WO2023206609A1 - 针对p-tau 217的抗体及其用途 - Google Patents

针对p-tau 217的抗体及其用途 Download PDF

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WO2023206609A1
WO2023206609A1 PCT/CN2022/092435 CN2022092435W WO2023206609A1 WO 2023206609 A1 WO2023206609 A1 WO 2023206609A1 CN 2022092435 W CN2022092435 W CN 2022092435W WO 2023206609 A1 WO2023206609 A1 WO 2023206609A1
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antibody
antigen
binding fragment
sequence
seq
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PCT/CN2022/092435
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French (fr)
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赵颖俊
许华曦
张登虹
张云武
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厦门大学
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07—ORGANIC CHEMISTRY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
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    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present application belongs to the field of biomedical technology. More specifically, the present application relates to antibodies or antigen-binding fragments thereof that can specifically bind to p-tau 217 (tau protein phosphorylated at amino acid 217), as well as multispecific molecules containing the same. , pharmaceutical compositions and kits, this application also relates to the use of the antibody or its antigen-binding fragment in the preparation of kits or medicines.
  • AD Alzheimer’s disease
  • AD The main pathological features of AD include neuritic plaques formed by the deposition of ⁇ -amyloid protein (A ⁇ ) and neurofibrillary tangles (NFTs) formed by the accumulation of hyperphosphorylated tau protein, microglia and Activation of astrocytes, loss of synapses and neuronal death, etc.
  • Clinical symptoms include progressive memory decline, impaired executive function, and difficulty with daily activities. The early onset of AD often manifests as changes in thinking or unconscious behavior, memory impairment of new information, and changes in language dysfunction, etc. Patients with advanced AD will suffer from severe memory loss, hallucinations, disorientation and lack of self-care ability, which are ultimately life-threatening. Due to the complex causes of AD and the lack of clear pathological mechanisms, there is currently no intervention method that can prevent or reverse the progression of AD. It can only temporarily improve or slow down the development of symptoms. Therefore, it is urgent to find new clinical treatments.
  • AD pathogenesis hypotheses such as A ⁇ cascade hypothesis, cholinergic hypothesis, abnormal tau protein phosphorylation hypothesis, neuroinflammation hypothesis, metal ion disorder hypothesis, etc. Based on these hypotheses, many drug researchers have developed drugs targeting different pathways and conducted corresponding clinical trials, but with little success.
  • the anti-AD drugs currently on the market mainly include 4 categories, cholinesterase inhibitors (such as tacrine, donepezil, galantamine and rivastigmine, etc.), NMDA receptor antagonists (such as memantine), A ⁇ and tau Antibodies or inhibitors (such as Aducanumab, solanezumab and TPI-287, etc.) and intestinal flora modulators (such as GV-971).
  • cholinesterase inhibitors such as tacrine, donepezil, galantamine and rivastigmine, etc.
  • NMDA receptor antagonists such as memantine
  • a ⁇ and tau Antibodies or inhibitors such as Aducanumab, solanezumab and TPI-287, etc.
  • intestinal flora modulators such as GV-971
  • AD Alzheimer's disease
  • the current targets for AD treatment mainly include pathogenic A ⁇ and tau proteins.
  • a large number of drugs targeting A ⁇ failed in the clinical trial stage.
  • clinical trials of gamma secretase inhibitors did not show efficacy and were forced to stop due to increased incidences of skin cancer and infection.
  • Anti-A ⁇ vaccine AN1792 was discontinued due to serious adverse effects of aseptic meningitis. Therefore, researchers turned their attention to tau protein because it mediates neuronal dysfunction and death caused by A ⁇ .
  • Tau is a microtubule-related protein (encoded by the MAPT gene) involved in microtubule stability. It is mainly concentrated around neuronal axons.
  • tau protein-targeted vaccines small molecule tau aggregation inhibitors, antisense oligonucleotides targeting the gene encoding tau protein, and anti-tau protein antibodies.
  • Tau protein is an intracellular protein, but it can be discharged into the gaps between brain cells in free form or in the form of extracellular vesicles, where it can diffuse, leading to abnormal tau protein aggregation and irreversible damage.
  • Immunoantibody therapies bind to diffuse tau protein and slow or prevent neurodegeneration.
  • N-terminal epitopes such as AC Immune’s semorinemab (recognizes various forms of full-length tau protein), Biogen’s gosuranemab and AbbVie’s ABBV-8E12 (recognizes N-terminal epitopes).
  • AC Immune semorinemab
  • Biogen s gosuranemab
  • AbbVie AbbVie
  • ABBV-8E12 recognizes N-terminal epitopes
  • these antibodies did not specifically target the pathogenic tau protein (such as phosphorylated tau protein), failed to exert the maximum therapeutic effect, and most of them ended in clinical failure. Therefore, the development of antibodies against pathogenic tau protein is of great exploratory significance for the treatment of AD or other tau protein diseases.
  • AD Late intervention in AD is another important reason for poor AD treatment results, so early diagnosis of AD is particularly important.
  • Clinical diagnosis of AD is mainly based on clinical symptoms, neuropsychological tests, neuroimaging examinations, and cerebrospinal fluid testing.
  • a ⁇ positron emission tomography (amyoid-PET-CT) is the gold standard for AD diagnosis, but due to price, instrument and location constraints, it is not easy to conduct population screening.
  • neuropsychological tests are easily affected by factors such as the subject's life experience, education level, race, and gender, and are usually used as auxiliary testing methods.
  • the clinical symptoms of early-stage AD patients are not obvious and almost undetectable, making it easy to miss the best opportunity for diagnosis and treatment.
  • AD biomarker detection into routine screening of the general population is crucial for the prevention and treatment of AD.
  • Many countries in the world have included AD biomarker screening in AD prevention and treatment guidelines. Among them, changes in the content of AD-related biomarkers (A ⁇ 40, A ⁇ 42, t-tau, p-tau, etc.) in cerebrospinal fluid can more directly reflect neuronal damage and precede clinical symptoms by about 20 years.
  • AD-related biomarkers A ⁇ 40, A ⁇ 42, t-tau, p-tau, etc.
  • p-tau181 threonine phosphorylated tau protein at position 181
  • p-tau 217 showed a stronger correlation with tau-PET and neocortical A ⁇ plaque load, and was more accurate. Distinguish between AD and non-AD, while the sensitivity and specificity of p-tau 217 is greater than 90%.
  • plasma p-tau 217 combined with APOE genotyping and cognitive examination can also be used in AD risk assessment models.
  • phosphorylated tau at position 217 can be detected in the early stages of AD, and a large amount of phosphorylated tau at position 217 is detected in the insoluble fraction of brain tissue extracts from patients with AD or other tauopathies, targeting Phosphorylated tau protein at position 217 may be able to intervene in the disease process in the early stages of AD or other tau protein diseases, which is of great significance.
  • one of the main purposes of this application is to provide an antibody that specifically binds to p-tau 217 protein.
  • the present application also provides preparation methods and uses of the antibodies.
  • the anti-p-tau 217 protein antibodies of the present application can be used to detect, prevent and/or treat tau protein diseases, especially AD.
  • the application provides an antibody or an antigen-binding fragment thereof that specifically binds to p-tau 217 protein, the antibody or an antigen-binding fragment thereof comprising:
  • VH Heavy chain variable region containing the following three complementarity determining regions (CDRs):
  • VH CDR1 which consists of the following sequence: SEQ ID NO: 3, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or add) sequence,
  • VH CDR2 which consists of the following sequence: SEQ ID NO: 4, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or add) sequence, and
  • VH CDR3 which consists of the following sequence: SEQ ID NO: 5, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or add) sequence;
  • VL light chain variable region containing the following three complementarity determining regions (CDRs):
  • VL CDR1 which consists of the following sequence: SEQ ID NO: 6, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or add) sequence,
  • VL CDR2 which consists of the following sequence: SEQ ID NO: 7, or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions) or addition) sequence
  • VL CDR3 which consists of the following sequence: SEQ ID NO: 8, or has one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions).
  • substitutions of any one of (i)-(vi) are conservative substitutions.
  • the CDR of any of (i)-(vi) is defined according to the Kabat, Chothia or IMGT numbering system.
  • the CDR of any of (i)-(vi) is defined according to the IMGT numbering system.
  • the antibody or antigen-binding fragment thereof comprises the following 3 heavy chain CDRs: VH CDR1 as shown in SEQ ID NO:3, VH CDR2 as shown in SEQ ID NO:4, SEQ ID VH CDR3 shown in NO:5; and/or, the following 3 light chain CDRs: VL CDR1 shown in SEQ ID NO:6, VL CDR2 shown in SEQ ID NO:7, SEQ ID NO:8 VL CDR3 shown.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH Heavy chain variable region comprising an amino acid sequence selected from the following:
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain framework sequence and/or a light chain framework sequence derived from a human immunoglobulin.
  • the antibody or antigen-binding fragment thereof comprises: a VH having the sequence set forth in SEQ ID NO: 1 and a VL having the sequence set forth in SEQ ID NO: 2.
  • the antibody or antigen-binding fragment thereof comprises a constant region derived from a human immunoglobulin or a variant thereof.
  • the antibody or antigen-binding fragment thereof comprises:
  • CH heavy chain constant region
  • a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence from which it is derived (e.g. , substitution, deletion or addition of up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids. addition or any combination thereof); and/or
  • the heavy chain constant region is an IgG heavy chain constant region, such as an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region.
  • the light chain constant region is a kappa light chain constant region.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, BsFv, dsFv, (dsFv) 2 , dsFv-dsFv', scFv, scFv dimer, camelized single chain domain antibody, diabody, ds diabody, nanobody, single domain antibody (sdAb), bivalent domain antibody; and/ Alternatively, the antibody is a murine antibody, a chimeric antibody, a humanized antibody or a multispecific antibody.
  • the antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant technology.
  • DNA molecules encoding the heavy chain and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into an expression vector and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibody of the invention.
  • the antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
  • these antigen-binding fragments can also be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )).
  • Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab') 2 fragments can also be directly isolated from the recombinant host cell culture medium. Those of ordinary skill in the art are well aware of other techniques for preparing such antigen-binding fragments.
  • the antibody or antigen-binding fragment thereof is labeled.
  • the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or Biotin.
  • an enzyme e.g., horseradish peroxidase
  • a radionuclide e.g., a radionuclide
  • a fluorescent dye e.g., a fluorescent dye
  • a luminescent substance e.g., a chemiluminescent substance
  • the application provides an isolated nucleic acid molecule encoding an antibody as described above or an antigen-binding fragment thereof, or a heavy chain variable region and/or a light chain variable region thereof.
  • the nucleic acid molecule comprises a nucleotide sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13.
  • the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention and a first nucleotide sequence encoding said antibody or antigen-binding fragment thereof A second nucleotide sequence of a light chain or light chain variable region, wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different separate nucleic acid molecules.
  • the isolated nucleic acid molecule of the present invention includes a third nucleotide sequence containing the first nucleotide sequence. a nucleic acid molecule and a second nucleic acid molecule containing said second nucleotide sequence.
  • the present application provides a vector comprising a nucleic acid molecule as described above.
  • the vector is a cloning vector or an expression vector.
  • the vector comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention and a light chain encoding said antibody or antigen-binding fragment thereof. or a second nucleotide sequence of the light chain variable region, wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different vectors.
  • the vector of the present invention includes a first vector containing the first nucleotide sequence and a vector containing the A second vector for a second nucleotide sequence.
  • the present application provides a host cell comprising a nucleic acid molecule as described above or a vector as described above.
  • Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., small mouse cells, human cells, etc.).
  • prokaryotic cells such as bacterial cells (e.g., E. coli cells)
  • eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., small mouse cells, human cells, etc.).
  • the application provides a method for preparing an antibody or an antigen-binding fragment thereof as described above, which includes culturing a host cell as described above under conditions that allow expression of the antibody or an antigen-binding fragment thereof, and The antibody or antigen-binding fragment thereof is recovered from the cultured host cell culture.
  • the host cell is a mammalian cell.
  • the application provides multispecific molecules comprising an antibody or an antigen-binding fragment thereof as described above.
  • the multispecific molecule specifically binds p-tau 217 protein and additionally specifically binds one or more other targets.
  • the multispecific molecule further comprises at least one molecule with a second binding specificity for a second target (eg, a second antibody or antigen-binding fragment thereof).
  • a second target eg, a second antibody or antigen-binding fragment thereof.
  • the multispecific molecule comprises an antibody, or antigen-binding fragment thereof, as described above, and a second antibody, or antigen-binding fragment thereof.
  • the multispecific molecule comprises an antibody or antigen-binding fragment thereof as described above, and a second antibody or antigen-binding fragment thereof linked to the antibody or antigen-binding fragment thereof.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described above, or a multispecific molecule as described above, and a pharmaceutically acceptable carrier and/or excipient. agent.
  • compositions further comprise additional pharmaceutically active agents.
  • the additional pharmaceutically active agent is a drug active in treating tauopathies (eg, AD).
  • the pharmaceutically acceptable carrier and/or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution).
  • a sterile injectable liquid such as an aqueous or non-aqueous suspension or solution.
  • such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • the present application provides a kit containing the antibody or antigen-binding fragment thereof as described above.
  • the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or Biotin.
  • a detectable label such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or Biotin.
  • the kit further includes a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof as described above.
  • the second antibody further includes a detectable label, such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.
  • a detectable label such as an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), or biotin.
  • the kit is used to detect the presence or amount of p-tau 217 in a sample.
  • the sample is cerebrospinal fluid, whole blood, serum or plasma obtained from a subject.
  • the kit further includes a reagent to dilute the sample (eg, horse serum).
  • a reagent to dilute the sample eg, horse serum
  • the second antibody is coated on magnetic beads.
