as heterogeneous polylysine, fixed-length oligopeptides, and polyethylenimine
  • Vectors used to construct the recombinant vector of the present invention include (but are not limited to) MarEx expression vector produced by Celltrion Inc. (South Korea); pCDNA vectors widely available on the market; F, R1, RP1, Col, pBR322, ToL, Ti Vector; cosmid; phage, such as ⁇ phage, ⁇ -shaped phage, M13 phage, Mu phage, P1 phage, P22 phage, Q ⁇ phage, T-even phage, T2 phage, T4 phage, T7 phage, etc.; plant viruses. Any of a variety of vectors known to those skilled in the art may be used in the present invention, and the choice of vector depends on the nature of the selected cells.
  • the introduction of vectors into cells can be achieved by (but not limited to) calcium phosphate transfection, viral infection, DEAE-dextran mediated transfection, lipofection or electroporation, and any person skilled in the art can Select and use an introduction method appropriate for the vector and cells used.
  • the above-mentioned vector contains one or more selectable markers, but is not limited thereto, and a vector that does not contain a selectable marker can also be used.
  • selectable marker may depend on the cells selected (as is known to those skilled in the art), but this is not critical to the invention.
  • cells containing the nucleic acid or the recombinant vector are also provided.
  • the cells include prokaryotic cells and eukaryotic cells.
  • the prokaryotic cells include bacterial cells.
  • the eukaryotic cells include protist cells, animal cells, plant cells, and fungal cells.
  • the animal cells include mammalian cells, avian cells, and insect cells.
  • the antigen polypeptide or the fusion or the composition or the nucleic acid molecule or the recombinant vector or the cell are used in preparing reagents for detecting ADAP autoantibodies or application in the kit.
  • antigen polypeptide or the fusion or the composition or the nucleic acid molecule or the recombinant vector or the cells and/or ADAP autoantibodies are used in the preparation of Alzheimer's disease. Application in early diagnostic reagents or kits.
  • the test sample is serum or plasma.
  • a reagent or kit for detecting ADAP autoantibodies and/or Alzheimer's disease including the antigen polypeptide or the fusion or the composition or the Nucleic acid molecule or said recombinant vector or said cell.
  • the antigen polypeptide or the fusion or the composition is coated in an enzyme plate.
  • the coating concentration of the antigen polypeptide or the fusion or the composition in the enzyme plate is 5.0 ⁇ g/ml.
  • it also includes at least one of a coating solution, a positive control, a negative control, a washing buffer, a sample dilution analysis solution, a secondary antibody standard solution, a stop buffer, and a substrate chromogenic solution.
  • Polypeptides useful in the present invention may be prepared using any suitable means known in the art. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means and methods for preparing such polypeptides are well known in the art.
  • the polypeptide is or comprises a recombinant polypeptide.
  • Methods suitable for generating such recombinant polypeptides are well known in the art, such as expression in prokaryotic or eukaryotic cells (see, e.g., Sambrook and Russell, 2000, Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor, New York, the relevant disclosures of which are incorporated herein by reference).
  • Polypeptides of the present invention may also be produced using commercial in vitro translation systems, such as rabbit reticulocyte lysate or wheat germ lysate (obtained from Promega) to produce polypeptides of the present invention.
  • the translation system is rabbit reticulocyte lysate.
  • the translation system can be coupled to a transcription system, such as the TNT transcription-translation system (Promega). This system has the advantage of generating appropriate mRNA transcripts from the encoding DNA polynucleotide in the same reaction as translation.
  • polypeptides of the invention can be prepared by any of a variety of techniques.
  • the polypeptide can be produced by cell culture techniques, including production of the polypeptide by conventional techniques, or by transfection of the nucleic acid molecule of the polypeptide into a suitable bacterial or mammalian cell host to allow production of the polypeptide, wherein the polypeptide can be recombinant of.
  • transfection are intended to include various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc.
  • polypeptides of the present invention may be expressed in prokaryotic or eukaryotic cells, expression of the polypeptides in eukaryotic cells is preferred, and expression in mammalian cells is most preferred because such eukaryotic cells (especially mammalian cells) are more likely to Than prokaryotic cells assemble and secrete correctly folded polypeptides.
  • a recombinant expression vector for a nucleic acid molecule encoding a polypeptide is introduced into a mammalian cell, the polypeptide is cultured by culturing the cell for a period of time sufficient to allow expression of the polypeptide in the cell or, more preferably, secretion of the polypeptide into the medium in which the cell is cultured.
  • Polypeptides can be recovered from the culture medium using standard protein purification methods.
  • antigen polypeptide or the fusion or the composition or the pharmaceutical composition or the nucleic acid molecule or the recombinant vector or the cell or ADAP itself is provided.
  • an in vitro ADAP autoantibody detection and/or Alzheimer's disease diagnosis method including using the antigen polypeptide or the fusion or the composition or the kit for detection.
  • ADAP autoantibodies in the sample preferably, the detection method is for non-diagnostic purposes.
  • the method for detecting the formation of a complex containing the antigen polypeptide or the fusion or the composition includes Kjeldahl method, fluorescence detection technology, isotope tracing method, and chemiluminescence.
  • Detection method electrophoresis method, colorimetric method, enzyme-linked immunosorbent assay, chromatography, electrochemical method, chromatography technology, western blot analysis, immunohistochemistry, biuret method, Folin-phenol reagent method, OPA method (O-phthalaldehyde method), acid-base titration method, UV absorption method, UV graduation spectrometry, potency determination method, mass spectrometry and/or high-pressure liquid chromatography, time-of-flight mass spectrometry (MALDI-TOF) method, nuclear magnetic resonance H spectrum and C spectrum of wave spectroscopy.
  • MALDI-TOF time-of-flight mass spectrometry
  • polypeptide refers to the ability to bind to (eg, immunoreact with) a given target (eg, ADAP autoantibody).
  • a polypeptide may be monospecific and contain one or more binding sites that specifically bind a target, or a polypeptide may be multispecific and contain two or more binding sites that specifically bind the same or different targets.
  • the present invention has the following beneficial effects:
  • the present invention uses ADAP antigen polypeptide to detect specific autoantibodies in the serum and plasma of AD patients, and the reaction has high specificity and sensitivity.
  • This autoantibody can be used as an immune marker to evaluate the risk of Alzheimer's disease. and the progression of cognitive impairment associated with dementia.
  • This antigenic polypeptide and its antibody can be used to prepare early diagnostic reagents for Alzheimer's disease and develop targeted drugs to treat the disease.
  • the amino acid sequence contained in the antigen polypeptide is a simple linear polypeptide amino acid sequence. Compared with the existing recombinant protein antigens, the acquisition cost is significantly reduced, and the binding specificity to ADAP autoantibodies is strong.
  • Figure 8 ROC curve of joint diagnosis of 8 ADAP autoantibodies
  • the antigenic polypeptide containing the amino acid sequence shown in SEQ ID NO.1-10 contains specific antigenic determinants, which enables it to specifically recognize and bind to the ADAP autoantibody antigen-binding site, thereby improving the interaction between the above-mentioned antigenic polypeptide and the sample to be tested.
  • the specific reaction between the ADAP autoantibodies contained in it and improve the specific binding rate of the two. In this way, after specifically identifying and combining it with the ADAP autoantibodies in the sample to be tested, such as serum or plasma, the content level of ADAP autoantibodies in the sample to be tested can be detected. By judging the content level of the autoantibodies, This can indirectly determine the risk and progression of Alzheimer's disease in the source of the sample to be tested.
  • ADAP autoantibodies may be ADAP-specific auto-IgG antibodies.
  • amino acid sequences shown in SEQ ID NO.1-10 contained in the above-mentioned antigen polypeptides are simple linear polypeptide amino acid sequences. Compared with the antigens of existing recombinant proteins, the acquisition cost is significantly reduced, and the combination with ADAP autoantibodies is Strong specificity. In view of this, while disclosing candidate detection epitopes, the present invention also improves the detection means of ELISA.
  • the inventors followed the following principles to design linear polypeptide antigens: 1) Select the surface area of cell membrane proteins; 2) Select sequences that do not form ⁇ -helix; 3) Avoid duplication within the protein; 4) Avoid peptides with strong homology; 5) Must Contains restricted epitopes of the human leukocyte class II antigen (HLA-II) system.
  • the present invention uses bioinformatics prediction methods combined with multiple epitope prediction simulation software to comprehensively analyze parameters related to antigenicity, and designs linear amino acid polypeptide sequences as shown in SEQ ID NO. 1-10, corresponding to 10 kinds of ADAP autoantibodies, the designed antigen polypeptides are completely complementary to the target antibodies in terms of spatial structure and configuration.
  • the sequence of SORL1 antigen protein (NP_003096.2sortilin-related receptor preproprotein [Homo sapiens]) is shown below as SEQ ID NO.11.
