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WO2025168025A1 - Agent de liaison à la pompe à protons et son utilisation dans la préparation d'un réactif - Google Patents

Agent de liaison à la pompe à protons et son utilisation dans la préparation d'un réactif

Info

Publication number
WO2025168025A1
WO2025168025A1 PCT/CN2025/076106 CN2025076106W WO2025168025A1 WO 2025168025 A1 WO2025168025 A1 WO 2025168025A1 CN 2025076106 W CN2025076106 W CN 2025076106W WO 2025168025 A1 WO2025168025 A1 WO 2025168025A1
Authority
WO
WIPO (PCT)
Prior art keywords
binding agent
atp6v1b2
amino acid
acid sequence
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/076106
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English (en)
Chinese (zh)
Inventor
李开诚
李震
攸璞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Quietd Biotechnology Co Ltd
Original Assignee
Shanghai Quietd Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Quietd Biotechnology Co Ltd filed Critical Shanghai Quietd Biotechnology Co Ltd
Publication of WO2025168025A1 publication Critical patent/WO2025168025A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present application relates to the field of biomedicine, and specifically to a proton pump binder and its use in preparing reagents.
  • AD Alzheimer's disease
  • the ATP6V1B2 protein encoded by the ATP6V1B2 gene, is a key structural component of the proton pump driven by ATP hydrolysis. It is widely distributed throughout human tissues, but is more abundant in the brain, kidneys, and osteoclasts. It plays a crucial role in synaptic transmission and lysosomal acidification. Mutations in the gene can cause DOORS syndrome (predisposed autosomal dominant congenital deafness with nail dysplasia syndrome). Reduced ATP6V1B2 protein expression may be associated with the development of AD.
  • the present application provides a proton pump binder, which can be used as a potential drug for improving a subject's learning ability, treating cognitive impairment, preventing and/or treating neurodegenerative diseases, and/or preventing and/or treating stroke.
  • the present application provides a binding agent comprising a molecule capable of binding to ATP6V1B2 and/or its functionally active fragment, wherein the molecule comprises the amino acid sequence shown in SEQ ID NO: 19 and/or its variant, wherein X is any amino acid.
  • the molecule comprises the amino acid sequence shown in any one of SEQ ID NOs: 20-24 and/or its variants, wherein X is any amino acid.
  • the molecule comprises the amino acid sequence shown in any one of SEQ ID NOs: 25-30 and/or its variants, wherein X is any amino acid.
  • the molecule comprises the amino acid sequence shown in any one of SEQ ID NOs: 31-35 and/or its variants.
  • the molecule comprises the amino acid sequence shown in any one of SEQ ID NOs: 36-41 and/or its variants.
  • the molecule comprises the amino acid sequence shown in any one of SEQ ID NOs: 42-48 and/or its variants.
  • the molecules comprise proteins and/or polypeptides.
  • the molecule comprises a multimer.
  • the multimer comprises a homodimer.
  • cysteine residues in the amino acid sequence of the molecule do not have sulfhydryl blocking modifications.
  • the serine in the amino acid sequence of the molecule does not have a phosphorylation modification.
  • the molecule comprises a fusion protein and/or a fusion polypeptide.
  • the fusion protein and/or fusion polypeptide comprises a molecule capable of being transported across the blood-brain barrier to the brain and/or a molecule capable of crossing a cell membrane.
  • the molecule capable of being transported across the blood-brain barrier to the brain and/or the molecule capable of crossing the cell membrane comprises a polypeptide.
  • the molecule capable of being transported across the blood-brain barrier to the brain and/or the molecule capable of crossing the cell membrane comprises a cell-penetrating peptide.
  • the membrane-penetrating peptide comprises an amino acid sequence as shown in any one of SEQ ID NOs: 79-84 and/or its variants.
  • it comprises an amino acid sequence as shown in any one of SEQ ID NOs: 49-71 and/or a variant thereof, wherein X is any amino acid.
  • the functionally active fragment of ATP6V1B2 has the ability to specifically bind to the amino acid sequence shown in SEQ ID NO: 5.
  • it comprises the amino acid sequence shown in any one of SEQ ID NOs: 42-48 and/or its variants.
  • the molecule that is transported across the blood-brain barrier to the brain and/or the molecule that is capable of crossing cell membranes comprises a polypeptide.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the binding agent described herein, the polypeptide described herein, the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein and/or the cell described herein, and optionally a pharmaceutically acceptable carrier.