  • the subject is a mammal, such as a human.
  • the application provides a method for preventing and/or treating tauopathy in a subject (e.g., a human), the method comprising administering to a subject in need thereof an effective amount of, e.g. The aforementioned antibody or its antigen-binding fragment, or the aforementioned multispecific molecule, or the aforementioned pharmaceutical composition.
  • the tauopathies include, but are not limited to, Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • AD Alzheimer's disease
  • primary age-related tauopathies include, but are not limited to, Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • the tauopathies are selected from the group consisting of Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • AD Alzheimer's disease
  • primary age-related tauopathies chronic traumatic encephalopathy
  • Pick's disease Pick's disease
  • corticobasal degeneration corticobasal degeneration
  • the tauopathy is AD.
  • the subject's cerebrospinal fluid contains p-tau 217 protein.
  • the subject is a mammal, such as a human.
  • the methods further comprise administering an additional agent having activity in preventing and/or treating AD.
  • the application provides the use of an antibody or an antigen-binding fragment thereof as described above, or a multispecific molecule as described above, or a pharmaceutical composition as described above, in the preparation of a medicine.
  • the medicament is used to prevent and/or treat tauopathy in a subject (e.g., a human);
  • the tauopathies include, but are not limited to, Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • AD Alzheimer's disease
  • primary age-related tauopathies include, but are not limited to, Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • the tauopathies are selected from the group consisting of Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • AD Alzheimer's disease
  • primary age-related tauopathies chronic traumatic encephalopathy
  • Pick's disease Pick's disease
  • corticobasal degeneration corticobasal degeneration
  • the tauopathy is AD.
  • the medicament further comprises an additional pharmaceutically active agent active in treating tauopathies (e.g., AD); in certain embodiments, the subject's cerebrospinal fluid contains p-tau 217 protein.
  • an additional pharmaceutically active agent active in treating tauopathies e.g., AD
  • the subject's cerebrospinal fluid contains p-tau 217 protein.
  • the subject is a mammal, such as a human.
  • the present application provides an antibody or an antigen-binding fragment thereof as described above, or a multispecific molecule as described above, or a pharmaceutical composition as described above, for use in a subject Prevention and/or treatment of tauopathies (e.g., humans).
  • the tauopathies include, but are not limited to, Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • AD Alzheimer's disease
  • primary age-related tauopathies include, but are not limited to, Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • the tauopathies are selected from the group consisting of Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • AD Alzheimer's disease
  • primary age-related tauopathies chronic traumatic encephalopathy
  • Pick's disease Pick's disease
  • corticobasal degeneration corticobasal degeneration
  • the tauopathy is AD.
  • the antibody or antigen-binding fragment thereof as described above, or the multispecific molecule as described above, or the pharmaceutical composition as described above is combined with another treatment for tauopathy (e.g., AD ) active pharmaceutically active agents.
  • AD tauopathy
  • the subject's cerebrospinal fluid contains p-tau 217 protein.
  • the subject is a mammal, such as a human.
  • the present application provides a method for detecting the presence or amount of p-tau 217 protein in a sample, which includes the following steps:
  • the antibody or antigen-binding fragment thereof is detectably labeled.
  • the methods are performed in vivo or in vitro in a subject.
  • the present application provides the use of the antibody or antigen-binding fragment thereof as described above, or the multispecific molecule as described above, in the preparation of a reagent for detecting whether a subject is suffering from the disease. tauopathies, or used to distinguish patients with Alzheimer's disease (AD) from patients with other tauopathies.
  • AD Alzheimer's disease
  • the tauopathies include, but are not limited to, Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • AD Alzheimer's disease
  • primary age-related tauopathies include, but are not limited to, Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • the tauopathies are selected from the group consisting of Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • AD Alzheimer's disease
  • primary age-related tauopathies chronic traumatic encephalopathy
  • Pick's disease Pick's disease
  • corticobasal degeneration corticobasal degeneration
  • the tauopathy is AD.
  • the reagent detects the amount of p-tau 217 protein in a sample by a method as previously described, to detect whether the subject suffers from tauopathy, or to distinguish Alzheimer's disease. AD) or a patient suffering from other tauopathies, and the sample is obtained from the subject or patient.
  • AD Alzheimer's disease
  • the sample is a blood sample (eg, whole blood, serum, plasma).
  • a blood sample eg, whole blood, serum, plasma.
  • the present application provides a method for detecting whether a subject suffers from tauopathies, or for distinguishing patients suffering from Alzheimer's disease (AD) or patients suffering from other tauopathies, the method comprising:
  • the method further includes comparing the detected amounts of p-tau 217 protein in different samples to detect whether the subject suffers from Alzheimer's disease (AD), or to distinguish between Alzheimer's disease and Alzheimer's disease. (AD) patients or patients with other tauopathies.
  • AD Alzheimer's disease
  • the methods include:
  • the sample is a blood sample (eg, whole blood, serum, plasma).
  • a blood sample eg, whole blood, serum, plasma.
  • the tauopathies include, but are not limited to, Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • AD Alzheimer's disease
  • primary age-related tauopathies include, but are not limited to, Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • the tauopathies are selected from the group consisting of Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • AD Alzheimer's disease
  • primary age-related tauopathies chronic traumatic encephalopathy
  • Pick's disease Pick's disease
  • corticobasal degeneration corticobasal degeneration
  • the tauopathy is AD.
  • the present application provides an antibody or an antigen-binding fragment thereof as described above, or a multispecific molecule as described above, for detecting whether a subject suffers from tauopathy, or for distinguishing patients with Alzheimer's disease. Patients with Alzheimer's disease (AD) or other tauopathies.
  • AD Alzheimer's disease
  • the tauopathies include, but are not limited to, Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • AD Alzheimer's disease
  • primary age-related tauopathies include, but are not limited to, Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • the tauopathies are selected from the group consisting of Alzheimer's disease (AD), primary age-related tauopathies, chronic traumatic encephalopathy, Pick's disease, and corticobasal degeneration.
  • AD Alzheimer's disease
  • primary age-related tauopathies chronic traumatic encephalopathy
  • Pick's disease Pick's disease
  • corticobasal degeneration corticobasal degeneration
  • the tauopathy is AD.
  • the reagent detects whether the subject suffers from Alzheimer's disease (AD) by detecting the amount of p-tau 217 protein in the sample, or to distinguish the subject from Alzheimer's disease (AD). ) or a patient suffering from other tauopathies, and the sample is obtained from said subject or patient.
  • AD Alzheimer's disease
  • the reagent detects whether the subject suffers from Alzheimer's disease (AD) by detecting the amount of p-tau 217 protein in the sample, or to distinguish the subject from Alzheimer's disease (AD). ) or a patient suffering from other tauopathies, and the sample is obtained from said subject or patient.
  • p-tau 217 protein refers to a phosphorylated tau protein that is phosphorylated at the amino acid position 217 corresponding to native tau protein. Since pathological p-tau 217 can be detected early in the onset of AD, and a large amount of p-tau 217 is detected in the insoluble components of brain tissue extracts from AD patients, antibodies targeting p-tau 217 are important in AD. It has great application potential in prevention, detection and treatment.
  • native tau protein has the amino acid sequence set forth in SEQ ID NO: 10.
  • native tau protein refers to biologically active, naturally occurring tau protein.
  • the amino acid sequence of native tau protein can be readily obtained from various public databases (eg, GenBank database).
  • native tau protein has the amino acid sequence set forth in SEQ ID NO: 10.
  • the amino acid at position 127 of the natural tau protein refers to the amino acid at position 127 of the protein shown in SEQ ID NO: 10.
  • natural tau protein can have multiple versions that have essentially the same primary structure (i.e., amino acid sequence) and higher-order structure (i.e., spatial structure), as well as essentially the same biological function. , but they can still have slight differences in amino acid sequences from each other. Therefore, in this application, natural tau protein is not limited to the protein shown in SEQ ID NO: 10, but is intended to cover all known natural tau proteins.
  • natural tau protein shall include various naturally occurring tau proteins with biological functions, including, for example, the tau protein shown in SEQ ID NO: 10 and its naturally occurring variants. And, when describing the amino acid position of tau protein, it includes not only the specific amino acid position in SEQ ID NO: 10, but also includes the amino acid position corresponding to the specific amino acid position in its natural variant.
  • amino acid at position 127 of native tau protein includes the amino acid at position 127 of SEQ ID NO: 10, and the corresponding amino acid position in its natural variant.
  • corresponding amino acid positions means amino acid positions in the compared sequences that are equivalent when the sequences are optimally aligned, ie when the sequences are aligned to obtain the highest percent identity.
  • corresponding to position 127 of SEQ ID NO:10 means that when a sequence is optimally aligned with SEQ ID NO:10, that is, when a sequence is optimally aligned with SEQ ID NO:10 When aligning for the highest percent identity, the amino acid position in the sequence that is identical to position 127 of SEQ ID NO:10 is compared.
  • identity is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of two DNA molecules is occupied by adenine, or two A certain position in each polypeptide is occupied by lysine)
  • Percent identity between two sequences is a function of the number of matching positions common to the two sequences divided by the number of positions compared ⁇ 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences are 60% identical.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6 positions).
  • comparisons are made when two sequences are aligned to yield maximum identity.
  • alignment can be accomplished using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the PAM120 weight residue table using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0).
  • the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) algorithm can be used using the Blossum62 matrix or PAM250 in the GAP program integrated into the GCG software package (available at www.gcg.com) Matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences.
  • tauopathies refers to diseases resulting from abnormalities (eg, abnormal aggregation) of the microtubule-associated protein tau.
  • tauopathies are diseases resulting from abnormal aggregation or deposition of pathological tau protein within neurons or glial cells. Alzheimer's disease is the most representative tau disease.
  • phosphorylation refers to the addition of a phosphate group to an amino acid residue of a protein.
  • amino acid residues such as threonine, serine, and tyrosine have hydroxyl groups and are therefore easily phosphorylated.
  • antibody refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC).
  • Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q).
  • VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antigen-binding site.
  • the assignment of amino acids to each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901 Definition of -917; Chothia et al. (1989) Nature 342:878-883.
  • CDR complementarity determining region
  • the variable regions of the heavy chain and light chain each contain three CDRs, named CDR1, CDR2 and CDR3.
  • CDR1, CDR2 and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
  • the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art.
  • the CDRs contained in the antibodies of the invention, or antigen-binding fragments thereof are preferably determined by the Kabat, Chothia, or IMGT numbering systems.
  • framework region or "FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
  • the terms “monoclonal antibody”, “monoclonal antibody” and “mAb” have the same meaning and are used interchangeably and refer to one from a group of highly homologous antibody molecules.
  • An antibody or a fragment of an antibody that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously.
  • Monoclonal antibodies are highly specific for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen.
  • the modifier "monoclonal” merely indicates that the antibody is characterized as being obtained from a highly homologous population of antibodies and is not construed as requiring any specific method to prepare the antibody.
  • Monoclonal antibodies of the invention can be prepared by a variety of techniques, such as hybridoma technology (see, eg, Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, eg, U.S. Patent Application 4,816,567), or phage Antibody library technology (see, eg, Clackson et al. Nature 352: 624-628, 1991, or Marks et al. J. Mol. Biol. 222: 581-597, 1991).
  • antigen-binding fragment of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody Specific binding to an antigen, which is also called an "antigen-binding moiety.”
  • an antigen-binding moiety which is also called an "antigen-binding moiety.”
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabody, single domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis), probody and such polypeptides, which contain sufficient to confer specificity to the polypeptide At least a portion of an antibody with antigen-binding capabilities.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.
  • full-length antibody means an antibody consisting of two “full-length heavy chains” and two “full-length light chains.”
  • “full-length heavy chain” refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), and a heavy chain in the direction from the N end to the C end. It consists of a constant region CH2 domain and a heavy chain constant region CH3 domain; and, when the full-length antibody is of IgE isotype, optionally also includes a heavy chain constant region CH4 domain.
  • a "full-length heavy chain” is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction.
  • a "full-length light chain” is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction.
  • the two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains.
  • the full-length antibody of the present invention can be from a single species, such as human; it can also be a chimeric antibody or a humanized antibody.
  • the full-length antibody of the present invention contains two antigen-binding sites formed by VH and VL pairs respectively, and these two antigen-binding sites specifically recognize/bind the same antigen.
  • the term “Fd” means an antibody fragment consisting of VH and CH1 domains
  • the term “dAb fragment” means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 ( 1989));
  • the term “Fab fragment” means an antibody fragment consisting of VL, VH, CL and CH1 domains;
  • the term “F(ab') 2 fragment” means an antibody fragment consisting of two fragments connected by a disulfide bridge on the hinge region An antibody fragment of a Fab fragment;
  • the term “Fab'fragment” means the fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and the Fd of the heavy chain. Fragment (consisting of VH and CH1 domains).
  • Fv means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three CDRs specific for the antigen) can recognize and bind the antigen, although its affinity may be lower than that of the intact binding site.
  • Fc means a region formed by disulfide bonding of the second and third constant regions of the first heavy chain of an antibody to the second and third constant regions of the second heavy chain.
  • Antibody fragments The Fc fragment of an antibody has many different functions but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain comprising VL and VH domains connected by a linker (see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Volume 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al.
  • scFv can form di-scFv, which refers to two or more individual scFvs connected in series to form an antibody.
  • scFv can form (scFv)2, which refers to two or more individual scFvs joining in parallel to form an antibody.
  • single-domain antibody has the meaning commonly understood by those skilled in the art, which refers to an antibody composed of a single monomeric variable domain (e.g., a single heavy chain variable An antibody fragment consisting of a region) that retains the ability to specifically bind to the same antigen that the full-length antibody binds.