  • the boxed part is the antigen polypeptide fragment and its position in the SORL1 protein:
  • TMEM163 antigen protein (NP_112185.1 transmembrane protein 163 [Homo sapiens]) is shown below as SEQ ID NO.12.
  • the boxed part is the antigen polypeptide fragment and its position in the TMEM163 protein:
  • SPRED2 antigen protein (NP_861449.2 sprouty-related, EVH1 domain-containing protein 2 isoform a [Homo sapiens]) is shown below as SEQ ID NO.13.
  • the boxed part is the antigen polypeptide fragment and its position in the SPRED2 protein :
  • the sequence of the CLU antigen protein (NP_001822.3 clusterin isoform 1 preproprotein [Homo sapiens]) is shown below as SEQ ID NO.14.
  • the boxed part is the antigen polypeptide fragment and its position in the CLU protein:
  • PTK2B antigen protein AAH42599.1 PTK2B protein tyrosine kinase 2 beta [Homo sapiens]
  • SEQ ID NO.15 The sequence of PTK2B antigen protein (AAH42599.1 PTK2B protein tyrosine kinase 2 beta [Homo sapiens]) is shown below as SEQ ID NO.15.
  • the boxed part is the antigen polypeptide fragment and its position in the PTK2B protein:
  • TSPAN14 antigen protein (NP_001121781.1 tetraspanin-14 isoform 2 [Homo sapiens]) is shown below as SEQ ID NO.16.
  • the boxed part is the antigen polypeptide fragment and its position in the TSPAN14 protein:
  • NP_006823.1 fermitin family homolog 2 isoform 1 [Homo sapiens] The sequence of the FERMT2 antigen protein (NP_006823.1 fermitin family homolog 2 isoform 1 [Homo sapiens]) is shown below in SEQ ID NO.17.
  • the boxed part is the antigen polypeptide fragment and its position in the FERMT2 protein:
  • ADAM10 antigen protein (NP_001101.1 disintegrin and metalloproteinase domain-containing protein 10 isoform 1 preproprotein [Homo sapiens]) is as follows SEQ ID NO.18.
  • the boxed part is the antigen polypeptide fragment and its position in the ADAM10 protein:
  • TREM2 antigen protein (NP_061838.1 triggering receptor expressed on myeloid cells 2 precursor isoform 1 precursor [Homo sapiens]) is as follows: SEQ ID NO.19.
  • the boxed part is the antigen polypeptide fragment and its position in the TREM2 protein:
  • VKORC1 antigen protein NP_001298240.1 vitamin K epoxide reductase complex subunit 1 isoform 3 precursor [Homo sapiens]
  • SEQ ID NO. 20 The sequence of the VKORC1 antigen protein (NP_001298240.1 vitamin K epoxide reductase complex subunit 1 isoform 3 precursor [Homo sapiens]) is shown in SEQ ID NO. 20.
  • the framed part is the antigen polypeptide fragment and its position in the VKORC1 protein:
  • the antigen polypeptide can also be chemically modified to increase the antigenicity of the ADAP antigen polypeptide and facilitate the coating process of the polypeptide.
  • the above-mentioned antigen polypeptide can be obtained through chemical synthesis or through genetic engineering technology. This technology is well known to those skilled in the art. Those skilled in the art can understand that the above-mentioned ADAP antigen polypeptide can be synthesized effectively through conventional synthesis methods, thereby replacing the biosynthesis method of recombinant expression.
  • Subject information 56 peripheral blood samples from AD patients were collected and matched with 56 samples from the healthy group; the samples were matched by gender and age and were comparable (P>0.05). All subjects were enrolled after passing MMSE cognitive assessment and 11 C-labeled Pittsburgh Compound-B (PiB) PET test. Basic demographic information is shown in Table 1.
  • Table 1 Basic demographic information of subjects
  • ELISA plate design Each plasma sample has two replicate wells for human ADAP antigen peptide, two replicate wells for goat peptide control antigen (PC), and two replicate wells for negative control (NC).
  • PC antigen has no homology with the human proteome, the purpose is to reduce the interference of nonspecific binding reactions, and the working concentration range of PC is 10-20 ⁇ g/ml.
  • the antigen polypeptide is coated on a 96-well enzyme plate (COSTAR, USA) with a coating solution (pH 7.4 0.01M PBS/0.1% NaN 3 ), 100 ⁇ l per well.
  • the coating concentration of ADAP antigen polypeptide is 5.0 ⁇ g/ml
  • the coating concentration of PC antigen is 15 ⁇ g/ml, overnight at 4°C.
  • Plasma incubation Wash each well 3 times with 0.01M PBS/0.005% TWEEN-20, dilute the plasma 1:500 with analysis solution (0.01M PBS+1% BSA), and leave 100 ⁇ l per well overnight at 4°C.
  • Detection Add 50 ⁇ l 10% H 2 SO 4 to each well as the reaction stop solution, and use a microplate reader (BioTeck EL x800, USA) to detect the OD value within 10 minutes.
  • the detection wavelength is 450 nm and the reference wavelength is 630 nm.
  • the rank sum test was used to compare the differences between different groups.
  • the average SBI value was ⁇ 2 times the variance (SD) and the first-class error level a ⁇ 0.05 was used as the positive judgment threshold.
  • the diagnostic performance was evaluated using the receiver characteristic curve (ROC). Pearson correlation was used to analyze the correlation between IgG antibody levels and subjects' cognitive scores.
  • the ROC curve is drawn based on a series of different binary classification methods (cut-off value or judgment threshold), with the true positive rate (sensitivity) as the ordinate, the false positive rate (1-specificity) as the abscissa, and the area under the curve (AUC ) to determine diagnostic efficacy.
  • the AUC value range is 0.5-1.0. The closer it is to 1, the stronger the diagnostic performance.
  • Graphpad Prism 7.0 Graphpad Software, United States
  • the antibody level at 95% specificity was used as the positive threshold to determine the sensitivity. greater than 20%.
  • the above data fully demonstrate that there is a statistically significant difference between the autoantibody IgG levels of AD patients detected using the antigen polypeptides designed in the present invention and that of normal healthy controls.
  • Multi-target joint diagnosis can effectively improve the diagnostic efficacy of biomarkers in diseases.
  • the present invention fits the detection values of 8 plasma anti-ADAP IgG antibodies (SORL1, TMEM163, SPRED2, CLU, PTK2B, TSPAN14, FERMT2, ADAM10) by establishing a logistic regression linear model.
  • the area under the curve AUC of the ROC analysis after fitting was 0.89 (see Figure 8).
  • the antibody level at 95% specificity as the positive threshold was 73.2%, and the diagnostic performance was much higher than that of single-target applications.