  • the neurodegenerative disease includes a neurodegenerative disease caused by neuronal death and glial cell homeostasis, a neurodegenerative disease caused by aging, a neurodegenerative disease caused by affected CNS cell function, a neurodegenerative disease caused by abnormal intercellular communication and/or a neurodegenerative disease caused by impaired cell motility.
  • the ischemic stroke comprises cerebral infarction.
  • the ischemic stroke is caused by factors including thrombosis, embolism and/or hypotension.
  • the thrombus is caused by factors including atherosclerosis, aneurysm, vascular malformation, arteritis and/or vasospasm.
  • the hemorrhagic stroke is caused by factors including: vascular malformation, aneurysm, blood disease, cerebral amyloid angiopathy, abnormal vascular network at the base of the brain, cerebral arteritis, anticoagulant or thrombolytic therapy and/or tumor stroke.
  • the lesions observed by imaging include dense artery sign, island band sign, blurred outline or decreased density of the lenticular nucleus, occlusion of cerebral perforating arterioles, edema and/or mass.
  • the learning ability comprises cognitive ability, motor ability, memory ability and/or spatial exploration ability.
  • the improvement in learning ability comprises an improvement in the subject's learning ability assessment score by at least about 50% compared to the subject's original learning ability assessment score.
  • the assessment score of learning ability is measured by performing a test selected from the group consisting of a novel object recognition test and a water maze test.
  • the novel object recognition test assesses the cognitive ability, motor ability and/or spatial exploration ability.
  • the water maze test evaluates the memory ability, motor ability and/or spatial exploration ability.
  • the subject comprises a mammal.
  • the subject comprises a human.
  • the subject comprises a non-cognitive disorder patient, a non-neurodegenerative disease patient, and/or a non-stroke patient.
  • the subject comprises a patient with cognitive impairment, a patient with a neurodegenerative disease, and/or a patient with a stroke.
  • the subject comprises an Alzheimer's disease patient.
  • the subject is elderly.
  • the agent is formulated for oral administration and/or injection.
  • the agent is formulated for intravenous injection.
  • the present application provides a binding agent described herein, a polypeptide described herein, a fusion polypeptide described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, a drug combination described herein and/or a pharmaceutical composition described herein for improving learning ability, treating cognitive disorders, treating neurodegenerative diseases, preventing and/or treating stroke.
  • the present application provides a method for improving learning ability, treating cognitive disorders, treating neurodegenerative diseases, preventing and/or treating stroke, which comprises administering the binding agent described herein, the polypeptide described herein, the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, the drug combination described herein and/or the pharmaceutical composition described herein to a subject in need.
  • the present application provides a binding agent comprising two parts, wherein the first part comprises a molecule that binds to ATP6V1B2 and/or a functionally active fragment thereof, and the second part comprises a molecule that delivers the first part.
  • the second portion functions to deliver the first portion into cells and/or transport it across the blood-brain barrier to the brain.
  • the second moiety is capable of delivering the first moiety into a cell expressing ATP6V1B2.
  • the second moiety comprises a modifying group, a small molecule, a nanoparticle, an exosome, a virus, a nucleic acid, a protein and/or a polypeptide.
  • the second portion includes a group for PEGylation, lipidation, and/or glycosylation of the first portion.
  • the nanoparticles comprise liposomes.
  • the liposomes include modified liposomes, and the modifications include polypeptide modification, antibody modification, glycosyl modification, ligand modification, nucleic acid aptamer and/or multi-targeting modification.
  • the second moiety comprises a molecule that interferes with the physiological barrier function of the blood-brain barrier and/or a binding agent for a transporter protein on the blood-brain barrier.
  • the molecule that interferes with the physiological barrier function of the blood-brain barrier comprises an efflux inhibitor.
  • the transport proteins on the blood-brain barrier include insulin receptor (IR), transferrin receptor (TfR), low density lipoprotein receptor (LDLR), low density lipoprotein receptor-related protein 1 (LRP1) and LRP2, and diphtheria toxin receptor (DTR).
  • IR insulin receptor
  • TfR transferrin receptor
  • LDLR low density lipoprotein receptor
  • LRP1 low density lipoprotein receptor-related protein 1
  • DTR diphtheria toxin receptor
  • the second moiety comprises a blood-brain barrier transporter binding protein.