  • Single domain antibodies are also called nanobodies.
  • Each of the above antibody fragments retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
  • Antigen-binding fragments of an antibody can be obtained from a given antibody (e.g., the antibodies provided by the invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods) ), and the antigen-binding fragments of the antibody are screened for specificity in the same manner as for intact antibodies.
  • antibody includes not only intact antibodies but also antigen-binding fragments of the antibodies, unless the context clearly indicates otherwise.
  • chimeric antibody refers to an antibody in which a portion of the light chain or/and heavy chain is derived from an antibody (which may originate from a specific species or belong to a specific species). a specific antibody class or subclass), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but regardless of However, it still retains the binding activity to the target antigen (U.S.P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • the term “chimeric antibody” may include antibodies in which the heavy and light chain variable regions of the antibody are derived from a first antibody and the heavy and light chain constant regions of the antibody are derived from a second antibody.
  • variant in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing substitutions, deletions, or additions of amino acid residues. In some cases, the term “variant” also refers to a polypeptide or peptide that has been modified (ie, by covalently linking any type of molecule to the polypeptide or peptide).
  • polypeptides may be modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, Attached to cellular ligands or other proteins, etc.
  • Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.
  • a variant has a similar, identical or improved function to the polypeptide or peptide from which it is derived.
  • the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed.
  • the strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) of the interaction.
  • KD refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • the specific binding properties between two molecules can be determined using methods known in the art.
  • One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate.
  • Both the "association rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187).
  • the ratio kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990;59:439-473).
  • KD, kon and kdis values can be measured by any valid method.
  • dissociation constants can be measured in Biacore using surface plasmon resonance (SPR).
  • bioluminescence interferometry or Kinexa can be used to measure dissociation constants.
  • a detectable label of the invention may be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means.
  • labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides fluorescein (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin ( PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa 750)), luminescent substances (such as chemiluminescent substances, such as acridinium est
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyomavacuolating viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses poxviruses
  • baculoviruses papillomaviruses
  • papillomaviruses papillomaviruses
  • Polyomavacuolating viruses such as SV40.
  • a vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as E. coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acids, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • amino acids involved in this article have been prepared following conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.
  • polypeptide and “protein” have the same meaning and are used interchangeably.
  • amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers Agents, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffer.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc.
  • the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions).
  • such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution e.g. 0.9% (w/v) NaCl
  • dextrose solutions eg 5% glucose
  • surfactant containing solutions eg 0.01% polysorbate 20
  • pH buffer solutions eg phosphate buffer solution
  • Ringer's solution any combination thereof.
  • prevention refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom in a subject.
  • treatment refers to a method performed to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., no worsening) of the state of the disease, delaying or slowing the progression of the disease, ameliorating or alleviating the disease. status, and relief of symptoms (whether partial or complete), whether detectable or undetectable.
  • treatment may also refer to prolonging survival compared to expected survival if not receiving treatment.
  • the term "subject” refers to a mammal, such as a human, a cynomolgus monkey, a mouse.
  • the subject e.g., human, cynomolgus monkey, mouse
  • has a disease associated with TIGIT e.g., involving TIGIT-positive infiltrating T cells and/or NK cells, and/or Tumors involving tumor cells that are positive for TIGIT ligands (e.g., CD155 and/or CD112)
  • TIGIT e.g., involving TIGIT-positive infiltrating T cells and/or NK cells, and/or Tumors involving tumor cells that are positive for TIGIT ligands (e.g., CD155 and/or CD112)
  • an effective amount to prevent disease e.g., a tumor involving TIGIT-positive infiltrating T cells and/or NK cells, and/or involving TIGIT ligand (e.g., CD155 and/or CD112)-positive tumor cells
  • an effective amount to prevent disease is one that is sufficient to prevent , the amount that prevents, or delays the occurrence of the disease
  • the effective amount for treating the disease refers to the amount that is sufficient to cure or at least partially prevent the disease and its complications in patients who already have the disease. Determining such effective amounts is well within the capabilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other treatments administered concurrently etc.
  • the monoclonal antibodies of the present application can bind to p-tau 217 protein with high specificity.
  • the monoclonal antibody of the present application can also detect the content of p-tau 217 protein (for example, the content of p-tau 217 in the subject's cerebrospinal fluid), so the monoclonal antibody can be used to identify or detect AD patients, and differentiate AD patients and other tauopathies.
  • the monoclonal antibody administered to subjects can improve the behavior and abilities of subjects with tauopathy (for example, the time spent exploring new objects is significantly increased, the perception of surrounding light and spatial avoidance abilities are improved, and spatial learning and memory are improved) ability improvement), inhibit the hippocampal atrophy of subjects, and improve the pathological changes in brain tissue. Therefore, the monoclonal antibody (eg, 2A7 antibody) of the present application has high clinical application value in the detection, prevention, and treatment of AD and other tauopathies.
  • Figure 1 shows the immunoblotting results of antibodies 2A7 and GAPDH (internal reference) on different proteins in Example 2.1.
  • Figure 2 shows the results of tissue immunofluorescence staining of different proteins by the antibody 2A7 in Example 2.1; among them, the binding area of 2A7 and irrelevant monoclonal antibodies is shown in green, the NeuN-specific antibody binding area is shown in red, and the DAPI binding area is shown in blue.
  • Figure 3 shows the immunoblotting results of antibodies 2A7 and GAPDH (internal reference) on different proteins in Example 2.2.
  • Figure 4 shows the detection results of p-tau 217 content in AD patient cerebrospinal fluid and PS19 mouse cerebrospinal fluid samples by the 2A7 antibody in Example 3.
  • Figure 5 shows the behavioral improvement results of 10.5-month-old PS19 mice after treatment with 2A7 antibody.
  • Figure 5A shows that the time spent exploring new objects by mice in the PS19-2A7 group increased significantly compared with PS19-IgG in the new object recognition test
  • Figure 5B shows that in the open field experimental test, after treatment with 2A7 antibody, PS19 mice The time spent staying in the middle of the box was significantly reduced, indicating that the mice's perception of surrounding light and spatial avoidance abilities were improved
  • Figure 5C and Figure 5D show the spatial learning and memory abilities of mice in the PS19-2A7 group and PS19-IgG in the Morris water maze experiment There is a significant improvement compared to.
  • Figure 6 shows the inhibition results of hippocampal atrophy in 10.5-month-old PS19 mice after treatment with 2A7 antibody.
  • Figure 6A shows the results of nuclear magnetic resonance detection of the axial anatomy (T1) and 3D reconstruction of the brain of mice in the WT-IgG, PS19-IgG and PS19-2A7 groups
  • Figure 6B shows the results of WT-IgG, PS19-IgG and PS19 -Statistical results of hippocampal volume of mice in the -2A7 group.
  • Figure 7 shows the results of pathological reduction in 10.5-month-old PS19 mice after treatment with 2A7 antibody.
  • Figure 7A immunofluorescence staining results show that the loss of neurons (NeuN) in mice in the PS19-2A7 group is significantly reduced
  • Figure 7B shows the statistical results of NeuN fluorescence intensity in mice in the WT-IgG, PS19-IgG and PS19-2A7 groups
  • Figure 7C shows The staining signal of p-tau 217 (2A7) in mice in the PS19-2A7 group was significantly reduced
  • Figure 7D shows the statistical results of fluorescence intensity of 2A7 in mice in the WT-IgG, PS19-IgG and PS19-2A7 groups
  • Figure 7E shows the PS19-2A7
  • the proliferation of microglia (IBA1) in the mice in the group was significantly inhibited
  • Figure 7F shows the statistical results of the fluorescence intensity of IBA1 in mice in the WT-IgG, PS19-IgG and PS19
  • peptides phosphorylated at amino acid position 217 were used for immunization, and non-phosphorylated peptides at position 217 were used for differential screening to screen specific antibodies.
  • the immunogen is KLH-C-RSRTPSLPT(p)PPTREP, which is a phosphorylated polypeptide whose amino acid sequence corresponds to the 209-223rd amino acid sequence of the natural tau protein (its amino acid sequence is shown in SEQ ID NO: 9).
  • the peptides used in this study were obtained through chemical contracts and synthesized by Nanjing Zhongding Biotechnology Co., Ltd.
  • Mouse immunization First, mix and emulsify 100ug of polypeptide with complete Freund's adjuvant (CFA) in equal volumes, and then inject multiple points into the muscles of the limbs to immunize the mice for the first time. Secondly, 50ug of polypeptide and Freund's incomplete adjuvant (IFA) were mixed and emulsified in equal volumes, and then the mice were boosted on days 14, 28, and 42 after the first immunization. Finally, on the 56th day after the first immunization, the mice were given intraperitoneal immunization booster, and the immunogen was an equal volume mixture of 50ug polypeptide and PBS. Three days after the completion of immunization, the mouse spleens were taken for fusion experiments.
  • CFA complete Freund's adjuvant
  • Cell fusion Take the mouse spleen, grind it to obtain a spleen cell suspension, and then mix it with mouse myeloma cells SP2/0 in the logarithmic growth phase, and perform cell fusion under the action of PEG1500. The fused cells were resuspended in 300 mL of fusion medium (RPMI-1640 medium containing HAT and 20% FBS), and distributed into 15 96-well cell culture plates for culture.
  • fusion medium RPMI-1640 medium containing HAT and 20% FBS
  • Hybridoma culture The stable hybridoma monoclonal antibody cell line is amplified and cultured in a carbon dioxide incubator, and then transferred from the 96-well plate to the 24-well plate, 6-well plate, and 10cm cell plate. The cells in the cell plate were then collected and injected into the peritoneal cavity of mice. After 7 to 10 days, ascites containing monoclonal antibodies was aspirated from the peritoneal cavity of mice.
  • the obtained monoclonal antibody was PCR amplified, and the PCR product was sent to the company for sequencing to obtain the sequence.
  • the obtained antibody was named 2A7, and its specific sequence is shown in Table 1.
  • the CDR sequence of the antibody is determined by the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).
  • the first part was added with phosphatase inhibitors and stored at -80°C for later use; the second part was Add phosphatase inhibitor, mix evenly, take out a portion and incubate at 37°C for 1 hour; add alkaline phosphatase to the third portion, mix evenly, take out a portion and incubate at 37°C for 1 hour. More than 15 ⁇ g of mouse brain lysates were taken for WB to verify the reactivity and specificity of 2A7 to natural tau, using GAPDH as the internal reference.
  • the concentration of 2A7 used was 1 ⁇ g/mL, and HRP-labeled horse anti-mouse IgG (HAM-HRP, CST, 7076S) diluted 1:1000 was used for development (Shanghai Qinxiang, ChemiScope 6200).
  • the reactivity and specificity of 2A7 were verified by tissue immunofluorescence staining.
  • the experiment was divided into 6 groups, using AD-unrelated IgG antibodies (corresponding to the second column of IgG in Figure 2), 2A7 antibodies (corresponding to the fourth column of p-tau 217 in Figure 2), and polypeptide blocking phosphorylation at position 217.
  • the final 2A7 antibody (corresponding to the 217 peptide block in the fifth column in Figure 2) was used to stain PS19 mouse brain tissue sections; AD-independent IgG antibodies (corresponding to the first column of IgG in Figure 2) and 2A7 antibodies (corresponding to the The third column in Figure 2 p-tau 217) stains WT mouse brain tissue sections.
  • the dosage of the above antibodies is 1 ⁇ g, and the dosage of the peptide is 5 ⁇ g.
  • the brain tissues of 13-month-old WT and PS19 mice were taken for frozen section. Two pieces of WT mouse brain tissue and four pieces of PS19 mouse brain tissue were washed three times with PBST for 10 minutes each time. Then they were blocked with PBST containing 10% donkey serum (Solarbo, SL050) and 0.3% Triton X-100, and incubated at room temperature for 1 hour. Add the above antibodies to 200 ⁇ L of blocking solution, add them to the corresponding mouse brain tissue, and incubate at 4°C overnight. The above antibodies were recovered and the brain slices were washed three times with PBST for 10 minutes each time.
  • the 2A7 antibody can specifically recognize the tau protein in the hippocampus and cortex of PS19. After incubation with the peptide phosphorylated at position 217, the 2A7 antibody can specifically recognize the tau protein in PS19. The binding capacity of tau protein is reduced in the hippocampus and cortical areas.
  • CTR4-Tau and CTR4-T217A vectors with 217 phosphorylation site mutations respectively.
  • the insert fragment of CTR4-Tau vector is the nucleotide sequence encoding tau protein (as shown in SEQ ID NO:10).
  • the CTR4-T217A vector is The inserted fragment is the nucleotide sequence encoding the tau protein mutated at position 217 (shown in SEQ ID NO:11).
  • 293T cells were transfected at the same time, and the cells were collected after 48 hours, and PLCDH-GFP was used as a control.
  • Use TNEN (containing protease inhibitors and phosphatase inhibitors) for lysis. After lysis is completed, centrifuge at 12,000 rpm for 10 minutes at 4°C. Take the supernatant and measure the concentration and store it at -80°C for later use.
  • the 217th amino acid of the eukaryotic expressed tau protein will be phosphorylated by intracellular kinases, so it can be recognized by the 2A7 antibody; and the mutation at position 217 is to mutate the amino acid T at position 217 to A. After the mutation, the amino acid at this position Cannot be phosphorylated.
  • the results are shown in Figure 3.
  • the reactivity of 2A7 to eukaryotic expressed tau protein (lane 2 in Figure 3) is higher than that of tau protein after mutation of the 217 site (lane 3 in Figure 3).
  • This example uses tauopathy model mice, namely PS19 mice, for experiments.
  • the cerebrospinal fluid (CSF) of 13-month-old PS19 mice was collected.