  • Table 2 Comparative analysis results of ADAP auto-IgG antibody detection in plasma of AD patients and healthy controls

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Abstract

用于检测阿尔兹海默病疾病相关蛋白(Alzheimer's Disease Associated Protein,ADAP)自身抗体的抗原多肽及其应用，属于免疫学技术领域。提供了10条ADAP抗原多肽分别对应10种ADAP自身抗体。应用ADAP抗原多肽检测阿尔兹海默病患者外周血中相应的特异性自身抗体，这种自身抗体可作为免疫标志物评估阿尔兹海默病的发病风险和痴呆相关的认知损害发展程度。这种抗原多肽及其抗体可用于制备阿尔兹海默病早期诊断试剂和开发治疗该疾病的靶向药物。

Description

用于检测阿尔兹海默病自身抗体的蛋白抗原组合及其应用 技术领域
本发明涉及免疫学技术领域，具体涉及用于检测ADAP自身抗体的抗原多肽及其应用。
背景技术
阿尔兹海默病(AD)是老年人痴呆的最常见原因，其在全球范围内发病率逐年上升。但至今为止，基于认知评定的临床方式仍是AD临床上鉴别诊断的最主要方式。淀粉样蛋白-β(Aβ)假说被广泛认为是AD最重要的发病机制。据信，Aβ异常积累到细胞外毒性斑块中是AD中神经变性和导致痴呆的重要基础。随着技术的进步，近年来涌现了一批围绕Aβ展开的AD新型诊断技术，如Aβ_PET-CT、脑脊液的Aβ水平测定、Aβ超高灵敏度质谱等。但这些技术存在一定的局限性：1)都是在Aβ沉积产生并积累一段时间后才能进行检测，这意味着由Aβ引起的神经退行性病变已经发生且不可逆转，无法做到疾病早期的风险预警；2)基于PET-CT或超高分辨率质谱等检测方式前期投入巨大，传导至受试者身上的单位检测价格很高，难以做到应检尽检；3)脑脊液检测需进行腰椎穿刺，受试者痛苦较大，且存在一定手术风险，因此临床上接受度不高。
研究证据表明，生物体的免疫机制在Aβ的清除中起重要作用。全基因组关联研究(GWAS)已经确定了130多个与AD相关的易感性位点，这些遗传发现提示了先天性和适应性免疫在AD发病机制中的作用，并表明细胞介导的Aβ清除的系统性失能促进了AD的发病和进展。围绕Aβ产生和积累过程中相关信号通路或生物学过程上关键靶点的自身免疫反应影响了Aβ的清除效率，在AD相关的神经退行性病变中发挥了关键作用。人体内源性蛋白抗原诱导产生的抗体被称为自身抗体，能够识别内源性蛋白分子，引起一系 列免疫级联反应。在人体大脑中，小胶质细胞是清除Aβ的关键细胞。小胶质细胞与外周巨噬细胞同源，保留了较强的免疫应答功能。有研究表明，一些内源性蛋白分子的自身抗体能够激活小胶质细胞的吞噬功能。国外一些著名药企已开展AD相关的疫苗和治疗单抗临床研究(如罗氏的Aβ单抗crenezumab等)，但由于关注点都集中于Aβ本身，靶点选择面较窄，实际效果并不尽如人意。
本发明涉及10个阿尔兹海默病疾病相关蛋白(Alzheimer’s Disease Associated Protein,ADAP)自身抗体(SORL1，TMEM163，SPRED2，CLU，PTK2B，TSPAN14，FERMT2，ADAM10，TREM2，VKORC1)：
Sortilin(SORL1)是一种在人类中由1号染色体上的SORT1基因编码的蛋白质。该蛋白是分选受体的液泡蛋白分选10蛋白(Vps10)家族中的一种I型膜糖蛋白。SORL1在许多组织中普遍表达，但在中枢神经系统中最为丰富。在细胞水平上，SORL1在高尔基体、内体、溶酶体和质膜之间的蛋白质转运中发挥作用。这种分子功能使SORL1能够参与各种生物过程，包括响应胰岛素将GLUT4转运到脂肪和骨骼肌细胞的质膜；介导proNGF和p75NTR:sortilin复合物之间的相互作用，通过充当共同受体来发出细胞死亡信号；对神经元存活所必需的脑源性神经营养因子(BDNF)具有精细调节功能。在AD中，SORL1是Vps10家族中首个被GWAS证明是迟发性AD风险基因的成员，其在迟发性AD和轻度认知障碍患者的大脑中表达水平降低。SORL1是逆转录酶的货物受体，能够将APP蛋白从内质网系统和BACE蛋白高活性的环境中转运至反面高尔基体进行重新修饰，也能直接调控APP的寡聚化来调控其蛋白水解过程。SORL1表达水平或活性的降低能够促进Aβ的积累。
TMEM163锌离子跨膜转运蛋白，是散发AD的一个GWAS风险基因。生物学功能上，TMEM163负责神经元突触上锌离子的内外流过程，包括参与突触小泡的释放。
SPRED2属于出芽蛋白家族Sprouty/SPRED family成员，参与由生长因子诱导的MAPK激酶级联过程的激活。遗传学上，SPRED2被证明是散发性AD的一个风险基因。本发明通过自行设计的SPRED2抗原表位多肽，检测 AD患者血清及血浆中自身抗体水平并开发响应试剂，预测AD发生风险及病程发展程度，为临床治疗提供可靠参考。
CLU是脑内仅次于载脂蛋白APOE的第二大脂蛋白，是散发性AD的GWAS风险基因。生物学功能上，CLU可能参与β-淀粉样蛋白在脑与血浆之间的转运，在AD的致病机制中有助于调节大脑中β-淀粉样蛋白的清除。而β-淀粉样蛋白产生和清除的失衡是阿尔兹海默病发展的核心因素。
PTK2B是一种细胞质蛋白酪氨酸激酶，参与钙诱导的离子通道调节和激活图谱激酶信号通路。PTK2B作为代表增加钙通量的神经肽激活受体或神经递质之间的重要信号中间体，以及调节神经元活性的下游信号。其响应于细胞内钙浓度、烟碱乙酰胆碱受体激活、膜去极化或蛋白激酶C活化的增加，而经历快速的酪氨酸磷酸化和活化。在遗传学上，PTK2B是散发性AD的GWAS风险基因。磷酸化的PTK2B可与AD患者及转基因Tau鼠脑中磷酸化的Tau共定位，参与Tau的磷酸化和聚集等过程，是Tau毒性的早期标记物和体内调节剂。
TSPAN14是一个细胞膜表面四次跨膜蛋白，是散发AD的一个GWAS风险基因。生物学功能上，TSPAN14能够影响细胞膜内侧相关信号通路激酶的活性，包括对Notch信号通路的激活调控功能等。分布上，TSPAN14高表达于中枢神经系统和外周免疫细胞当中，具有调节神经免疫机制的功能。
FERMT2是细胞膜表面的粘着斑蛋白，是散发AD的一个GWAS风险基因。生物学功能上，FERMT2参与调控细胞骨架微管蛋白的结合活性，影响磷脂酰肌醇的结合活性，以及转化生长因子β受体的活性。在生物学过程中，FERMT2通过影响多个细胞表面受体参与的信号通路，参与如细胞分化、细胞局部合成等重要事件。FERMT2的功能调控异常在AD的疾病发展中起了关键作用。
ADAM10基因编码一个含有去整合素和金属蛋白酶结构域的蛋白质分子，是散发AD的一个GWAS风险基因，家族性AD中也发现多个ADAM10基因的功能性突变。在神经元中，ADAM10是最重要的酶之一，具有α-分泌酶活性，可用于淀粉样前体蛋白的蛋白水解加工。ADAM10与ADAM17一起切割骨髓细胞2(TREM2)上表达的触发受体的胞外域，产生可溶性 TREM2(sTREM2)，是脑内Aβ42清除的关键分子之一。ADAM10的功能调控异常在AD的疾病发展中起了关键作用。
TREM2是一个髓系细胞表面应激表达受体，是散发AD的一个GWAS风险基因，家族性AD中也发现多个TREM2基因的功能性突变。生物学功能上，该基因编码一种与TYRO蛋白酪氨酸激酶结合蛋白形成受体信号复合物的膜蛋白，免疫反应中起作用，并且可能通过触发组成性炎性细胞因子的产生而参与慢性炎症。在大脑中，TREM2是小胶质细胞监视并响应神经退行性病变信号的关键受体之一。在动物实验中，发现TREM2的抗体能够抑制小胶质细胞的炎性功能亢进状态，并修复其正常的吞噬功能。TREM2的功能调控异常在AD的疾病发展中起了关键作用。
VKORC1编码维他命K氧化还原酶复合物亚基，是散发及家族性AD的一个GWAS风险基因。生物学功能上，VKORC1蛋白负责在内质网膜中将非活性维生素K 2,3-环氧化物还原为活性维生素K。维生素K是凝血酶中维生素K依赖性γ-羧化酶对谷氨酸残基进行羧化所必需的辅助因子。
本发明通过自行设计的抗原表位多肽，检测AD患者血清及血浆中自身抗体水平并开发响应试剂，预测AD发生风险及病程发展程度，为临床治疗提供可靠参考。
发明内容