  • the second portion comprises an antibody and/or an antigen-binding fragment thereof that targets a transporter protein on the blood-brain barrier.
  • the second portion comprises an antibody and/or an antigen-binding fragment thereof that targets transferrin receptor.
  • the second moiety comprises a binding agent for a protein that mediates endocytosis.
  • the second moiety comprises a cell-penetrating peptide.
  • the cell-penetrating peptide includes a cell-penetrating peptide that enters the cell via endocytosis and/or direct penetration.
  • the cell-penetrating peptide comprises: a cationic cell-penetrating peptide, an amphipathic cell-penetrating peptide and/or a hydrophobic cell-penetrating peptide.
  • the cell-penetrating peptide comprises a linear peptide and/or a cyclic peptide.
  • the cell-penetrating peptide comprises a cell-penetrating peptide derived from TAT protein.
  • the second part comprises the amino acid sequence shown in SEQ ID NO: 79 and/or its variants.
  • the second portion comprises the amino acid sequence shown in any one of SEQ ID NOs: 80-84 and/or its variants.
  • the first portion is connected to the second portion.
  • the first portion is directly or indirectly connected to the second portion.
  • the first portion comprises the amino acid sequence shown in SEQ ID NO: 85 and/or its variants.
  • the first portion comprises a protein and/or a polypeptide.
  • the first portion comprises a polymer
  • the multimer comprises a homodimer.
  • the cysteine in the first amino acid sequence does not have a sulfhydryl blocking modification.
  • the serine in the first amino acid sequence does not have a phosphorylation modification.
  • the first portion and the second portion form a fusion polypeptide.
  • the binding agent comprises the amino acid sequence shown in any one of SEQ ID NOs: 100-104 and/or its variants.
  • the ATP6V1B2 and/or its functionally active fragments are derived from mammals.
  • the ATP6V1B2 comprises the amino acid sequence shown in SEQ ID NO: 8 or 16.
  • the functionally active fragment of ATP6V1B2 has the ability to specifically bind to the amino acid sequence shown in SEQ ID NO: 5.
  • the functionally active fragment of ATP6V1B2 comprises at least a portion of the amino acid sequence from position 288 to position 512 of the human ATP6V1B2 protein.
  • the functionally active fragment of ATP6V1B2 comprises at least a portion of the amino acid sequence from position 288 to position 512 of the mouse ATP6V1B2 protein.
  • it can increase the expression level and/or activity of a proton pump-related protein in a subject.
  • the expression level of the proton pump-associated protein is measured by performing an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blotting, dot blotting, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, Western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blotting, dot blotting, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, Western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • the expression level of ATP6V1B2 includes the expression level of the ATP6V1B2 gene, the transcription level of the ATP6V1B2 gene and/or the expression level of the ATP6V1B2 protein.
  • the increase comprises an increase in the expression level and/or activity of ATP6V1B2 by at least about 10% compared to the expression level and/or activity of native ATP6V1B2 in the subject.
  • it is capable of increasing neuronal synaptic transmitter release.
  • the increase comprises an increase of at least about 10% compared to the level of synaptic transmitter release from native neurons in the subject.
  • it increases the firing frequency of excitatory postsynaptic currents.
  • the increase comprises an increase of at least about 10% compared to the level of native excitatory postsynaptic current firing frequency in the subject.
  • the present application provides a fusion polypeptide comprising an amino acid sequence shown in any one of SEQ ID NOs: 100-104, wherein X is any amino acid.
  • the present application provides an immunoconjugate comprising the binding agent described herein and/or the fusion polypeptide described herein.
  • the present application provides a nucleic acid molecule encoding the binding agent and/or the fusion polypeptide described in the present application.
  • the present application provides a vector comprising the nucleic acid molecule described in the present application.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the binding agent described herein, the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein and/or the cell described herein, and optionally a pharmaceutically acceptable carrier.
  • the present application provides a use of the binding agent described in the present application, the fusion polypeptide described in the present application, the immunoconjugate described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application, the drug combination described in the present application and/or the pharmaceutical composition described in the present application in preparing a kit.
  • the present application provides a binding agent described in the present application, a fusion polypeptide described in the present application, an immunoconjugate described in the present application, a nucleic acid molecule described in the present application, a vector described in the present application, a cell described in the present application, a drug combination described in the present application, a pharmaceutical composition described in the present application and/or a kit described in the present application, for use in detecting ATP6V1B2.