  • CSF cerebrospinal fluid
  • For the collection of cerebrospinal fluid refer to the optimized method of collecting mouse cerebrospinal fluid by Lim et al. (For details, please see Lim, N.K., V. Moestrup, X. Zhang, W.A. Wang, A. Moller and F.D. Huang (2016).
  • a detection kit (Cat. No.: Lite-P64050, Suzhou Yucei) to detect the content of p-tau 217 in the cerebrospinal fluid of AD and PS19 mice.
  • the mouse cerebrospinal fluid was diluted 5 times with horse serum, and AD cerebrospinal fluid samples were tested at the original times.
  • the sample reaction and analysis were all performed on the Ast-Dx90 single molecule immunodiagnostic instrument.
  • the reagent needle draws 10 ⁇ L of the detection antibody (Reagent 2) modified with a single-molecule signal marker from the kit into the reaction cup, mixes and incubates for 4 minutes.
  • Reagent 2 contains a single-molecule signal marker modified with the detection antibody, which can convert the target molecule. is a single molecule signal.
  • the detection needle transfers the reaction system to the flow cell, uses magnetic separation to attract the magnetic beads to the bottom of the flow cell and lays them flat on the surface of the detection well, washes to remove other components, and then uses an integrated fluorescence microscope to capture fluorescence images and analyze the individual beads through the machine. molecular signals and calculate biomarker concentrations using a pre-prepared standard curve.
  • Figure 4 shows that the 2A7 antibody can be used to detect the content of p-tau 217 in the cerebrospinal fluid of AD patients and the cerebrospinal fluid of PS19 mice.
  • This study used intranasal administration to detect the improvement effect of 2A7 antibody on the pathology and behavior of PS19 mice (tauopathy model mice).
  • mice in the experiment were all administered intranasally. After being anesthetized with isoflurane, they were placed on their backs. The antibody was slowly dripped into the nostrils with a microsyringe. The mouth was closed when instilling to facilitate the absorption of the solution.
  • the antibody was administered every 3 days for 5 months.
  • the dosage volume for each mouse was 20 ⁇ l, with 10 ⁇ g administered to each mouse in the first three months and 20 ⁇ g each in the next two months.
  • Example 4 Behavioral experiments on all mice treated in Example 4 used Smart Video Tracking Software (Panlab, Harvard Apparatus) for data collection and analysis. Animal behavior experiments are conducted between 9:00 a.m.-7:00 p.m. every day, and the light intensity in the laboratory is 650lux.
  • mice touched the mice three days before the start of the experiment, once a day, stroked one mouse at a time, gently grabbed the mouse, let the mouse stay on the tester's hand for 30 seconds, and put a marker on the tail of the mouse. Mark the mouse with a line. After marking the mouse, grab the tail and gently put it back into the cage;
  • mice to be tested On the day of the experiment, transfer the mice to be tested to the experimental room before the experiment to allow the mice to adapt to the surrounding environment and light; before starting the experiment, clean the box and maze used in the experiment with 75% ethanol. After each round of experiments, 75% ethanol was used to wipe the box and maze to remove the feces and urine excreted by the mice during the experiment to eliminate the interference of the residual odor of the mice on the test results.
  • the open field experiment is used to study the autonomous movement ability and anxiety behavior of mice, mainly based on the mice's avoidance of bright light and open spaces.
  • the new object recognition experiment is a learning and memory test method established based on the principle that rodents are naturally curious to explore new objects.
  • mice On the first day, put the mice into the center of the open field box (length 40cm x width 40cm x height 40cm). Place each mouse in the same position (same grid, same direction), and let the mice adapt to the training for 5 minutes; on the second day , place two identical objects A and B on one side of the open field box, and gently put the mouse into the open field box facing the wall of the box. The placement position is as close as possible to the distance between the two objects, and let the mouse explore freely for 8 minutes. ; On the third day, keep the position of the object unchanged and replace one of the old objects (A or B) with a new object C; put the mouse into the open field box facing the box arm, and put it in the same position as the distance between the two objects. Let the mice explore freely for 8 minutes. When the mouse's nose touches an object or its nose points to an object within 2 cm of the object, it is considered exploratory behavior; a camera system is used to record the time the mouse spends exploring familiar objects and exploring new objects.
  • the Morris water maze test is used to study and evaluate the spatial learning and memory abilities of mice.
  • the water maze is conducted in a circular water tank (radius 60cm x height 100cm).
  • the height of the water in the water tank should be 2cm above the platform.
  • the temperature of the water in the water tank is set to 22°C.
  • Four icons of different shapes are posted in the four directions (E, S, W, N) within the maze arm as spatial positioning references.
  • the platform was 2cm below the water surface, and then the mice were put into the maze from the four water entry points and allowed to search for the platform for 60 seconds.
  • the experiment stopped when the mice stayed on the platform for 10 seconds. If the mouse cannot find the platform within 60 s, use a ruler to guide the mouse to the location of the platform and let the mouse stay on the platform for 10 s.
  • Each mouse was tested twice a day, entering the water from two different directions twice.
  • the interval between two experiments for each mouse was at least 1 hour. Record the latency of the mouse to find the platform (Escape latency), the total swimming distance and the average swimming speed of the mouse, and conduct learning training for 7 consecutive days.
  • Platform experiment On the 8th day, remove the platform, then gently put the mouse into the water from the diagonal position of the platform, let it search the area where the original platform is for 60 seconds, record the number of times the mouse shuttles in the area where the original platform is located (Number of crossing) and the swimming time of the mouse in the target quadrant of the platform and the other three different quadrants (Time in quadrants). The water in the maze was changed every day, and the number and position of surrounding objects and experimenters were fixed.
  • mice (10.5 months old) in the above experiment were subjected to magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • PS19 mice that are untreated or treated with ineffective antibodies will have significant shrinkage in hippocampal volume.
  • the therapeutic effect of the 2A7 antibody can be evaluated by the degree of hippocampal volume shrinkage in mice.
  • TR repetition time
  • TE echo time
  • SI layer spacing
  • FA inversion angle
  • Figure 6A shows the results of MRI detection of the axial anatomy (T1) and 3D reconstruction of the brains of mice in the WT-IgG, PS19-IgG and PS19-2A7 groups
  • Figure 6B shows the results of WT-IgG , statistical results of hippocampal volume of mice in PS19-IgG and PS19-2A7 groups.
  • the above results show that after treatment with 2A7 antibody, hippocampal atrophy in 10.5-month-old PS19 mice was significantly inhibited.
  • mice from the above experiment anesthetize them with 5% chloral hydrate, perfuse them with pre-cooled 1 ⁇ PBS and 4% PFA (in1 ⁇ PBS, pH7.4) respectively, peel off the brains, and place them in 4% PFA. Continue to fix at 4°C overnight;