抗原抗体的结合实际上只发生在抗原决定簇和抗体的抗原结合位点之间，二者在空间结构和空间构型上完全互补。因此抗原决定簇就可以代表整个蛋白与抗体结合的状态与亲和特征。传统酶联免疫法(ELISA)检测自身抗体主要以重组蛋白为抗原，需经过载体构建、转染、表达、筛选、纯化等一系列繁琐过程，生产成本昂贵。且完整蛋白空间结构复杂、抗原表位不易暴露，因此抗原抗体结合的特异性不强，导致信号检出率低。
基于此，有必要提供了检测阿尔兹海默病标志物ADAP自身抗体的抗原多肽。
为实现上述目的，本发明具体技术方案如下：
首先，提供了检测阿尔兹海默病标志物ADAP自身抗体的抗原多肽，所述抗原多肽包含如SEQ ID NO.1-10所示的氨基酸序列中的至少一种：
SORL1：CEVWTQRLHGGSAPLPQDRGFLVVQGDPR；
TMEM163：CIVVKAIHDLSTRLLPEVDDFLF；
SPRED2：MTEETHPDDDSYIVRVKAVVMTR；
CLU：TQGEDQYYLRVTTVASHTSDSDVPSG；
PTK2B：SGVSEPLSRVKLGTLRRPEGPAEPM；
TSPAN14：GVPFSCCVPDPAQKVVNTQCGYDVRIQ；
FERMT2：GIRMPDGCYADGTWELSVHVTDLNR；
ADAM10：FSDEFKVETSNKVLDYDTSHIYTGH；
TREM2：PLRLLILLFVTELSGAHNTTVFQG；
VKORC1：KAARARDRDYRALCDVGTAISCSRV；
或其片段、变体、融合物或衍生物、或所述其片段、变体或衍生物的融合物。
所述的其片段、变体、融合物或衍生物、或所述其片段、变体或衍生物的融合物保留SEQ ID NO.1-10的与ADAP自身抗体特异性结合的活性。
所述变体包括所述的抗原多肽经一个或多个氨基酸添加和/或替换后获得的变体。
优选的，所述变体包含与SEQ ID NO.1-10任一所示的氨基酸序列有至少55％，60％，65％，70％，75％，80％，85％，90％，95％，96％，97％，98％或99％同源性的氨基酸序列，纯度＞95％，pH＞7.0。
术语“氨基酸”包括标准的20种遗传编码的氨基酸及其相应的“D”形式的立体异构体(与天然的“L”形式相比)、Ω-氨基酸、其它天然存在的氨基酸、非常规的氨基酸(例如，α,α-双取代氨基酸、N-烃基氨基酸等)和经化学衍 生化的氨基酸。
在明确列举氨基酸，诸如“丙氨酸”或“Ala”或“A”时，术语指L-丙氨酸和D-丙氨酸两者，除非另有明确规定。其它非常规的氨基酸也可以是适合于本发明多肽的组分，只要多肽保留期望的功能性特性。对于显示的肽，每个编码的氨基酸残基(在适当的情况中)由对应于常规氨基酸俗名的单字母名称代表。
多肽的“变体”包括插入、缺失和取代，其或是保守的或是非保守的。例如，保守取代指将相同通用类别(例如酸性氨基酸、碱性氨基酸、非极性氨基酸、极性氨基酸或芳香族氨基酸)内的氨基酸用相同类别内的另一种氨基酸取代。如此，保守氨基酸取代和非保守氨基酸取代的意义是本领域中公知的。特别地，包括展现出可与ADAP自身抗体特异性结合的活性的多肽的变体。
所述衍生物包括所述的抗原多肽的修饰产物，其中，所述的修饰包括但不限于氨基化修饰、甲基化修饰、酰胺化修饰、羟基化修饰、羧基化修饰、羰基化修饰、烷基化修饰、乙酰化修饰、磷酸化修饰、硫酸化修饰、酯化修饰、糖基化修饰、环化修饰、生物素化修饰、荧光基团修饰、聚乙二醇PEG修饰、豆蔻酰化修饰、非金属化学元素修饰、固定化修饰。
如本领域中领会的，PEG化的蛋白质可以展现出降低的肾清除和蛋白水解、降低的毒性、降低的免疫原性和升高的溶解度。
为了获得具有最大延长的半衰期和保留的生物学活性的成功PEG化的蛋白质，可以影响后果的几项参数是重要的，并且应当进行考虑。PEG分子可以存在不同，并且已经用于蛋白质PEG化的PEG变体包括PEG及单甲氧基-PEG。另外，它们可以或是线性的或是分支的。
已经显示了提高PEG化的程度导致体内半衰期延长。然而，本领域技术人员应当领会，PEG化过程会需要以个体为基础对特定蛋白质进行优化。
PEG可以在天然存在的二硫键处偶联，如记载于WO 2005/007197的。可以经由添加不损害多肽结构的化学桥来稳定二硫键。这允许利用构成二硫 键的两个硫的缀合硫醇选择性来创建用于对PEG进行位点特异性附接的桥。由此，避开了需要将残基工程化改造到肽中以附接于靶分子。
优选的，所述抗原多肽包含SEQ ID NO.9所示的TREM2抗原多肽。
优选的，所述抗原多肽包含SEQ ID NO.10所示的VKORC1抗原多肽。
进一步地，提供了一种融合物，所述融合物包含所述的抗原多肽或其片段、变体或衍生物、或所述其片段、变体或衍生物的融合物，或包含荧光标记、his-tag、CPP、连接肽。
多肽的“融合物”包括对应于与任何其它多肽融合的参照序列(例如SEQ ID NO.1-10，或其片段或变体)的氨基酸序列。例如，所述多肽可以与多肽诸如谷胱甘肽-S-转移酶(GST)或蛋白A融合以便于纯化所述多肽。此类融合物的例子是本领域技术人员公知的。类似地，所述多肽可以与寡组氨酸标签诸如His6或被抗体识别的表位诸如公知的Myc标签表位融合。另外，可以使用包含疏水性寡肽末端标签的融合物。本发明的范围中还包括与所述多肽的任何变体或衍生物的融合物。
融合物可以包含对本发明的所述多肽赋予期望特征的别的部分；例如，所述部分可用于检测或分离多肽，或促进多肽的细胞摄取。所述部分可以例如是生物素模块、链霉亲合素模块、放射性模块、荧光模块，例如小荧光团或绿色荧光蛋白(GFP)荧光团，如本领域技术人员公知的。模块可以是免疫原性标签，例如Myc标签，如本领域技术人员已知的，或者可以是能够促进多肽的细胞摄取的亲脂性分子或多肽域，如本领域技术人员已知的。
进一步地，提供了一种组合物，所述组合物包含所述的抗原多肽或其片段、变体、融合物或衍生物、或所述其片段、变体或衍生物的融合物，或包含荧光标记、his-tag、CPP、连接肽。
优选的，所述组合物包含SEQ ID NO.1-10或SEQ ID NO.1-8所示的抗原多肽或其片段、变体、融合物或衍生物，
优选的，所述组合物包含SEQ ID NO.9所示的抗原多肽或其片段、变体、融合物或衍生物，或与SEQ ID NO.1-8、SEQ ID NO.10所示的至少一条抗原多肽或其片段、变体、融合物或衍生物的组合，
优选的，所述组合物包含SEQ ID NO.10所示的抗原多肽或其片段、变体、融合物或衍生物，或与SEQ ID NO.1-9所示的至少一条抗原多肽或其片段、变体、融合物或衍生物的组合。
进一步地，提供了一种药物组合物，所述药物组合物包括所述的抗原多肽或所述的融合物或所述的组合物。
优选的，所述的药物组合物还包括药学上可接受的缓冲液、载体或赋形剂。
此类药学可接受缓冲液、载体或赋形剂是本领域中公知的(见Reming to n’s Pharmaceutical Sciences,第18版,A.R Gennaro编,Mack Publishing Company(1990)及handbook of Pharmaceutical Excipients,第3版,A.Kibbe编,Pharmaceutical Press(2000)。
术语“缓冲液”意图指以稳定pH为目的的含有酸-碱混合物的水溶液。缓冲液的例子是Trizma、Bicine、Tricine、MOPS、MOPSO、MOBS、Tris、Hepes、HEPBS、MES、磷酸盐、碳酸盐、乙酸盐、柠檬酸盐、乙醇酸盐(glycolate)、乳酸盐、硼酸盐、ACES、ADA、酒石酸盐、AMP、AMPD、AMPSO、BES、CABS、卡可酸盐(cacodylate)、CHES、DIPSO、EPPS、乙醇胺、甘氨酸、HEPPSO、咪唑、咪唑乳酸、PIPES、SSC、SSPE、POPSO、TAPS、TABS、TAPSO和TES。
本发明所述的载体包括抗微生物剂、等渗试剂、抗氧化剂、局部麻醉剂、悬浮剂、分散剂、乳化剂、螯合剂、增稠剂或增溶剂。
赋形剂可以是下列一种或多种：碳水化合物、聚合物、脂质和矿物。碳水化合物的例子包括乳糖、蔗糖、甘露醇、和环状糊精，其被添加至组合物，例如以便于冻干。聚合物的例子是不同程度水解的淀粉、纤维素醚、纤维素、 羧甲基纤维素、羟丙基甲基纤维素、羟乙基纤维素、乙基羟乙基纤维素、乙基纤维素、甲基纤维素、丙基纤维素、藻酸盐(alginates)、角叉菜胶(carageenans)、透明质酸及其衍生物、聚丙烯酸、聚磺酸盐(polysulphonate)、聚乙二醇/聚氧化乙烯、聚氧化乙烯/聚环氧丙烷共聚物、聚乙烯醇/聚乙烯乙酸酯、聚(乳酸)、聚(乙醇酸)或具有各种组成的其共聚物、及聚乙烯吡咯烷酮(它们都具有不同分子量)，其被添加至组合物，例如以控制粘性，实现生物粘着，或保护活性成分免于化学和蛋白水解降解。脂质的例子是脂肪酸、磷脂、甘油单、二和三酯、神经酰胺、鞘脂和糖脂(均具有不同酰基链长度和饱和度)、卵磷脂(egg lecithin)、大豆卵磷脂、氢化的卵磷脂和大豆卵磷脂，其出于与聚合物相似的原因被添加至组合物。矿物的例子是滑石、氧化镁、氧化锌和氧化钛，其被添加至组合物以获得诸如降低液体积累或有利的色素特性等益处。