  • Figure 7A and B immunofluorescence staining results show that the neuronal loss of mice in the PS19-2A7 group is significantly reduced;
  • Figure 7C and D show the phosphorylated p-tau 217 signal of mice in the PS19-2A7 group.
  • Figure 7E and F show that microglial proliferation in mice in the PS19-2A7 group was significantly inhibited.
  • Experimental results showed that after treatment with 2A7 antibody, the pathology of 10.5-month-old PS19 mice was significantly reduced.

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Abstract

本申请属于生物医药技术领域，更具体地，本申请涉及能够特异性结合p-tau 217的抗体或其抗原结合片段，以及包含其的多特异性分子、药物组合物和试剂盒，本申请还涉及所述抗体或其抗原结合片段在制备试剂盒或药物中的用途。本申请的单克隆抗体（例如，2A7抗体）在AD的检测、预防、以及AD与其它tau蛋白病的治疗中具有较高的临床应用价值。

Description

针对p-tau 217的抗体及其用途 技术领域
本申请属于生物医药技术领域，更具体地，本申请涉及能够特异性结合p-tau 217(第217位氨基酸磷酸化的tau蛋白)的抗体或其抗原结合片段，以及包含其的多特异性分子、药物组合物和试剂盒，本申请还涉及所述抗体或其抗原结合片段在制备试剂盒或药物中的用途。
背景技术
阿尔茨海默病(Alzheimer’s disease,AD)是一种最常见的与年龄相关的神经退行性疾病，随着年龄的增加，患病率逐渐上升。根据美国阿尔茨海默病协会2021年的年度报告，目前美国65岁以上的人群中约有620万AD患者，预计到2060年，将达到1380万。随着我国人口的老龄化，AD以及其他形式的痴呆症也正在急剧增加。根据山西医科大学研究人员2020年的荟萃分析(meta-analysis)，目前我国AD的综合患病率约为4％，同时发现其患病率与性别、年龄、受教育程度和地区密切相关。AD不仅严重损害老年人的健康与生活质量，还给家庭和社会都带来巨大的经济负担。
AD的主要病理特征包括β淀粉样蛋白(β-amyloid protein,Aβ)沉积形成的神经炎性斑块和过度磷酸化tau蛋白聚集形成的神经纤维缠结(neurofibrillary tangles,NFTs)、小胶质和星形胶质细胞的激活、神经突触的丢失及神经元死亡等。其临床症状包括进行性记忆衰退、执行功能受损和日常活动困难。AD发病的早期常表现为思维或无意识行为的改变，对新信息的记忆障碍，以及语言功能障碍性改变等等。晚期AD患者会出现严重的记忆丧失、幻觉、定向障碍和缺乏自理能力，最终危及生命。由于AD致病原因复杂，而且缺乏明确的病理机制，目前还没有任何干预方法能阻止或逆转AD的进展，仅能暂时改善或减缓症状发展，因此寻找新的临床治疗药物迫在眉睫。
AD的发病机制尚不明确，科学家提出多种AD发病机制假说，如Aβ级联假说、胆碱能假说、tau蛋白异常磷酸化假说、神经炎症假说、金属离子紊乱假说等。根据这些假说，许多药物研究者研发出针对不同途径的药物，并进行相应的临床实验，但都收效甚微。目前上市的抗AD药物主要包括4类，胆碱酯酶抑制剂(如他克林、多奈哌齐、加兰他敏和卡巴拉汀等)、NMDA受体拮抗剂(如美金刚)、Aβ和tau的抗体或抑制剂(如Aducanumab、solanezumab和TPI-287等)以及肠道菌群调节剂(如GV-971)。这些药物对于初期和中期AD患者具有一定的治疗作用，可以维持患者认知状态。但 是，这些药物并不能改善AD患者病情发展，而且基本都具有严重副作用，如他克林具有肝毒性，卡巴拉汀有恶心、呕吐等胃肠道不良反应等。许多医药公司的研发重点主要为症状明显的临床治疗阶段，对于预防研究相对较少。当AD患者出现早期临床症状时，体内病理早已出现。AD疗效差一个最主要的原因是临床治疗干预时间太晚目前的治疗往往在患者出现明显的临床症状的中晚期才开始，从而导致治疗效果非常不想理。因此早期检测、诊断以及有效的风险预测对于AD的预防和治疗至关重要。
目前AD治疗的靶点主要包括致病性Aβ和tau蛋白。但大量靶向Aβ的药物在临床试验阶段失败，如γ分泌酶抑制剂(semagacestat和avagacestat)的临床试验未显示出疗效，因增加皮肤癌和感染的发生率而被迫停止。抗Aβ疫苗AN1792由于无菌性脑膜炎的严重不良反应而终止。因此，研发人员将注意力转向了tau蛋白，原因在于其介导了Aβ引发的神经元功能紊乱和死亡。tau是一种参与微管稳定的微管相关蛋白(由MAPT基因编码)，主要富集于神经元轴突周围，其主要功能包括调节、维持微管稳定性、辅助神经元轴突的运输功能等。tau蛋白的过度磷酸化会导致其从微管解离，并聚集成具有神经毒性的寡聚体和/或纤维，引发神经元功能障碍和死亡。值得注意的是，除了AD之外，磷酸化tau蛋白的沉积还是进行性核上性麻痹(progressive superanuclear palsy,PSP)、皮质基底节变性(corticobasal degeneration,CBD)额颞叶痴呆(frontotemporal dementia,FTD)等多种神经退行性疾病的主要病理特征和致病因子，这类疾病又被称为tau蛋白病。因此靶向tau蛋白的治疗策略可以被用于这一大类神经退行性疾病。目前针对tau蛋白的治疗药物主要包括tau蛋白靶向疫苗、小分子类的tau聚集抑制剂、靶向编码tau蛋白基因的反义寡核苷酸及抗tau蛋白抗体类。tau蛋白是一种细胞内蛋白，但它可以以游离形式或细胞外囊泡的形式排到脑细胞之间的空隙中，进行扩散，导致异常tau蛋白聚集和不可逆损伤。免疫抗体疗法会结合扩散的tau蛋白从而延缓或防止神经退行性病变。但目前抗tau蛋白抗体大部分靶向N端抗原表位，如AC Immune公司的semorinemab(识别各种形式全长的tau蛋白)、Biogen公司的gosuranemab和AbbVie的ABBV-8E12(识别N末端附近的一个表位)，这些抗体没有特意靶向致病性的tau蛋白(如磷酸化tau蛋白)，未能发挥最大的治疗效果，大多以临床失败告终。因此开发针对致病性tau蛋白抗体对AD或其它tau蛋白病的治疗具有重要的探索意义。
对AD干预过晚是导致AD治疗效果不佳的另一大重要原因，因此对AD的早期诊断显得尤为重要。临床上对于AD的诊断主要基于临床症状、神经心理学测试、神经影像学检查、和脑脊液检测。其中Aβ正电子发射断层扫描(amyoid-PET-CT)是AD诊断的金标准，但受价格、仪器以及场所等限制，不易进行人群筛查。同时神经心理学测试极 易受到受试者生活经历、教育程度、种族和性别等因素的影响，通常作为辅助检测手段。早期AD患者的临床症状表现不显著，几乎无法察觉，容易错过最佳诊疗时机。因此，将AD生物标志物检测纳入普通人群常规筛查对于AD的防治至关重要。国际上许多国家已把AD生物标志物筛查列入AD防治指南。其中，脑脊液中AD相关生物标志物(Aβ40、Aβ42、t-tau以及p-tau等)的含量变化能够较直接反应神经元的损伤情况，并且早于临床症状20年左右。目前181位的苏氨酸磷酸化的tau蛋白(p-tau181)是使用最广泛的p-tau检测靶标，并能够实现血清标本的检测。2020年，Janelidze和Barthélemy等的研究发现，在脑脊液中，与p-tau181相比，p-tau 217与tau-PET、新脑皮层Aβ斑块负荷表现出更强的相关性，更能准确的区分AD和非AD，同时p-tau 217的灵敏度和特异度大于90％。血浆中p-tau 217区分临床AD患者和其他神经退行性疾病患者的准确性(AUC＝0.96)显著高于p-tau 181、NfL以及MRI检测(AUC为0.50-0.81)。除此之外，血浆p-tau 217联合APOE基因分型以及认知检查还可用于AD的风险评估模型。
研究表明，聚合tau蛋白由于蛋白酶作用，易造成N端截断，因此靶向中段tau蛋白的抗体可能具有更大的优势。由于217位磷酸化tau蛋白在AD发病早期就可以被检测出来，且在AD或其它tau蛋白病的患者脑组织提取物的不可溶组分中检测到大量217位磷酸化tau蛋白，因此靶向217位点磷酸化tau蛋白可能在AD或其它tau蛋白病的发病早期阶段就能干预疾病进程，具有重要意义。
但迄今为止，以p-tau 217作为AD及其它tau蛋白病的早期诊断标准以及特异性识别病理性tau蛋白217位点磷酸化的单抗治疗药物未见报道。故以病理性p-tau 217为靶点建立高效、高灵敏度、高特异性、操作简便的AD或其它tau蛋白病的早期诊断方法以及制备更多特异性识别、阻断致病性tau 217位点磷酸化的抗体治疗药物，将为AD等tau蛋白病的治疗提供更多的可能性，同时也为患者带来新的曙光。
发明内容
基于现有技术的不足，本申请的主要目的之一在于，提供一种特异性结合p-tau 217蛋白的抗体。本申请还提供了所述抗体的制备方法和用途，本申请的抗p-tau 217蛋白的抗体可以用于检测、预防和/或治疗tau蛋白病，特别是AD。
因此，在第一方面，本申请提供了一种特异性结合p-tau 217蛋白的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
(a)包含下述3个互补决定区(CDRs)的重链可变区(VH)：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:3，或与其相比具有一个或几个氨基 酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:4，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:5，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
和/或，
(b)包含下述3个互补决定区(CDRs)的轻链可变区(VL)：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:6，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:7，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和(vi)VL CDR3，其由下述序列组成：SEQ ID NO:8，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，(i)-(vi)中任一项所述的置换为保守置换。
在某些实施方案中，(i)-(vi)中任一项所述的CDR根据Kabat、Chothia或IMGT编号系统定义。
在某些实施方案中，(i)-(vi)中任一项所述的CDR根据IMGT编号系统定义。
在某些实施方案中，所述抗体或其抗原结合片段包含如下3个重链CDRs：如SEQ ID NO:3所示的VH CDR1、如SEQ ID NO:4所示的VH CDR2、如SEQ ID NO:5所示的VH CDR3；和/或，如下3个轻链CDRs：如SEQ ID NO:6所示的VL CDR1、如SEQ ID NO:7所示的VL CDR2、如SEQ ID NO:8所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO:1所示的序列；
(ii)与SEQ ID NO:1所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO:1所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和/或
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
(iv)SEQ ID NO:2所示的序列；
(v)与SEQ ID NO:2所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(vi)与SEQ ID NO:2所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的重链框架区序列和/或轻链框架区序列。
在某些实施方案中，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:1所示的序列的VH和具有如SEQ ID NO:2所示的序列的VL。
在某些实施方案中，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的恒定区或其变体。
在某些实施方案中，所述抗体或其抗原结合片段包含：
(a)人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)；和/或
(b)人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)。
在某些实施方案中，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区。
在某些实施方案中，所述轻链恒定区是κ轻链恒定区。
在某些实施方案中，所述抗原结合片段选自Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、BsFv、dsFv、(dsFv) 2、dsFv-dsFv'、scFv、scFv二聚体、骆驼化单域抗体(camelized single chain domain antibody)、双抗体(diabody)、ds双功能抗体(ds diabody)、纳米抗体、单域抗体(sdAb)、双价域抗体；和/或，所述抗体为鼠源抗体、嵌合抗体、人源化抗体或多特异性抗体。
本发明的抗体可以用本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本发明抗体的重链和轻链基因的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗体。
本发明的抗原结合片段可以通过水解完整的抗体分子获得(参见Morimoto et al.,J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81(1985))。另外，这些抗原结合片段也可以直接由重组宿主细胞产生(reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999)；Little et al.,Immunol.Today,21:364-370(2000))。比如，Fab’片段可以直接从宿主细胞中获得；可以将Fab’片段化学偶联形成F(ab’)2片段(Carter et al.,Bio/Technology,10:163-167(1992))。另外，Fv、Fab或F(ab’) 2片段也可以直接从重组宿主细胞培养液中直接分离得到。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
在某些实施方案中，所述抗体或其抗原结合片段带有标记。在某些实施方案中，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
在另一方面，本申请提供了分离的核酸分子，其编码如前所述的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区。
在某些实施方案中，所述核酸分子包含如SEQ ID NO:12或SEQ ID NO:13中所示的核苷酸序列。
在某些实施方案中，所述分离的核酸分子包含编码本发明的抗体或其抗原结合片段的重链或重链可变区的第一核苷酸序列和编码所述抗体或其抗原结合片段的轻链或轻链可变区的第二核苷酸序列，其中所述第一核苷酸序列和所述第二核苷酸序列存在于相同或不同的分离的核酸分子上。当所述第一核苷酸序列和所述第二核苷酸序列存在于不同的分离的核酸分子上时，本发明所述的分离的核酸分子包含含有所述第一核苷酸序列的第一核酸分子以及含有所述第二核苷酸序列的第二核酸分子。
在另一方面，本申请提供了一种载体，其包含如前所述的核酸分子。在某些实施方案中，所述载体为克隆载体或表达载体。
在某些实施方案中，所述载体包含编码本发明的抗体或其抗原结合片段的重链或重链可变区的第一核苷酸序列和编码所述抗体或其抗原结合片段的轻链或轻链可变区的第二核苷酸序列，其中所述第一核苷酸序列和所述第二核苷酸序列存在于相同或不同的载 体上。当所述第一核苷酸序列和所述第二核苷酸序列存在于不同的载体上时，本发明所述的载体包含含有所述第一核苷酸序列的第一载体以及含有所述第二核苷酸序列的第二载体。
在另一方面，本申请提供了一种宿主细胞，其包含如前所述的核酸分子或如前所述的载体。
此类宿主细胞包括但不限于，原核细胞例如细菌细胞(如大肠杆菌细胞)，以及真核细胞例如真菌细胞(例如酵母细胞)，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。
在另一方面，本申请提供了制备如上所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养如上所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。在某些实施方案中，所述宿主细胞是哺乳动物细胞。
在另一方面，本申请提供了多特异性分子，其包含如前所述的抗体或其抗原结合片段。