药物组合物也可以含有一种或多种单糖或二糖，诸如木糖醇、山梨糖醇、甘露醇、乳糖醇、异麦芽酮糖醇(isomalt)、麦芽糖醇或木糖苷、和/或单酰甘油，诸如甘油一月桂酸酯(monolaurin)。
载体的特征取决于施用路径。一种施用路径是表面施用。例如，对于表面施用，一种优选的载体是含有活性肽的乳化乳剂，但是可以使用其它常见的载体诸如某些基于矿脂(petrolatum)/矿物的和基于植物的软膏剂，以及聚合物凝胶、液体晶相和微乳。
本发明的药物组合物也可以为脂质体形式，其中在其它药学可接受载体外，组合多肽与以聚集形式作为微团、不溶性单层和液态晶体存在的两亲剂(amphipathic agent)诸如脂质。适合于脂质体配制剂的脂质包括但不限于甘油一酯、甘油二酯、硫苷脂、溶血卵磷脂、磷脂、皂苷、胆汁酸等。
本发明的药物组合物也可以为生物可降解微球形式。已经在微球的生成中广泛使用脂肪族聚酯诸如聚(乳酸)(PLA)、聚(乙醇酸)(PGA)、PLA和PGA的共聚物(PLGA)或聚(己内酯(carprolactone))(PCL)、和聚酸酐作为生物可降 解聚合物。此类微球的制备可参见US 5,851,451和EP 213 303。
本发明的药物组合物也可以为聚合物凝胶形式，其中使用聚合物诸如不同程度水解的淀粉、纤维素醚、纤维素、羧甲基纤维素、羟丙基甲基纤维素、羟乙基纤维素、乙基羟乙基纤维素、乙基纤维素、甲基纤维素、丙基纤维素、藻酸盐、壳聚糖、角叉菜胶、透明质酸及其衍生物、聚丙烯酸、聚乙烯咪唑、聚磺酸盐、聚乙二醇/聚氧化乙烯、聚氧化乙烯/聚环氧丙烷共聚物、聚乙烯醇/聚乙烯乙酸酯和聚乙烯吡咯烷酮来增稠含有肽的溶液。聚合物也可以包含明胶或胶原。
或者，可以将本发明的多肽在盐水、水、聚乙二醇、丙二醇、乙醇或油(诸如红花籽油、玉米油、花生油、棉籽油或芝麻油)、黄蓍胶和/或各种缓冲液中溶解。
本领域技术人员应当领会，可以局部或系统施用本发明的药物组合物。施用路径包括表面、眼、鼻、肺、含服、胃肠外(静脉内、皮下、和肌肉内)、口服、阴道和直肠。自植入物的施用也是有可能的。合适的制剂形式是例如颗粒剂、粉末、片剂、包衣片剂、(微)胶囊、栓剂、糖浆剂、乳剂、微乳(其定义为由水、油和表面活性剂组成的光学上各向同性的热力学稳定性体系)、液晶相(其定义为以长程有序但短程无序为特征的体系(例子包括薄片状、六边形和立方体相，或是水或是油连续的)、或其分散的对应物、凝胶、软膏剂、分散体、悬浮液、乳膏、气雾剂、微滴或安瓿形式的可注射溶液，而且还有具有活性化合物的延长释放的制剂，在该制剂中，通常使用赋形剂、稀释剂、或载体，如上文所描述的。药物组合物也可以在绷带、石膏或缝合线等中提供。
本发明的药物组合物在制造和储存条件下必须是无菌和稳定的。在用于制备无菌可注射溶液的无菌粉末的情况下，制备的优选方法是真空干燥和冷冻干燥，真空干燥和冷冻干燥从活性成分和其它期望成分的预先无菌过滤过的溶液产生活性成分和其它期望成分的粉末。可选择地，本发明的组合物可 在溶液中，且在递送之前或递送时可加入和/或混合适当的药学上可接受的赋形剂以提供可注射的单位剂型。优选地，本发明中使用的药学上可接受的赋形剂适用于高药物浓度，可保持适当的流动性，且如果需要可延迟吸收。
在优选实施方案中，药物组合物适合于肺施用或鼻施用。
例如，可以鼻内或通过吸入来施用本发明的药物组合物，并且方便地以干粉末吸入剂或气雾剂喷雾形式投递，所述气雾剂喷雾通过使用合适的推进剂，例如二氯二氟甲烷、三氯氟-甲烷、二氯四氟-乙烷、氢氟代烷诸如1,1,1,2-四氟乙烷(HFA 134A3或1,1,1,2,3,3,3-七氟丙烷(HFA 227EA3)、二氧化碳或其-它合适的气体自加压容器、泵、喷雾或喷雾器提供。在加压气雾剂的情况中，可以通过提供阀来决定剂量单位以投递计量的剂量。加压容器、泵、喷雾或喷雾器可以含有活性化合物的溶液或悬浮液，例如使用乙醇和推进剂混合物作为溶剂，其可以另外含有滑润剂，例如，山梨聚糖三油酸酯。吸入器或吹入器中使用的胶囊和筒(例如由明胶制成)可以配制成含有本发明多肽和合适的粉末基底诸如乳糖或淀粉的粉末混合物。
药物组合物会以药学有效剂量对患者施用。“药学有效剂量”意指就其施用所针对的状况而言足以产生期望的效果的剂量。精确的剂量取决于化合物的活性、施用方式、病症的性质和严重性、患者的年龄和体重，可能需要不同剂量。可以通过以个别剂量单位(否则，几个更小的剂量单位)形式的单次施用和还通过特定时间间隔的细分剂量的多次施用来实施剂量施用。
本发明的药物组合物的最佳给药途径的选择会受到几个因素的影响，包含组合物中活性分子的物理化学性质、临床表现的紧迫性和活性分子的血浆浓度与期望的治疗效果之间的关系。例如，可与载剂一起制备本发明的多肽，其中载剂将保护它们以防止快速释放(诸如控释制剂)，该载剂包含植入物、透皮贴剂和微胶囊化的递送系统。可在本发明中使用生物可降解的、生物相容的聚合物，诸如乙烯醋酸乙烯酯、聚酸酐、聚乙醇酸、胶原、聚正酯和聚乳酸。进一步地，多肽可包被有防止多肽失活的材料或化合物、或与这样的 材料或化合物同时给药。例如，多肽可与适当的载剂(例如脂质体或稀释剂)一起给药。
口服剂型可被配制成片剂、锭剂、糖锭、水性或油性悬浮液、分散的粉末或颗粒、乳剂、硬胶囊、软胶胶囊、糖浆或酏剂、丸剂、糖衣丸、液体、凝胶或膏剂。这些制剂可包含药学赋形剂，其中药学赋形剂包括但并不限于：成粒剂和崩解剂，结合剂，润滑剂，防腐剂，着色剂、调味剂或甜味剂，植物油或矿物油，湿润剂，和增稠剂。
由于胃肠外给药的制剂可为水性或非水性的等渗无菌无毒的注射或灌注溶液或悬浮液的形式。所述溶液或悬浮液可包括所采用的剂量和浓度对接受者无毒的试剂，诸如1，3-丁二醇、林格氏溶液(Ringer’s solution)、汉克溶液(Hank’s solution)、等渗的氯化钠溶液、油、脂肪酸、局部的麻醉剂、防腐剂、缓冲液、粘度或溶解性增高的试剂、水溶性的抗氧化剂、油溶性的抗氧化剂和金属螯合剂。
进一步地，还提供了编码所述抗原多肽的核酸分子。
目前，已经可以完全通过化学合成来得到编码本发明的多肽的核酸序列。然后可将该核酸序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明的多肽的序列中。
本文所用的术语“编码多肽的核酸”包括包含编码本发明多肽、尤其是具有SEQ ID NO.1-10中所示氨基酸序列的多肽的序列的核酸。所述术语也包括这样的核酸：所述核酸包含编码所述多肽的单个连续区或多个不连续区(例如，由于整合噬菌体、整合插入序列、整合载体序列、整合转座子序列或由于RNA编辑或基因组DNA重建而间断的多核苷酸)以及额外的区，所述额外的区也可包含编码序列和/非编码序列。
进一步地，还提供了含有所述核酸分子的重组载体。
如本文所用，术语“载体”是指包含完整复制子的非染色体核酸，使得当置于允许的细胞内时，载体可以被复制，例如通过转化过程。载体可以在 一种细胞类型(例如细菌)中复制，但是在另一种细胞(例如哺乳动物细胞)中复制的能力有限。载体可以是病毒的或非病毒的。用于递送核酸的示例性非病毒载体包括裸露的DNA；和单独或与阳离子聚合物结合的与阳离子脂质复合的DNA；阴离子和阳离子脂质体；DNA-蛋白质复合物和包含与阳离子聚合物(如异质聚赖氨酸，定长寡肽和聚乙烯亚胺)缩合的DNA的颗粒，在某些情况下还包含在脂质体中。
构建本发明的重组载体的载体包括(但不限于)由Celltrion Inc.(韩国)生产的MarEx表达载体；市场上可广泛买到的pCDNA载体；F、R1、RP1、Col、pBR322、ToL、Ti载体；粘粒；噬菌体，诸如λ噬菌体、λ形噬菌体、M13噬菌体、Mu噬菌体、P1噬菌体、P22噬菌体、Qμ噬菌体、T-偶数噬菌体、T2噬菌体、T4噬菌体、T7噬菌体等；植物病毒。本发明中可使用本领域技术人员已知的各种载体的任意一种，且载体的选择依赖于所选择的细胞的性质。细胞中载体的导入可通过(但并不限于)磷酸钙转染、病毒感染、DEAE-葡聚糖介导的转染、脂质体转染或电穿孔实现，且本领域的任何技术人员可选择和使用适用于所用的载体和细胞的导入方法。