在某些实施方案中，所述多特异性分子特异性结合p-tau 217蛋白，并且额外地特异性结合一个或多个其他靶标。
在某些实施方案中，所述多特异性分子还包含至少一种具有针对第二靶标的第二结合特异性的分子(例如第二抗体或其抗原结合片段)。
在某些实施方案中，所述多特异性分子包含如前所述的抗体或其抗原结合片段，以及第二抗体或其抗原结合片段。
在某些实施方案中，所述多特异性分子包含如前所述的抗体或其抗原结合片段，以及连接于所述抗体或其抗原结合片段的第二抗体或其抗原结合片段。
在另一方面，本申请提供了药物组合物，其包含如前所述的抗体或其抗原结合片段，或如前所述的多特异性分子，以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，药物组合物还包含另外的药学活性剂。
在某些实施方案中，所述另外的药学活性剂是具有治疗tau蛋白病(例如，AD)活性的药物。
在某些示例性实施方案中，所述药学上可接受的载体和/或赋形剂包含无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体 选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
在另一方面，本申请提供了试剂盒，其含有如前所述的抗体或其抗原结合片段。
在某些实施方案中，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
在某些实施方案中，所述试剂盒还包括第二抗体，其特异性识别如前所述的抗体或其抗原结合片段。
在某些实施方案中，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
在某些实施方案中，所述试剂盒用于检测样本中p-tau 217的存在或其含量。
在某些实施方案中，所述样本为获自受试者的脑脊液，全血，血清或血浆。
在某些实施方案中，所述试剂盒还包含稀释样本的试剂(例如，马血清)。
在某些实施方案中，所述第二抗体包被于磁珠上。
在某些实施方案中，所述受试者为哺乳动物，例如人。
在另一方面，本申请提供了一种用于在受试者(例如人)中预防和/或治疗tau蛋白病的方法，所述方法包括向有此需要的受试者施用有效量的如前所述的抗体或其抗原结合片段，或如前所述的多特异性分子，或如前所述的药物组合物。
在某些实施方案中，所述tau蛋白病包括但不限于阿尔兹海默症(AD)、原发性年龄相关的tau蛋白病、慢性创伤性脑病、Pick病和皮质基底节变性。
在某些实施方案中，所述tau蛋白病选自阿尔兹海默症(AD)、原发性年龄相关的tau蛋白病、慢性创伤性脑病、Pick病和皮质基底节变性。
在某些实施方案中，所述tau蛋白病是AD。
在某些实施方案中，所述受试者的脑脊液中含有p-tau 217蛋白。
在某些实施方案中，所述受试者为哺乳动物，例如人。
在某些实施方案中，所述方法还包括施用另外的具有预防和/或治疗AD活性的药物。
在另一方面，本申请提供了一种如前所述的抗体或其抗原结合片段，或如前所述的多特异性分子，或如前所述的药物组合物，在制备药物中的用途，所述药物用于在受试者(例如人)中预防和/或治疗tau蛋白病；
在某些实施方案中，所述tau蛋白病包括但不限于阿尔兹海默症(AD)、原发性年龄相关的tau蛋白病、慢性创伤性脑病、Pick病和皮质基底节变性。
在某些实施方案中，所述tau蛋白病选自阿尔兹海默症(AD)、原发性年龄相关的tau蛋白病、慢性创伤性脑病、Pick病和皮质基底节变性。
在某些实施方案中，所述tau蛋白病是AD。
在某些实施方案中，药物还包含另外的治疗tau蛋白病(例如，AD)活性的药学活性剂；在某些实施方案中，所述受试者的脑脊液中含有p-tau 217蛋白。
在某些实施方案中，所述受试者为哺乳动物，例如人。
在另一方面，本申请提供了一种如前所述的抗体或其抗原结合片段，或如前所述的多特异性分子，或如前所述的药物组合物，用于在受试者(例如人)中预防和/或治疗tau蛋白病。
在某些实施方案中，所述tau蛋白病包括但不限于阿尔兹海默症(AD)、原发性年龄相关的tau蛋白病、慢性创伤性脑病、Pick病和皮质基底节变性。
在某些实施方案中，所述tau蛋白病选自阿尔兹海默症(AD)、原发性年龄相关的tau蛋白病、慢性创伤性脑病、Pick病和皮质基底节变性。
在某些实施方案中，所述tau蛋白病是AD。
在某些实施方案中，如前所述的抗体或其抗原结合片段，或如前所述的多特异性分子，或如前所述的药物组合物与另外的治疗tau蛋白病(例如，AD)活性的药学活性剂联用。
在某些实施方案中，所述受试者的脑脊液中含有p-tau 217蛋白。
在某些实施方案中，所述受试者为哺乳动物，例如人。
在另一方面，本申请提供了一种检测p-tau 217蛋白在样品中的存在或其量的方法，其包括以下步骤：
(1)将所述样品与如前所述的抗体或其抗原结合片段接触；
(2)检测所述抗体或其抗原结合片段与p-tau 217蛋白之间复合物的形成或检测所述复合物的量。
在某些实施方案中，所述抗体或其抗原结合片段带有可检测的标记。
在某些实施方案中，所述方法在受试者体内或体外进行。
在另一方面，本申请提供了如前所述的抗体或其抗原结合片段，或如前所述的多特异性分子，在制备试剂中的用途，所述试剂用于检测受试者是否患tau蛋白病，或者用 于区分患阿尔兹海默症(AD)的患者或患其它tau蛋白病的患者。
在某些实施方案中，所述tau蛋白病包括但不限于阿尔兹海默症(AD)、原发性年龄相关的tau蛋白病、慢性创伤性脑病、Pick病和皮质基底节变性。
在某些实施方案中，所述tau蛋白病选自阿尔兹海默症(AD)、原发性年龄相关的tau蛋白病、慢性创伤性脑病、Pick病和皮质基底节变性。
在某些实施方案中，所述tau蛋白病是AD。
在某些实施方案中，所述试剂通过如前所述的方法检测p-tau 217蛋白在样品中的量，以检测受试者是否患tau蛋白病，或者以区分患阿尔兹海默症(AD)的患者或患其它tau蛋白病的患者，且所述样品获自所述受试者或患者。
在某些实施方案中，所述样品为血液样品(例如，全血，血清，血浆)。
在另一方面，本申请提供了一种检测受试者是否患tau蛋白病，或者区分患阿尔兹海默症(AD)的患者或患其它tau蛋白病的患者的方法，所述方法包括：
通过如前所述的抗体或其抗原结合片段检测p-tau 217蛋白在样品中的量，且所述样品获自所述受试者或患者；
任选地，所述方法还包括将不同样品中的p-tau 217蛋白的检测量进行比较，以检测受试者是否患阿尔兹海默症(AD)，或者以区分患阿尔兹海默症(AD)的患者或患其它tau蛋白病的患者。
在某些实施方案中，所方法包括：
(1)将所述样品与如前所述的抗体或其抗原结合片段接触；
(2)检测所述抗体或其抗原结合片段与p-tau 217蛋白之间复合物的形成或检测所述复合物的量。
在某些实施方案中，所述样品为血液样品(例如，全血，血清，血浆)。
在某些实施方案中，所述tau蛋白病包括但不限于阿尔兹海默症(AD)、原发性年龄相关的tau蛋白病、慢性创伤性脑病、Pick病和皮质基底节变性。
在某些实施方案中，所述tau蛋白病选自阿尔兹海默症(AD)、原发性年龄相关的tau蛋白病、慢性创伤性脑病、Pick病和皮质基底节变性。
在某些实施方案中，所述tau蛋白病是AD。
在另一方面，本申请提供了如前所述的抗体或其抗原结合片段，或如前所述的多特异性分子，用于检测受试者是否患tau蛋白病，或者用于区分患阿尔兹海默症(AD)的患者或患其它tau蛋白病的患者。
在某些实施方案中，所述tau蛋白病包括但不限于阿尔兹海默症(AD)、原发性年 龄相关的tau蛋白病、慢性创伤性脑病、Pick病和皮质基底节变性。
在某些实施方案中，所述tau蛋白病选自阿尔兹海默症(AD)、原发性年龄相关的tau蛋白病、慢性创伤性脑病、Pick病和皮质基底节变性。
在某些实施方案中，所述tau蛋白病是AD。
在某些实施方案中，所述试剂通过检测p-tau 217蛋白在样品中的量，以检测受试者是否患阿尔兹海默症(AD)，或者以区分患阿尔兹海默症(AD)的患者或患其它tau蛋白病的患者，且所述样品获自所述受试者或患者。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子遗传学、核酸化学、化学、分子生物学、生物化学、细胞培养、微生物学、细胞生物学、基因组学和重组DNA等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“p-tau 217蛋白”是指一种磷酸化的tau蛋白，其在对应于天然tau蛋白的第217位位置上的氨基酸被磷酸化。由于病理性p-tau 217在AD发病早期就可以被检测出来，且在AD患者脑组织提取物的不可溶组分中检测到大量p-tau 217，因此靶向p-tau 217的抗体在AD的预防、检测以及治疗中具有较大的应用潜能。在某些实施方案中，天然tau蛋白具有如SEQ ID NO:10所示的氨基酸序列。
如本文中所使用的，术语“天然tau蛋白”是指具有生物学活性的、天然存在的tau蛋白。可方便地从各种公共数据库(例如，GenBank数据库)获得天然tau蛋白的氨基酸序列。在某些实施方案中，天然tau蛋白具有如SEQ ID NO:10所示的氨基酸序列。
如本文中所使用的，当提及天然tau蛋白的氨基酸序列时，其使用SEQ ID NO：10所示的序列来进行描述。例如，表述“天然tau蛋白的第127位位置上的氨基酸”是指，SEQ ID NO：10所示的蛋白的第127位位置上的氨基酸。然而，本领域技术人员理解，天然tau蛋白可具有多种版本，它们具有基本上相同的一级结构(即，氨基酸序列)和高级结构(即，空间结构)，以及基本上相同的生物学功能，但是它们彼此之间在氨基酸序列上仍然可以存在微小差异。因此，在本申请中，天然tau蛋白并不局限于SEQ ID NO：10所示的蛋白，而意欲涵盖所有已知的天然tau蛋白。因此，在本申请中，术语“天然tau蛋白”应包括各种天然存在的、具有生物学功能的tau蛋白，包括例如SEQ ID NO：10所示的tau蛋白以及其天然存在的变体。并且，当描述tau蛋白的氨基酸位置时，其不仅包括SEQ ID NO：10中的特定氨基酸位置，还包括其天然变体中与所述特定氨基酸位置对应的氨 基酸位置。例如，表述“天然tau蛋白的第127位位置上的氨基酸”包括，SEQ ID NO：10的第127位位置上的氨基酸，以及其天然变体中的相应氨基酸位置。根据本申请，表述“相应氨基酸位置”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的氨基酸位置。类似地，表述“对应于SEQ ID NO:10的第127位位置”是指，当对某一序列与SEQ ID NO:10进行最优比对时，即当某一序列与SEQ ID NO:10进行比对以获得最高百分数同一性时，进行比较的该序列中位于与SEQ ID NO:10的第127位等同位置的氨基酸位置。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中所使用的，术语“tau蛋白病(tauopathies)”是指微管相关蛋白tau异常(例如，异常聚集)所导致的疾病。在某些实施方案中，tau蛋白病是由神经元或胶质细胞内病理性tau蛋白异常聚集或沉积所导致的疾病。阿尔茨海默症是最具有代表的tau蛋白病。
如本文中所使用的，术语“磷酸化”是指，在蛋白的氨基酸残基上加入磷酸基团。通常，苏氨酸，丝氨酸、酪氨酸等氨基酸残基具有羟基基团，因此容易被磷酸化。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条轻链 (LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987 and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDR，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。
在本发明中，本发明的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或IMGT编号系统确定。
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
如本文中所使用的，术语“单克隆抗体”、“单抗”、“mAb”具有相同的含义且可互换使用可互换，其是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即，除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。此外，修饰语“单克隆”仅表明该抗体的特征为从高度同源的抗体群中获得，不能理解为需要通过任何特定方法来制备所述抗体。
本发明的单克隆抗体可以通过多种技术进行制备，例如杂交瘤技术(参见，例如Kohler等人，Nature,256:495，1975)，重组DNA技术(参见，例如美国专利申请4,816,567)，或噬菌体抗体库技术(参见，例如Clackson等.Nature352：624-628,1991，或Marks等.J.Mol.Biol.222：581-597,1991)。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、F(ab’) 2、Fd、Fv、互补决定区(CDR)片段、scFv、双抗体(diabody)、单域抗体(single domain antibody)、嵌合抗体、线性抗体(linear antibody)、纳米抗体(技术来自Domantis)、、probody和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126-1136中。
如本文中所使用的，术语“全长抗体”意指，由两条“全长重链”和两条“全长轻链”组成的抗体。其中，“全长重链”是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域。优选地，“全长重链”是在N端到C端方向上由VH、CH1、HR、CH2和CH3组成的多肽链。“全长轻链”是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链。两对全长抗体链通过在CL和CH1之间的二硫键和两条全长重链的HR之间的二硫键连接在一起。本发明的全长抗体可以来自单一物种，例如人；也可以是嵌合抗体或人源化抗体。本发明的全长抗体包含分别 由VH和VL对形成的两个抗原结合部位，这两个抗原结合部位特异性识别/结合相同的抗原。
如本文中所使用的，术语“Fd”意指由VH和CH1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’) 2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’) 2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。
如本文中所使用的，术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
如本文中所使用的，术语“Fc”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键。在本发明的某些实施方案中，scFv可形成di-scFv，其指的是两个或两个以上单个scFv串联而形成抗体。在本发明的某些实施方案中，scFv可形成(scFv)2，其指的是两个或两个以上单个scFv并联而形成抗体。
如本文中所使用的，术语“单域抗体(single-domain antibody,sdAb)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，其保持特异性结合全长抗体所结合的相同抗原的能力。单域抗体也称为纳米抗体(nanobody)。
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“嵌合抗体(Chimeric antibody)”是指，这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567 to Cabilly et al.；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。在某些实施方案中，术语“嵌合抗体”可包括这样的抗体，其中抗体的重链和轻链可变区来自第一抗体，而抗体的重链和轻链恒定区来自第二抗体。
如本文中所使用的，术语“变体”，在多肽的情境中(包括多肽)也指包含已通过引入氨基酸残基置换、缺失或添加改变的氨基酸序列的多肽或肽。在某些情况下，术语“变体”还指已被修饰(即，通过将任何类型的分子共价连接至多肽或肽)的多肽或肽。例如，但非限制性地，多肽可以被修饰，例如通过糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过已知保护/封闭基团进行的衍生化、蛋白水解切割、连接至细胞配体或其它蛋白质等。衍生多肽或肽可使用本领域技术人员已知的技术通过化学修饰来产生，所述技术包括但不限于特异性化学切割、乙酰化、甲酰化、衣霉素的代谢合成等。此外，变体具有与其所源自的多肽或肽相似、相同或改善的功能。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该 相互作用的平衡解离常数(KD)表示。在本发明中，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,Nature,1993,361:186-187)。kdis/kon的比率等于解离常数KD(参见Davies等人,Annual Rev Biochem,1990；59:439-473)。可用任何有效的方法测量KD、kon和kdis值。在某些实施方案中，可以使用表面等离子体共振术(SPR)在Biacore中来测量解离常数。除此以外还可用生物发光干涉测量法或Kinexa来测量解离常数。