优选的，上述载体包含一种或多种选择标记，但并不限于此，且还可使用不包含选择标记的载体。选择标记的选择可依赖于选择的细胞(如本领域的技术人员公知的)，但这对于本发明并不是关键性的。
进一步地，还提供了包含所述核酸或所述重组载体的细胞。
优选的，所述细胞包括原核细胞、真核细胞。
优选的，所述原核细胞包括细菌细胞。
优选的，所述真核细胞包括原生生物细胞、动物细胞、植物细胞、真菌细胞。
优选的，所述动物细胞包括哺乳动物细胞、禽类细胞、昆虫细胞。
进一步地，提供了所述的抗原多肽或所述的融合物或所述的组合物或所 述的核酸分子或所述的重组载体或所述的细胞在制备用于检测ADAP自身抗体的试剂或试剂盒中的应用。
进一步地，所述的抗原多肽或所述的融合物或所述的组合物或所述的核酸分子或所述的重组载体或所述的细胞和/或ADAP自身抗体在制备阿尔兹海默病早期诊断试剂或试剂盒中的应用。
优选的，检测样本为血清或血浆。
进一步地，还提供了一种用于检测ADAP自身抗体和/或阿尔兹海默病的试剂或试剂盒，包括所述的抗原多肽或所述的融合物或所述的组合物或所述的核酸分子或所述的重组载体或所述的细胞。
优选的，所述抗原多肽或所述的融合物或所述的组合物是被包被于酶标板中。
优选的，所述抗原多肽或所述的融合物或所述的组合物在酶标板中包被浓度为5.0μg/ml。
优选的，还包括包被液、阳性对照、阴性对照、洗涤缓冲液、样品稀释分析液、二抗标准液、终止缓冲液、底物显色液中的至少一种。
可以使用本领域已知的任何合适的方式制备可用于本发明的多肽。此类多肽包括分离的天然存在的多肽、重组产生的多肽、合成产生的多肽、或通过这些方法的组合产生的多肽。用于制备此类多肽的手段和方法是本领域熟知的。
便利地，多肽是或包含重组多肽。适合于生成此类重组多肽的方法是本领域中公知的，诸如在原核或真核细胞中表达(例如见Sambrook和Russell,2000,Molecular Cloning,A Laboratory Manual,第三版,Cold Spring Harbor,New York,在此通过提及而收录该文件的相关公开内容)。
也可以使用商品化的体外翻译系统来生成本发明所述的多肽，诸如兔网织红细胞裂解物或小麦胚芽裂解物(获自Promega)来生成本发明的多肽。优选 地，翻译系统是兔网织红细胞裂解物。便利地，翻译系统可以与转录系统偶联，诸如TNT转录-翻译系统(Promega)。此系统具有在与翻译相同的反应中自编码DNA多核苷酸生成合适的mRNA转录物的优点。
本发明的多肽可以通过多种技术中的任一种制备。通常，多肽可以通过细胞培养技术产生，包括通过常规技术产生多肽，或通过将多肽的核酸分子转染到合适的细菌或哺乳动物细胞宿主中，以允许多肽的产生，其中所述多肽可以是重组的。术语“转染”的各种形式意在包括通常用于将外源DNA引入原核或真核细胞的各种技术，例如电穿孔，磷酸钙沉淀，DEAE-葡聚糖转染等。尽管可以在原核或真核细胞中表达本发明的多肽，但是优选在真核细胞中表达多肽，并且最优选在哺乳动物细胞中表达，因为这种真核细胞(特别是哺乳动物细胞)更可能比原核细胞组装和分泌正确折叠的多肽。当将编码多肽的核酸分子的重组表达载体引入哺乳动物细胞时，通过将细胞培养足以允许多肽在细胞中表达的一段时间，或更优选地，将多肽分泌至培养细胞的培养基。可以使用标准蛋白纯化方法从培养基中回收多肽。
进一步地，还提供了所述的抗原多肽或所述的融合物或所述的组合物或所述的药物组合物或所述的核酸分子或所述的重组载体或所述的细胞或ADAP自身抗体在制备预防或治疗阿尔兹海默病药物中的应用。
进一步地，还提供了一种体外ADAP自身抗体检测和/或阿尔兹海默病诊断方法，包括使用所述的抗原多肽或所述的融合物或所述的组合物或所述的试剂盒检测样品中的ADAP自身抗体，优选的，所述检测方法为非诊断目的。
优选的，包括以下步骤：
1)将所述样品与所述的抗原多肽或所述的融合物或所述的组合物接触；
(2)检测包含所述的抗原多肽或所述的融合物或所述的组合物的复合物的形成；
优选的，所述的检测包含所述的抗原多肽或所述的融合物或所述的组合物的复合物的形成的方法包括凯氏定氮法、荧光检测技术、同位素示踪法、化学发光检测法、电泳法、比色法、酶联免疫吸附剂测定法、色谱法、电化学法、层析技术、免疫印迹分析、免疫组织化学，双缩脲法、Folin—酚试剂法、OPA法(邻苯二甲醛法)、酸碱滴度法、紫外吸收法、紫外分度光谱法、效价测定法、质谱和/或高压液相色谱、飞行时间质谱(MALDI-TOF)法、核磁共振波谱法的H谱和C谱。
术语“特异性”是指结合(例如，与之免疫反应)给定靶标(例如ADAP自身抗体)的能力。多肽可以是单特异性的并且含有一个或多个特异性结合靶标的结合位点，或者多肽可以是多特异性的并且含有两个或更多个特异性结合相同或不同靶标的结合位点。
基于上述技术方案，本发明具有以下有益效果：
本发明应用ADAP抗原多肽检测到AD患者血清及血浆中的特异性自身抗体，并且该反应具有高特异性和高灵敏性，这种自身抗体可作为免疫标志物评估阿尔兹海默病的发病风险和痴呆相关的认知损害发展程度。这种抗原多肽及其抗体可用于制备阿尔兹海默病早期诊断试剂和开发治疗该疾病的靶向药物。抗原多肽所含的氨基酸序列为简单的线性多肽氨基酸序列，其对于现有重组蛋白的抗原而言，获得成本显著降低，而且与ADAP自身抗体的结合特异性强。
附图说明
图1 AD患者与健康对照其中8种血浆ADAP自身IgG抗体检测水平；
图2 AD患者与健康对照血浆TREM2自身IgG抗体检测水平；
图3 AD患者与健康对照血浆VKORC1自身IgG抗体检测水平；
图4其中8种血浆ADAP自身IgG抗体水平与受试者MMSE认知评分相关性；
图5血浆TREM2自身IgG抗体水平与受试者MMSE认知评分相关性；
图6血浆VKORC1自身IgG抗体水平与受试者MMSE认知评分相关性；
图7其中8种ADAP自身抗体单靶点诊断ROC曲线；
图8其中8种ADAP自身抗体联合诊断ROC曲线；
图9 AD患者与健康对照血浆TREM2自身IgG抗体检测ROC曲线；
图10 AD患者与健康对照血浆VKORC1自身IgG抗体检测ROC曲线；
图11 10种ADAP自身抗体联合诊断ROC曲线。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
下述实施例所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所有的材料、试剂等，如无特殊说明，均可从商业途径获得。
含有SEQ ID NO.1-10所示氨基酸序列的抗原多肽由于含有特定的抗原决定簇，使其能够与ADAP自身抗体抗原结合位点发生特异识别和结合，从而提高了上述抗原多肽与待测样本中所含的ADAP自身抗体之间的特异反应，并提高两者的特异结合率。这样，将其与待测样本——如血清或血浆——中的ADAP自身抗体特异识别和结合后，能够检测出待测样本中ADAP自身抗体的含量水平，通过对自身抗体的含量水平判断，从而间接判断待测样本来源体的阿尔兹海默病发病风险和进展。例如ADAP自身抗体水平降低预示患者可能出现了有ADAP介导的Aβ清除失能，可以预测AD的发生风险及发生进程，指导临床医生对AD的早期诊断。其中，ADAP自身抗体可以是ADAP的特异性自身IgG抗体。
另外，上述抗原多肽所含的SEQ ID NO.1-10所示氨基酸序列为简单的线性多肽氨基酸序列，其对于现有重组蛋白的抗原而言，获得成本显著降低，而且与ADAP自身抗体的结合特异性强。鉴于此，本发明在公开候选检测表 位的同时，亦对ELISA的检测手段进行改进。发明人遵循以下原则设计线性多肽抗原：1)选择细胞膜蛋白表面区域；2)选择不形成α-helix的序列；3)避免蛋白内部重复；4)避免同源性强的肽段；5)必须包含人白细胞二类抗原(HLA-II)系统的限制性表位。
基于上述原则，本发明运用生物信息学预测手段结合多个表位预测模拟软件，综合分析与抗原性相关的参数，设计了如SEQ ID NO.1-10所示的线性氨基酸多肽序列，分别对应10种ADAP自身抗体，所设计的抗原多肽在空间结构和构型上与目标抗体完全互补。其中SORL1抗原蛋白(NP_003096.2sortilin-related receptor preproprotein[Homo sapiens])的序列如下SEQ ID NO.11所示，加框部分是抗原多肽片段以及在SORL1蛋白中的位置：