如本文中所使用的，本发明所述的可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的，其实例包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如，3H、125I、35S、14C或32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、发光物质(例如化学发光物质，如吖啶酯类化合物、鲁米诺及其衍生物、钌衍生物如三联吡啶钌)、磁珠(例如，
Figure PCTCN2022092435-appb-000001
)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始 位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离 子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。在某些示例性实施方案中，所述药学上可接受的载体或赋形剂包括无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括(但不限于)减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中所使用的，术语“受试者”是指哺乳动物，例如人、食蟹猴、小鼠。在某些实施方案中，所述受试者(例如人、食蟹猴、小鼠)患有与TIGIT相关的疾病(例如，涉及TIGIT阳性的侵润T细胞和/或NK细胞，和/或涉及TIGIT配体(例如CD155和/或CD112)阳性的肿瘤细胞的肿瘤)，或者，具有患有上述疾病的风险。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，涉及TIGIT阳性的侵润T细胞和/或NK细胞，和/或涉及TIGIT配体(例如CD155和/或CD112)阳性的肿瘤细胞的肿瘤)有效量是指，足以预防，阻止，或延迟所述疾病的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域 技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
发明的有益效果
本申请的单克隆抗体(例如，2A7抗体)能够以高特异性与p-tau 217蛋白结合。同时，本申请的单克隆抗体还能够检测p-tau 217蛋白的含量(例如，受试者脑脊液中p-tau 217的含量)，因此所述单抗能够用于鉴定或检测AD患者，并区分AD患者与其它tau蛋白病患者。
将所述单抗施用于受试者，能够改善患有tau蛋白病受试者的行为与能力(例如，探索新物体的时间明显增加，对周围光亮感知及空间趋避能力提高，空间学习及记忆能力提高)，抑制受试者海马萎缩情况，并改善脑组织病理变化。因此，本申请的单克隆抗体(例如，2A7抗体)在AD的检测、预防、以及AD与其它tau蛋白病的治疗中具有较高的临床应用价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了实施例2.1中抗体2A7和GAPDH(内部参照)对不同蛋白的免疫印迹结果。
图2显示了实施例2.1中抗体2A7对不同蛋白的组织免疫荧光染色结果；其中，2A7和无关单抗结合区域显示绿色，NeuN特异性抗体结合区显示红色，DAPI结合区显示蓝色。
图3显示了实施例2.2中抗体2A7和GAPDH(内部参照)对不同蛋白的免疫印迹结果。
图4显示了实施例3中2A7抗体在AD病人脑脊液和PS19小鼠脑脊液样品中对p-tau 217含量的检测结果。
图5显示了经2A7抗体治疗后10.5月龄PS19小鼠的行为学改善结果。其中，图5A 显示了新物体识测试中PS19-2A7组与PS19-IgG相比小鼠探索新物体的时间明显增加；图5B显示了旷场实验测试中，经2A7抗体治疗后，PS19小鼠在箱体中间时间停留明显减少，表明小鼠对周围光亮感知及空间趋避能力提高；图5C和图5D显示了Morris水迷宫实验中，PS19-2A7组小鼠空间学习及记忆能力与PS19-IgG相比有明显提高。
图6显示了经2A7抗体治疗后10.5月龄PS19小鼠海马萎缩的抑制结果。其中，图6A为核磁共振检测WT-IgG、PS19-IgG与PS19-2A7组小鼠脑轴向位解剖结构(T1)及3D重构的结果；图6B为WT-IgG、PS19-IgG与PS19-2A7组小鼠海马体积统计结果。
图7显示了经2A7抗体治疗后10.5月龄PS19小鼠病理减轻的结果。其中，图7A免疫荧光染色结果显示PS19-2A7组小鼠神经元(NeuN)丢失明显减少；图7B为WT-IgG、PS19-IgG与PS19-2A7组小鼠NeuN荧光强度统计结果；图7C显示了PS19-2A7组小鼠p-tau 217(2A7)的染色信号明显减少；图7D为WT-IgG、PS19-IgG与PS19-2A7组小鼠2A7荧光强度统计结果；图7E显示了PS19-2A7组小鼠小胶质细胞(IBA1)增生明显被抑制；图7F为WT-IgG、PS19-IgG与PS19-2A7组小鼠IBA1荧光强度统计结果。
注：量化结果显示为平均值±SEM。统计分析是用GraphPad Prism软件(9.0版，https://www.graphpad.com/)进行的。在适当情况下，通过非配对t检验或单因素方差分析评估差异。P值<0.05被认为是有统计学意义的。*代表P<0.05,**代表P<0.01,***代表P<0.001。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述
Figure PCTCN2022092435-appb-000002
Figure PCTCN2022092435-appb-000003
Figure PCTCN2022092435-appb-000004
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。例如，本发明中所使用的免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等常规技术，可参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis)，《分子克隆：实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL)，第2次编辑(1989)；《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑，(1987))；《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司)：《PCR 2：实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))，以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。
另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。例如，本发明中所使用的免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等常规技术，可参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis)，《分子克隆：实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL)，第 2次编辑(1989)；《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑，(1987))；《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司)：《PCR 2：实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))，以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。
另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1.特异性识别217磷酸化位点的单克隆抗体的制备
本研究采用第217位氨基酸磷酸化的多肽进行免疫，同时用217位点非磷酸化的多肽进行差异筛选进行特异性抗体的筛选。
1.1免疫原的制备
免疫原为KLH-C-RSRTPSLPT(p)PPTREP，其是一段磷酸化的多肽，氨基酸序列对应于天然tau蛋白的第209-223位氨基酸序列(其氨基酸序列如SEQ ID NO:9所示)。本研究所使用的多肽均通过化学合同得到，均由南京钟鼎生物技术有限公司合成。
1.2实验小鼠
6周龄SPF级雌性Balb/C小鼠。
1.3杂交瘤的制备
使用标准的体内免疫方式和PEG融合方法获得分泌单抗的杂交瘤细胞，详细方法参见Ed Harlow et al.,“Antibodies A Laboratory Manual”,Cold Spring Harbor Laboratory1988。简要过程如下：
1、小鼠免疫：首先将100ug多肽与弗式完全佐剂(CFA)等体积混合并乳化，然后经四肢肌肉多点注射对小鼠进行首次免疫。其次将50ug多肽与弗式不完全佐剂(IFA)等体积混合并乳化，然后分别于首次免疫后第14天、28天、42天对小鼠进行加强免疫。最后，于首次免疫后第56天对小鼠进行腹腔免疫加强，免疫原为50ug多肽与PBS的等体积混合液。免疫结束后3天，后取小鼠脾脏进行融合实验。
2、细胞融合：取小鼠脾脏，研磨得到脾脏细胞悬液，然后与处于对数生长期的小鼠 骨髓瘤细胞SP2/0混合，并在PEG1500作用下进行细胞融合。将融合后的细胞用300mL融合培养基(含HAT和20％FBS的RPMI-1640培养基)重悬，并分装到15块96孔细胞培养板中进行培养。
3、杂交瘤的筛选：将融合后的细胞在96孔细胞培养板上培养7到10天，然后吸取细胞上清进行ELISA检测。检测用的多肽为217位点磷酸化的多肽。对于ELISA检测阳性的细胞孔，用217位点非磷酸化的多肽进行ELISA差异检测。对筛选到的对217位点磷酸化的多肽有反应、对217位点非磷酸化的多肽无反应的阳性克隆进行3次克隆化(每次克隆化检测均进行差异筛选)，得到能稳定分泌抗体的杂交瘤细胞株。最终获得抗217位点磷酸化多肽的2A7细胞株。
4、杂交瘤的培养：将该稳定的杂交瘤单抗细胞株在二氧化碳培养箱中进行扩增培养，依次从96孔板转至24孔板、6孔板、10cm细胞板中。然后收集细胞板中的细胞，将其注射到小鼠腹腔内，7到10天后从小鼠腹腔中吸取含有单抗的腹水。
1.4单抗的纯化：将含有单抗的小鼠腹水用50％的饱和硫酸铵溶液进行处理。之后将获得沉淀溶解于PBS中，并使用Protein A柱纯化，以获得经纯化的单抗，并通过SDS-PAGE来鉴定所获得的单抗的纯度。
将获得的单抗进行PCR扩增，并将PCR产物送公司测序以获得序列，将获得的抗体命名为2A7，其具体序列如表1所示，其中，抗体的CDR序列由IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)确定。
实施例2. 2A7的特异性鉴定
2.1 2A7对天然tau蛋白的反应性及特异性
通过对PS19小鼠的脑组织进行蛋白质免疫印迹实验(Western Blot,WB)验证2A7对天然tau蛋白的反应性及特异性。分别取6月龄的野生型(Wild type,WT)小鼠(含有鼠源tau蛋白)、tau基因KO的野生型小鼠(Tau-KO)(不含有tau蛋白)以及PS19小鼠(过表达P301S突变tau蛋白(人源)的转基因小鼠，表达过度人源磷酸化的tau蛋白)的脑组织。其中WT和Tau-KO小鼠脑组织分别用TNEN裂解液(20mM Tris-HCl pH＝7.4,100mM NaCl,1mM EDTA,0.5％NP40)重悬，添加蛋白酶抑制剂和磷酸酶抑制剂后进行裂解。裂解完成后，4℃条件下12000rpm离心10min，取上清保存于-80℃备用。PS19小鼠脑组织同样使用TNEN裂解液重悬，添加蛋白酶抑制剂后进行裂解，离心后上清等分为三份，第一份加入磷酸酶抑制剂，存于-80℃备用；第二份加入磷酸酶抑制剂，混合均匀取出一部分37℃孵育1h；第三份加入碱性磷酸酶，混合 均匀取出一部分37℃孵育1h。分别取15μg以上鼠脑裂解液进行WB，以验证2A7对天然tau的反应性和特异性，以GAPDH作为内参。2A7的使用浓度为1μg/mL，并使用1:1000稀释的HRP标记的马抗鼠IgG(HAM-HRP,CST,7076S)进行显影(上海勤翔，ChemiScope 6200)。
结果如图1所示，在图1中，2A7对PS19鼠脑裂解液具有很强的反应性(如图1中泳道3(PS19)和泳道5(PS19，37℃)所示)，经碱性磷酸酶处理后，反应性显著降低(如图1中泳道4(PS19+CIP)所示)。同时，对WT小鼠、Tau基因KO的WT小鼠仅有弱反应性(分别如图1中泳道1(WT)、泳道2(Tau-KO)所示)。
通过组织免疫荧光染色对2A7的反应性及特异性进行验证。实验分为6组，分别使用AD无关IgG抗体(对应于图2中第二列IgG)、2A7抗体(对应于图2中第四列p-tau 217)、217位点磷酸化的多肽阻断后的2A7抗体(对应于图2中第五列217 peptide block)对PS19小鼠脑组织切片进行染色；分别使用AD无关IgG抗体(对应于图2中第一列IgG)以及2A7抗体(对应于图2中第三列p-tau 217)对WT小鼠脑组织切片进行染色。其中，以上抗体使用量均为1μg，多肽使用量均为5μg。取13月龄的WT及PS19小鼠脑组织进行冰冻切片。将两片WT小鼠脑组织、四片PS19小鼠脑组织分别用PBST洗3遍，每次洗涤10分钟。之后分别用含有10％的驴血清(Solarbo,SL050)以及0.3％Triton X-100的PBST封闭，室温孵育1小时。将以上抗体分别加入200μL封闭液中，并加入到对应的小鼠脑组织中，4℃孵育过夜。回收以上抗体，用PBST洗涤脑片3次，每次洗涤10分钟。用封闭液将荧光二抗以及DAPI分别按照1:500、1:1000的比例稀释，并加入到对应的小鼠脑组织切片中，室温避光孵育1小时。之后除去荧光标记的二抗，避光条件下，PBST洗涤脑片3次，每次洗涤10分钟。用抗荧光淬灭剂进行封片，4度避光保存。最后用Zeiss 880进行拍照。
结果如图2所示，与WT小鼠以及无关抗体相比，2A7抗体可以特异性的识别PS19海马区和皮层区的tau蛋白，经与217位点磷酸化的多肽孵育后，2A7抗体对PS19海马区和皮层区tau蛋白的结合能力降低。
2.2 2A7对天然tau蛋白217位点突变体的反应性及特异性
分别构建CTR4-Tau以及217磷酸化位点突变的CTR4-T217A载体，CTR4-Tau载体的插入片段为编码tau蛋白(如SEQ ID NO:10所示)的核苷酸序列，CTR4-T217A载体的插入片段为编码第217位突变的tau蛋白(如SEQ ID NO:11所示)的核苷酸序列。同时转染293T细胞，48h后收集细胞，以PLCDH-GFP作为对照。利用TNEN(含蛋白酶抑制剂和磷酸酶抑制剂)进行裂解，裂解完成后在4℃条件下12000rpm离心10 min,取上清测浓度后保存于-80℃备用。
以上清测定浓度后，分别取15μg进行WB，以验证2A7的特异性，以β-actin作为内参。2A7的使用浓度为1ug/mL，并使用1:1000稀释的GAM-HRP作为二抗，进行显影。
真核表达的tau蛋白第217位氨基酸会被细胞内的激酶磷酸化，因此可以被2A7抗体所识别；而217位点的突变是将217位的氨基酸T突变为A，突变后该位点氨基酸不能被磷酸化。结果如图3所示，2A7对真核表达的tau蛋白(图3中的泳道2)的反应性高于217位点突变后的tau蛋白(图3中的泳道3)。
实施例3. 2A7抗体对AD病人及PS19小鼠脑脊液中p-tau 217的检测
本实施例采用tau蛋白病的模型小鼠，即PS19小鼠进行实验。取13月龄的PS19小鼠的脑脊液(CSF)。对于脑脊液的采集，参考Lim等优化的采集小鼠脑脊液的方法(具体请参见Lim,N.K.,V.Moestrup,X.Zhang,W.A.Wang,A.Moller and F.D.Huang(2018)."An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice."J Vis Exp(133))，即麻醉小鼠，通过手术暴露出小鼠枕大池上的硬脑膜，在体视镜下，避开血管，使用玻璃毛细管尖端刺破硬脑膜吸取脑脊液，然后将脑脊液收集于加有蛋白酶抑制剂和磷酸酶抑制剂的1.5mL离心管中，存于-80℃备用。16例AD病人脑脊液，来源于苏州宇测生物有限公司。
使用检测试剂盒(货号：Lite-P64050，苏州宇测)，对AD及PS19小鼠脑脊液中p-tau 217的含量进行检测。其中小鼠脑脊液用马血清稀释5倍，AD脑脊液样本原倍检测，样本反应及分析均在Ast-Dx90单分子免疫诊断仪进行。
按Sc-lite单分子免疫检测仪的说明书操作步骤完成试剂校准质控流程。将样本、试剂按顺序装载至指定位置，准备就绪后启动测试，设备自动将样本送入上样位置，同时将反应杯载入孵育盘中，取样针从96孔板中吸取25μL样品加入反应杯，试剂针从试剂盒中吸取25μL包被有捕获抗体的磁珠溶液(试剂1)加入反应杯，混匀孵育6min。
试剂针从试剂盒中吸取10μL修饰有单分子信号标记物的检测抗体(试剂2)加入反应杯，混匀孵育4min，试剂2中含有修饰检测抗体的单分子信号标记物，能够将靶标分子转化为单分子信号。
检测针将反应体系转移至流通池中，利用磁分离将磁珠吸引至流通池底部并平铺于检测孔的表面，洗涤去除其他组分，然后用集成荧光显微镜拍摄荧光图像，通过机器分析单分子信号，并使用预先准备的标准曲线计算生物标志物浓度。
实验结果如图4所示，图4显示使用2A7抗体可以检测出AD病人脑脊液及PS19小鼠脑脊液等样品中的p-tau 217的含量。
实施例4. PS19小鼠2A7抗体的鼻腔给药
本研究采用鼻腔给药的方式，检测2A7抗体对PS19小鼠(tau蛋白病的模型小鼠)的病理及行为学的改善作用。
4.1抗体准备
将制备好的2A7抗体以及与AD无关IgG抗体用PBS稀释至浓度为0.5μg/μl及1μg/μl，分装后于-80度保存。