Figure PCTCN2022124874-appb-000001
Figure PCTCN2022124874-appb-000002
Figure PCTCN2022124874-appb-000003
其中TMEM163抗原蛋白(NP_112185.1transmembrane protein 163[Homo sapiens])的序列如下SEQ ID NO.12所示，加框部分是抗原多肽片段以及在TMEM163蛋白中的位置：
Figure PCTCN2022124874-appb-000004
其中SPRED2抗原蛋白(NP_861449.2 sprouty-related,EVH1 domain-containing protein 2 isoform a[Homo sapiens])的序列如下SEQ ID NO.13所示，加框部分是抗原多肽片段以及在SPRED2蛋白中的位置：
Figure PCTCN2022124874-appb-000005
其中CLU抗原蛋白(NP_001822.3 clusterin isoform 1 preproprotein[Homo sapiens])的序列如下SEQ ID NO.14所示，加框部分是抗原多肽片段以及在CLU蛋白中的位置：
Figure PCTCN2022124874-appb-000006
Figure PCTCN2022124874-appb-000007
其中PTK2B抗原蛋白(AAH42599.1 PTK2B protein tyrosine kinase 2 beta[Homo sapiens])的序列如下SEQ ID NO.15所示，加框部分是抗原多肽片段以及在PTK2B蛋白中的位置：
Figure PCTCN2022124874-appb-000008
Figure PCTCN2022124874-appb-000009
其中TSPAN14抗原蛋白(NP_001121781.1 tetraspanin-14 isoform 2[Homo sapiens])的序列如下SEQ ID NO.16所示，加框部分是抗原多肽片段以及在TSPAN14蛋白中的位置：
Figure PCTCN2022124874-appb-000010
其中FERMT2抗原蛋白(NP_006823.1 fermitin family homolog 2 isoform 1[Homo sapiens])的序列如下SEQ ID NO.17所示，加框部分是抗原多肽片段以及在FERMT2蛋白中的位置：
Figure PCTCN2022124874-appb-000011
其中ADAM10抗原蛋白(NP_001101.1 disintegrin and metalloproteinase domain-containing protein 10 isoform 1 preproprotein[Homo sapiens])的序列如下SEQ ID NO.18所示，加框部分是抗原多肽片段以及在ADAM10蛋白中的位置：
Figure PCTCN2022124874-appb-000012
其中TREM2抗原蛋白(NP_061838.1 triggering receptor expressed on myeloid cells 2 precursor isoform 1 precursor[Homo sapiens])的序列如下SEQ ID NO.19所示，加框部分是抗原多肽片段以及在TREM2蛋白中的位置：
Figure PCTCN2022124874-appb-000013
其中VKORC1抗原蛋白(NP_001298240.1 vitamin K epoxide reductase complex subunit 1 isoform 3 precursor[Homo sapiens])的序列如下SEQ ID NO.20所示，加框部分是抗原多肽片段以及在VKORC1蛋白中的位置：
Figure PCTCN2022124874-appb-000014
在上文的基础上，本领域技术人员可以理解的是，还可以对所述的抗原多肽进行化学修饰，以便增加ADAP抗原多肽的抗原性和有助于多肽的包被处理。
上述抗原多肽可通过化学合成得到，也可以通过基因工程技术得到，此技术为本领域技术人员所熟知。本领域技术人员可以理解的是，可以有效地通过常规合成方法，合成上述ADAP抗原多肽，从而代替重组表达的生物合成方式。
实施例1
1.受试者信息：样本收集56份AD患者外周血样本，匹配健康组56份；性别、年龄匹配，具有可比性(P＞0.05)。所有受试者均经MMSE认知评定和 11C-labeled Pittsburgh Compound-B(PiB)PET检测后入组。基本人口学信息见表1。
表1：受试者基本人口学信息
Figure PCTCN2022124874-appb-000015
2.利用抗原多肽进行检测：
(1)酶标板设计：每份血浆样本各设人ADAP抗原多肽双复孔，山羊多肽对照抗原(PC)双复孔和阴性对照双复孔(NC)。PC抗原与人类蛋白质组无同源性，目的是减低非特异性结合反应的干扰，PC的工作浓度范围10-20μg/ml。
(2)包被：抗原多肽用包被液(pH 7.4 0.01M PBS/0.1％NaN 3)包被于96孔酶标板(COSTAR，美国)，每孔100μl。ADAP抗原多肽包被浓度5.0μg/ml，PC抗原包被浓度为15μg/ml，4℃过夜。
(3)血浆孵育：0.01M PBS/0.005％TWEEN-20清洗每孔3遍，利用分析液(0.01M PBS+1％BSA)将血浆1:500稀释，每孔100μl，4℃过夜。
(4)二抗孵育：0.01M PBS/0.005％TWEEN-20清洗每孔5遍，利用分析液(同前)稀释辣根过氧化物酶标记的羊抗人IgG(美国，Sigma)，每孔加200μl，25℃孵育2h。辣根过氧化物酶标记的羊抗人IgG ELISA工作范围：1:30000～1:50000。
(5)显色：0.01M PBS/0.005％TWEEN-20清洗每孔5遍，每孔加100μl3,3',5,5'-四甲基联苯胺(TMB)过氧化物酶底物(lnvtrogen,美国)，室温避光15～30min。
(6)检测：每孔加50μl 10％H 2SO 4为反应终止液，10min内使用酶标仪(BioTeck EL x800,美国)检测OD值，检测波长为450nm，参考波长为630nm。
(7)数据分析：采用SPSS或类似统计学软件进行数据统计分析处理。所有检测样品取双复孔均值进行计算。采用阳性样品比值(PSR)来判定抗原抗体的结合程度。PSR计算方法如下:
PSR＝[OD(ADAP)-OD(NC)]/[OD(PC)-OD(NC)]
获得各检测样本PSR数值后，采用秩和检验比较不同组别间的差异，以SBI平均值≥2倍方差(SD)，一类错误水平a<0.05为阳性判定阈值。采用受试者特征曲线(ROC)进行诊断效能评价。采用皮尔森相关性分析IgG抗体水平与受试者认知评分间的相关性。ROC曲线是根据一系列不同的二分类方式(分界值或判定阈值)，以真阳性率(灵敏度)为纵坐标，假阳性率(1- 特异性)为横坐标绘制，以曲线下面积(AUC)判定诊断效能。AUC取值范围为0.5-1.0，越趋近于1，表征诊断效能越强。本发明采用统计学计算分析软件Graphpad Prism 7.0(Graphpad Software，美国)软件绘制ROC曲线，计算曲线下面积(AUC)，以健康人PSR平均值-2SD判为阳性，判定灵敏度和特异度；进行Fisher's精确检验，检验一类错误水准为a＝0.05。
(8)质控：各检测样本设置双复孔，取平均OD值。OD值离散度判定：离散度＝(OD1-OD2)/(OD1+OD2)≤0.1，为有效结果；离散度>0.1，为无效结果。取100份健康人血浆等体积混合作为质控血(QC)，代表人群本底水平。每个检测板设置2个QC血浆孔，以QC变异水平判定结果的稳定性。批内变异CV＝每日各板QC孔SD值/每日各板QC孔均值×100％；批内变异需<10％。批间变异CV＝所有批次QC孔SD值/所有批次QC孔均值×100％；批间变异需<20％。
3.检测结果：
如表2和图1-3所示，AD患者血浆中与10种ADAP抗原多肽结合的IgG抗体阳性率均显著低于健康对照组(P<0.001)。如表3和图4-6所示，血浆抗ADAP IgG抗体水平与受试者MMSE认知评分呈现显著正相关关系(P<0.001)，即血浆抗ADAP IgG抗体可以预测受试者AD相关的痴呆表型发展程度相关，抗体水平越低，认识损害程度越高。如表4和图7、图9、图10所示，AD患者血浆抗ADAP IgG抗体ROC曲线下面积(AUC)均大于0.7，以95％特异性时抗体水平作为阳性阈值判定，得到的灵敏度均大于20％。以上数据充分表明，利用本发明所设计的抗原多肽检测得到的AD患者自身抗体IgG水平与正常健康对照组有显著性统计学差异。
多靶点的联合诊断能有效提升生物标志物在疾病中的诊断效力。本发明通过建立逻辑回归线性模型，将其中8个血浆抗ADAP IgG抗体(SORL1，TMEM163，SPRED2，CLU，PTK2B，TSPAN14，FERMT2，ADAM10)检测值进行拟合。拟合后ROC分析的曲线下面积AUC为0.89(见图8)，以95％特异性时抗体水平作为阳性阈值判定，得到的灵敏度为73.2％，诊断效能表现远高于单靶点的应用。将全部10个血浆抗ADAP IgG抗体(SORL1，TMEM163，SPRED2，CLU，PTK2B，TSPAN14，FERMT2，ADAM10，TREM2， VKORC1)检测值进行拟合。拟合后ROC分析的曲线下面积AUC为0.9825(见图11)，以95％特异性时抗体水平作为阳性阈值判定，得到的灵敏度为91.07％，诊断效能表现远高于所有单靶点的应用和以上8个靶点的联合诊断应用。
表2：AD患者与健康对照血浆ADAP自身IgG抗体检测对比分析结果
Figure PCTCN2022124874-appb-000016
表3：血浆ADAP自身IgG抗体水平与受试者MMSE认知评分相关性分析结果
Figure PCTCN2022124874-appb-000017
表4：AD患者与健康对照血浆ADAP自身IgG抗体检测ROC曲线分析结果
Figure PCTCN2022124874-appb-000018
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (16)

  1. 检测阿尔兹海默病标志物ADAP自身抗体的抗原多肽，其特征在于，所述抗原多肽包含如SEQ ID NO.1-10所示的氨基酸序列中的至少一种：
    SORL1：CEVWTQRLHGGSAPLPQDRGFLVVQGDPR；
    TMEM163：CIVVKAIHDLSTRLLPEVDDFLF；
    SPRED2：MTEETHPDDDSYIVRVKAVVMTR；
    CLU：TQGEDQYYLRVTTVASHTSDSDVPSG；
    PTK2B：SGVSEPLSRVKLGTLRRPEGPAEPM；
    TSPAN14：GVPFSCCVPDPAQKVVNTQCGYDVRIQ；
    FERMT2：GIRMPDGCYADGTWELSVHVTDLNR；
    ADAM10：FSDEFKVETSNKVLDYDTSHIYTGH；
    TREM2：PLRLLILLFVTELSGAHNTTVFQG；
    VKORC1：KAARARDRDYRALCDVGTAISCSRV；
    或其片段、变体、融合物或衍生物、或所述其片段、变体或衍生物的融合物。
  2. 根据权利要求1所述的抗原多肽，其特征在于，所述抗原多肽包含SEQ ID NO.9所示的TREM2抗原多肽。
  3. 根据权利要求1所述的抗原多肽，其特征在于，所述抗原多肽包含SEQ ID NO.10所示的VKORC1抗原多肽。
  4. 根据权利要求1-3任一所述的抗原多肽，其特征在于，所述衍生物包括权利要求1所述的抗原多肽的修饰产物，
    优选的，所述修饰包括氨基化修饰、甲基化修饰、酰胺化修饰、羟基化修饰、羧基化修饰、羰基化修饰、烷基化修饰、乙酰化修饰、磷酸化修饰、 硫酸化修饰、酯化修饰、糖基化修饰、环化修饰、生物素化修饰、荧光基团修饰、聚乙二醇PEG修饰、豆蔻酰化修饰、非金属化学元素修饰、固定化修饰，
    优选的，所述修饰为氨基化修饰。
  5. 根据权利要求1所述的抗原多肽，其特征在于，所述变体包括权利要求1所述的抗原多肽经一个或多个氨基酸添加和/或替换后获得的变体，
    优选的，所述变体包含与SEQ ID NO.1-10任一所示的氨基酸序列有至少55％，60％，65％，70％，75％，80％，85％，90％，95％，96％，97％，98％或99％同源性的氨基酸序列。
  6. 一种融合物，其特征在于，所述融合物包含权利要求1-3任一所述的抗原多肽或其片段、变体或衍生物、或所述其片段、变体或衍生物的融合物，或包含荧光标记、his-tag、CPP、连接肽。
  7. 一种组合物，其特征在于，所述组合物包含权利要求1-3任一所述的抗原多肽或其片段、变体、融合物或衍生物、或所述其片段、变体或衍生物的融合物，或包含荧光标记、his-tag、CPP、连接肽，
    优选的，所述组合物包含SEQ ID NO.1-10或SEQ ID NO.1-8所示的抗原多肽或其片段、变体、融合物或衍生物，
    优选的，所述组合物包含SEQ ID NO.9所示的抗原多肽或其片段、变体、融合物或衍生物，或与SEQ ID NO.1-8、SEQ ID NO.10所示的至少一条抗原多肽或其片段、变体、融合物或衍生物的组合，
    优选的，所述组合物包含SEQ ID NO.10所示的抗原多肽或其片段、变体、融合物或衍生物，或与SEQ ID NO.1-9所示的至少一条抗原多肽或其片段、变体、融合物或衍生物的组合。
  8. 一种药物组合物，其特征在于，所述药物组合物包括权利要求1-3任一所述的抗原多肽或权利要求6所述的融合物或权利要求7所述的组合物。
  9. 编码权利要求1-3任一所述抗原多肽的核酸分子。
  10. 含有权利要求9所述核酸分子的重组载体。
  11. 包含权利要求9所述核酸分子或权利要求10所述重组载体的细胞，
    优选的，所述细胞包括原核细胞、真核细胞，
    优选的，所述原核细胞包括细菌细胞，
    优选的，所述真核细胞包括原生生物细胞、动物细胞、植物细胞、真菌细胞，
    优选的，所述动物细胞包括哺乳动物细胞、禽类细胞、昆虫细胞。
  12. 根据权利要求1-3任一所述的抗原多肽或权利要求6所述的融合物或权利要求7所述的组合物或权利要求9所述的核酸分子或权利要求10所述的重组载体或权利要求11所述的细胞在制备用于检测ADAP自身抗体的试剂或试剂盒中的应用。
  13. 根据权利要求1-3任一所述的抗原多肽或权利要求6所述的融合物或权利要求7所述的组合物或权利要求9所述的核酸分子或权利要求10所述的重组载体或权利要求11所述的细胞和/或ADAP自身抗体在制备用于阿尔兹海默病早期诊断的试剂或试剂盒中的应用。
  14. 一种用于检测ADAP自身抗体和/或阿尔兹海默病的试剂或试剂盒，其特征在于，包括权利要求1-3任一所述的抗原多肽或权利要求6所述的融合物或权利要求7所述的组合物或权利要求9所述的核酸分子或权利要求10所述的重组载体或权利要求11所述的细胞。
  15. 根据权利要求1-3任一所述的抗原多肽或权利要求6所述的融合物或权利要求7所述的组合物或权利要求8所述的药物组合物或权利要求9所述的核酸分子或权利要求10所述的重组载体或权利要求11所述的细胞或ADAP自身抗体在制备预防和/或治疗阿尔兹海默病药物中的应用。
  16. 一种体外ADAP自身抗体检测和/或阿尔兹海默病诊断方法，其特征 在于，包括使用权利要求1-3任一所述的抗原多肽或权利要求6所述的融合物或权利要求7所述的组合物或权利要求14所述的试剂盒检测样品中的ADAP自身抗体，优选的，所述检测方法为非诊断目的，
    优选的，包括以下步骤：
    (1)将所述样品与权利要求1-3所述的抗原多肽或权利要求6所述的融合物或权利要求7所述的组合物接触；
    (2)检测包含权利要求1-3所述的抗原多肽或权利要求6所述的融合物或权利要求7所述的组合物的复合物的形成；
    优选的，所述的检测包含权利要求1-3所述的抗原多肽或权利要求6所述的融合物或权利要求7所述的组合物的复合物的形成的方法包括凯氏定氮法、荧光检测技术、同位素示踪法、化学发光检测法、电泳法、比色法、酶联免疫吸附剂测定法、色谱法、电化学法、层析技术、免疫印迹分析、免疫组织化学，双缩脲法、Folin—酚试剂法、OPA法(邻苯二甲醛法)、酸碱滴度法、紫外吸收法、紫外分度光谱法、效价测定法、质谱和/或高压液相色谱、飞行时间质谱(MALDI-TOF)法、核磁共振波谱法的H谱和C谱。
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