4.2实验小鼠及分组
5月龄SPF级雄性PS19小鼠，20～25g。实验分为3组：WT小鼠经鼻腔给IgG抗体组(WT-IgG)、PS19小鼠经鼻腔给IgG抗体组(PS19-IgG)及PS19小鼠经鼻腔给2A7抗体组(PS19-2A7)，每组13只小鼠。
4.3抗体鼻腔给药、剂量
实验的小鼠均采用鼻腔给药，用异氟烷麻醉后将其仰卧，用微量注射器缓慢将抗体滴入鼻孔，滴入时将嘴闭合，以利于溶液吸收。
每3天给抗体一次，连续给药5个月。每只小鼠给药体积为20μl，前3个月每只给药10μg，后两个月每只给药20μg。
实施例5. PS19小鼠经2A7抗体治疗后行为学变化
实施例4中处理过的所有小鼠的行为学实验均采用Smart Video Tracking Software(Panlab,Harvard Apparatus)进行数据采集及分析处理。动物行为学实验于每天9：00a.m.－7：00p.m.之间进行，实验室内的光强度为650lux。
a.测试者于实验开始前三天接触抚摸小鼠，每天一次，每次抚摸一只小鼠，轻柔地抓取小鼠，让小鼠在测试者手上停留30s，并用记号笔在鼠尾上划线标记，标记完小鼠后抓取尾巴，轻柔地将其放回鼠笼；
b.实验当天，在实验前先将待测试小鼠转移至实验间，让小鼠适应周围环境及光线；准备开始实验前，用75％乙醇清洁实验所用的箱体和迷宫。每轮实验结束后使用75％乙醇对箱体和迷宫进行擦拭，清除实验过程中小鼠排泄的粪便和尿液，排除小鼠残留的气味对测试结果的干扰。
5.1旷场
旷场实验用于研究小鼠的自主运动能力以及焦虑行为，主要基于小鼠对于亮光以及开阔空间的趋避性。将旷场箱内部场地(长40cmx宽40cmx高40cm)分为16小格，周边12格定义为周边区，中间4格定义为中央区(Center)；将小鼠放入旷场箱内正中央，每只小鼠放置位置要相同(同一格、同一方向)；让小鼠在迷宫里自由探索5min，记录小鼠在迷宫中总的运动距离以及在迷宫中间区域活动的时间(Time in center)。
5.2新物体识
新物体识别实验是利用啮齿类动物天性对于新物体有好奇探索的原理而建立的学习记忆测试方法。
第一天，将小鼠放入旷场箱(长40cmx宽40cmx高40cm)内正中央，每只小鼠放置位置相同(同一格、同一方向)，让小鼠适应性训练5min；第二天，在旷场箱内一侧放置两个相同的物体A和B，将小鼠面向箱壁轻轻放入旷场箱内，放置位置与两物体间的尽量距离相同，让小鼠自由探索8min；第三天，保持物体位置不变，用新物体C更换其中一个旧物体(A或B)；将小鼠面向箱臂放入旷场箱内，放入位置与两物体间的距离相同，让小鼠自由探索8min小鼠鼻子触及物体或者鼻子在距离物体2cm内指向物体都被视为探索行为；用摄像系统记录小鼠探索熟悉物体和探索新物体的时间。
5.3水迷宫测试
Morris水迷宫实验用于研究评价小鼠的空间学习、记忆能力。
水迷宫在一个圆形的水箱(半径60cmx高100cm)中进行，水箱中注水的高度以超过平台2cm为宜，水箱内水的温度设定为22℃。迷宫臂内四个方向(E、S、W、N)分别贴有四个不同形状的图标作为空间定位参照物。在训练实验中，平台在水面下2cm，然后将小鼠从迷宫的四个入水点放入，让小鼠寻找平台60s，以小鼠停留在平台上10s为实验停止的标准。若小鼠在60s内没法找不平台，就用尺子引导小鼠到平台所在的位置，并让小鼠在平台上面停留10s。每只小鼠每天测试2次，2次分别从两个不同的方位入水，每只小鼠两次实验的间隔时间至少要1h。记录小鼠找到平台的潜伏时间(Escape latency)，小鼠总的游泳距离和游泳的平均速度，如此连续进行7天的学习训练。
平台实验：第8天，撤掉平台，然后从平台对角线的位置将小鼠轻轻放入水中，让其搜索原来平台所在区域60s，记录小鼠在原来平台所在区域穿梭的次数(Number of crossing)以及小鼠在平台所在目标象限以及其他三个不同象限的游泳时间(Time in quadrants)。每天更换迷宫中的水，周围物体和实验人员数量和位置固定不变。
实验结果如图5所示，经2A7抗体治疗后，10.5月龄PS19小鼠行为学有明显改 善。具体来说，新物体识测试中PS19-2A7组与PS19-IgG相比小鼠探索新物体的时间明显增加(图5A)；旷场实验测试中，经2A7抗体治疗后，PS19小鼠在箱体中间时间停留明显减少，表明小鼠对周围光亮感知及空间趋避能力提高(图5B)。Morris水迷宫实验显示，PS19-2A7组小鼠空间学习及记忆能力与PS19-IgG相比有明显提高(图5C和图5D)。
实施例6.核磁共振检测PS19小鼠经2A7抗体治疗后海马体积变化
行为学结束后，将上述实验的小鼠(10.5月龄)进行磁共振成像(MRI)。未经治疗或经无效抗体治疗的PS19小鼠海马体积会有明显萎缩，2A7抗体的治疗效果可通过小鼠海马体积萎缩程度来进行评价。
磁共振成像数据采集与分析
所有实验动物MRI实验均在9.4 T Bruker小动物成像仪上进行，实验中所使用的线圈为小动物脑线圈。动物成像过程中全程使用1.5％异氟烷/氧气混合气体对小鼠进行麻醉，同时利用呼吸监控传感器实时监测小鼠的呼吸状况。使用扫描仪进行轴状位T1WI-3D解剖结构及T2WI-3D病理结构成像扫描；磁共振扫描序列及参数如下：(1)T1-weighted MRI：TR(重复时间)＝2500ms，TE(回波时间)＝33ms，field of view(扫描野)＝2×2cm，matrix(矩阵)＝256×256，SI(层间距)＝0.5mm，FA(反转角)＝180°，slices(层数)＝15。核磁共振扫描结果使用imageJ对图像进行分析，并对海马区进行3D重构。
实验结果如图6所示，图6A为核磁共振检测WT-IgG、PS19-IgG与PS19-2A7组小鼠脑轴向位解剖结构(T1)及3D重构的结果；图6B为WT-IgG、PS19-IgG与PS19-2A7组小鼠海马体积统计结果。上述结果显示，经2A7抗体治疗后，10.5月龄PS19小鼠海马萎缩情况明显被抑制。
实施例7. PS19小鼠经2A7抗体治疗后脑组织病理变化
7.1荧光抗体及使用比例
抗体X＝1:200；AT8＝1:200(Invitrogen，MN1020)；IBA1＝1:500(Wako，019-19741)；NeuN＝1:400(CST，24307)
7.2组织冰冻切片
1、取上述实验的小鼠，用5％水合氯醛麻醉，分别用预冷的1×PBS和4％PFA(in1×PBS，pH7.4)进行灌注，剥离取出大脑，并于4％PFA中继续固定，4℃过夜；
2、第二天，倾去固定液，换为25％蔗糖(in 1×PBS)于4℃进行脱水。
3、第三天，倾去25％蔗糖，换为30％蔗糖(in 1×PBS)于4℃进行脱水。每天更换蔗糖，30％蔗糖持续脱水3天。待组织脱水彻底后，用干净的滤纸吸去大脑组织表面液体，用德国徕卡Leica冰冻包埋剂(JUNG tissue freezing medium)包埋大脑，进行冰冻切片，切片厚度为40μm；
4、选取完好合适的切片，于1×PBS中，室温漂洗，10minx3次；
5、弃PBS，用含有10％的驴血清(Solarbio,SL050)以及0.3％Triton X-100的PBS封闭，室温孵育1小时。
6、吸去封闭液，孵育一抗；一抗用封闭液(含10％的驴血清+0.3％Triton X-100，in 1×PBS)稀释成所需浓度，4℃孵育过夜；
7、回收一抗，1×PBST(含0.3％Triton X-100的1×PBS)洗10minx3次；吸去PBST，孵育二抗；用封闭液将荧光二抗以及DAPI分别按照1:500、1:1000的比例稀释，并加入到对应的小鼠脑组织切片中，室温避光孵育1小时。
8、吸去二抗，1×PBST洗10minx3次；吸去PBST，将脑片用毛笔挑取放在1×PBS中，用粘附性玻片捞取脑片，晾干，用索莱宝抗荧光淬灭剂封片，然后用指甲油将盖玻片边缘封闭好，4度避光保存。
9、用倒置激光共聚焦(Zeiss 880)拍照分析。
实验结果如图7所示，其中，图7A、B免疫荧光染色结果显示PS19-2A7组小鼠神经元丢失明显减少；图7C、D显示了PS19-2A7组小鼠磷酸化p-tau 217信号明显减少；图7E、F显示了PS19-2A7组小鼠小胶质细胞增生明显被抑制。实验结果表明，经2A7抗体治疗后，10.5月龄PS19小鼠病理明显减轻。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (15)

  1. 特异性结合p-tau 217蛋白的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (a)包含下述3个互补决定区(CDRs)的重链可变区(VH)：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO:3，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO:4，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO:5，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    和/或，
    (b)包含下述3个互补决定区(CDRs)的轻链可变区(VL)：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO:6，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
    (v)VL CDR2，其由下述序列组成：SEQ ID NO:7，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
    (vi)VL CDR3，其由下述序列组成：SEQ ID NO:8，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    优选地，(i)-(vi)中任一项所述的置换为保守置换；
    优选地，(i)-(vi)中任一项所述的CDR根据Kabat、Chothia或IMGT编号系统定义；
    优选地，(i)-(vi)中任一项所述的CDR根据IMGT编号系统定义；
    优选地，所述抗体或其抗原结合片段包含如下3个重链CDRs：如SEQ ID NO:3所示的VH CDR1、如SEQ ID NO:4所示的VH CDR2、如SEQ ID NO:5所示的VH CDR3；和/或，如下3个轻链CDRs：如SEQ ID NO:6所示的VL CDR1、如SEQ ID NO:7所示的VL CDR2、如SEQ ID NO:8所示的VL CDR3。
  2. 权利要求1的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)重链可变区(VH)，其包含选自下列的氨基酸序列：
    (i)SEQ ID NO:1所示的序列；
    (ii)与SEQ ID NO:1所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO:1所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和/或
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
    (iv)SEQ ID NO:2所示的序列；
    (v)与SEQ ID NO:2所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (vi)与SEQ ID NO:2所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的重链框架区序列和/或轻链框架区序列；
    优选地，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:1所示的序列的VH和具有如SEQ ID NO:2所示的序列的VL。
  3. 权利要求1或2所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的恒定区或其变体；
    优选地，所述抗体或其抗原结合片段包含：
    (a)人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)；和/或
    (b)人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)；
    优选地，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区；
    优选地，所述轻链恒定区是κ轻链恒定区。
  4. 权利要求1-3任一项所述的抗体或其抗原结合片段，其中，所述抗原结合片段选自Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、BsFv、dsFv、(dsFv) 2、dsFv-dsFv'、 scFv、scFv二聚体、骆驼化单域抗体(camelized single chain domain antibody)、双抗体(diabody)、ds双功能抗体(ds diabody)、纳米抗体、单域抗体(sdAb)、双价域抗体；和/或，所述抗体为鼠源抗体、嵌合抗体、人源化抗体或多特异性抗体。
  5. 权利要求1-4任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段带有标记；优选地，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
  6. 分离的核酸分子，其编码权利要求1-5任一项所述的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区；
    优选地，所述核酸分子包含如SEQ ID NO:12或SEQ ID NO:13中所示的核苷酸序列。
  7. 载体，其包含权利要求6所述的核酸分子；优选地，所述载体为克隆载体或表达载体。
  8. 宿主细胞，其包含权利要求6的核酸分子或权利要求7的载体。
  9. 制备权利要求1-5任一项所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养权利要求8所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段；
    优选地，所述宿主细胞是哺乳动物细胞。
  10. 多特异性分子，其包含权利要求1-5任一项所述的抗体或其抗原结合片段；
    优选地，所述多特异性分子特异性结合p-tau 217蛋白，并且额外地特异性结合一个或多个其他靶标；
    优选地，所述多特异性分子还包含至少一种具有针对第二靶标的第二结合特异性的分子(例如第二抗体或其抗原结合片段)。
  11. 药物组合物，其包含权利要求1-5任一项所述的抗体或其抗原结合片段，或权利要求10所述的多特异性分子，以及药学上可接受的载体和/或赋形剂；
    优选地，药物组合物还包含另外的药学活性剂；
    优选地，所述另外的药学活性剂是具有治疗tau蛋白病(例如，AD)活性的药物。
  12. 试剂盒，其含有权利要求1-5任一项所述的抗体或其抗原结合片段；
    优选地，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素；
    优选地，所述试剂盒还包括第二抗体，其特异性识别权利要求1-5任一项所述的抗体或其抗原结合片段；
    优选地，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素；
    优选地，所述试剂盒用于检测样本中p-tau 217的存在或其含量；
    优选地，所述样本为获自受试者的脑脊液，全血，血清或血浆；
    优选地，所述试剂盒还包含稀释样本的试剂(例如，马血清)；
    优选地，所述第二抗体包被于磁珠上。
  13. 权利要求1-5任一项所述的抗体或其抗原结合片段，或权利要求10所述的多特异性分子，或权利要求11所述的药物组合物，在制备药物中的用途，所述药物用于在受试者(例如人)中预防和/或治疗tau蛋白病；
    优选地，所述tau蛋白病选自阿尔兹海默症(AD)、原发性年龄相关的tau蛋白病、慢性创伤性脑病、Pick病和皮质基底节变性；
    优选地，所述tau蛋白病是AD；
    优选地，药物还包含另外的治疗tau蛋白病(例如，AD)活性的药学活性剂；
    优选地，所述受试者的脑脊液中含有p-tau 217蛋白；
    优选地，所述受试者为哺乳动物，例如人。
  14. 一种检测p-tau 217蛋白在样品中的存在或其量的方法，其包括以下步骤：
    (1)将所述样品与权利要求1-5任一项所述的抗体或其抗原结合片段接触；
    (2)检测所述抗体或其抗原结合片段与p-tau 217蛋白之间复合物的形成或检测所述复合物的量；
    优选地，所述抗体或其抗原结合片段带有可检测的标记。
  15. 权利要求1-5任一项所述的抗体或其抗原结合片段，或权利要求10所述的多特异性分子，在制备试剂中的用途，所述试剂用于检测受试者是否患tau蛋白病，或者用于区分患阿尔兹海默症(AD)的患者或患其它tau蛋白病的患者；
    优选地，所述tau蛋白病选自阿尔兹海默症(AD)、原发性年龄相关的tau蛋白病、慢性创伤性脑病、Pick病和皮质基底节变性；
    优选地，所述tau蛋白病是AD；
    优选地，所述试剂通过检测p-tau 217蛋白在样品中的量，以检测受试者是否患tau蛋白病，或者以区分患阿尔兹海默症(AD)的患者或患其它tau蛋白病的患者，且所述样品获自所述受试者或患者；
    优选地，所述样品为获自受试者或患者的血液样品(例如，全血，血清，血浆)。
PCT/CN2022/092435 2022-04-28 2022-05-12 针对p-tau 217的抗体及其用途 WO2023206609A1 (zh)

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