  • the present application provides a method for detecting ATP6V1B2 in a sample, which comprises administering the binding agent described herein, the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, the drug combination described herein, the pharmaceutical composition described herein and/or the kit described herein.
  • the present application provides a use of the binding agent described in the present application, the fusion polypeptide described in the present application, the immunoconjugate described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application, the drug combination described in the present application and/or the pharmaceutical composition described in the present application in the preparation of a reagent.
  • the agent is used to improve learning ability, treat cognitive impairment, prevent and/or treat neurodegenerative diseases, and/or prevent and/or treat stroke.
  • the cognitive impairment comprises early cognitive impairment (MCI), moderate cognitive impairment, and severe cognitive impairment.
  • the cognitive impairment comprises cognitive impairment caused by normal aging, Lewis body dementia (LBD), frontotemporal dementia and/or vascular dementia.
  • LBD Lewis body dementia
  • frontotemporal dementia frontotemporal dementia
  • vascular dementia vascular dementia
  • the cognitive impairment-inducing disease comprises Alzheimer's disease, multi-infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).
  • the neurodegenerative disease includes acute neurodegenerative disease and chronic neurodegenerative disease.
  • the neurodegenerative disease includes a neurodegenerative disease caused by neuronal death and glial cell homeostasis, a neurodegenerative disease caused by aging, a neurodegenerative disease caused by affected CNS cell function, a neurodegenerative disease caused by abnormal intercellular communication and/or a neurodegenerative disease caused by impaired cell motility.
  • the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and/or Huntington's disease (HD).
  • MS multiple sclerosis
  • ALS amyotrophic lateral sclerosis
  • HD Huntington's disease
  • the stroke comprises ischemic stroke and/or hemorrhagic stroke.
  • the ischemic stroke comprises cerebral infarction.
  • the cerebral infarction comprises lacunar infarction, ischemic infarction and/or hemorrhagic infarction.
  • the ischemic stroke is caused by factors including thrombosis, embolism and/or hypotension.
  • the hemorrhagic stroke comprises intraparenchymal hemorrhage, intraventricular hemorrhage and/or subarachnoid hemorrhage.
  • the hemorrhagic stroke comprises an aneurysmal subarachnoid hemorrhage.
  • the lesions observed by imaging include intracerebral hematoma, intraventricular hemorrhage and/or subarachnoid hemorrhage.
  • the water maze test evaluates the memory ability, motor ability and/or spatial exploration ability.
  • the subject comprises a mammal.
  • the subject comprises a patient with cognitive impairment, a patient with a neurodegenerative disease, and/or a patient with a stroke.
  • the subject comprises an Alzheimer's disease patient.
  • the agent is formulated for intravenous injection.
  • the present application provides a method for improving learning ability, treating cognitive disorders, treating neurodegenerative diseases, preventing and/or treating stroke, which comprises administering the binding agent described herein, the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, the drug combination described herein and/or the pharmaceutical composition described herein to a subject in need.
  • FIG1 shows the results of immunoprecipitation of ATP6V1B2 protein by the ATP6V1B2 binding agent described in the present application.
  • FIG2 shows the effect of the ATP6V1B2 binder of the present application on spontaneous excitatory postsynaptic current.
  • FIG3 shows the effect of the ATP6V1B2 binder of the present application on spontaneous excitatory postsynaptic current.
  • binding agent generally refers to a natural or non-natural molecule that specifically binds to a target or a portion of a target.
  • Binding agents can include small molecule compounds, polymers, and/or biomacromolecules. Binding agents can include proteins, peptides, nucleic acids, sugars, lipids, and small molecule compounds.
  • a binding agent can include a polypeptide.
  • a binding agent can include a fusion polypeptide.
  • ATP6V1B2 generally refers to the ATPase H+ transporting V1 subunit B2 protein (also known as ATP6B2, DOOD, HO57, VATB, VPP3, Vma2 or ZLS2), and the gene encoding the protein.
  • the ATP6V1B2 can be a multi-subunit enzyme that mediates acidification of organelles in eukaryotic cells.
  • the ATP6V1B2 can affect the acidity of lysosomes.
  • the ATP6V1B2 can participate in processes such as protein sorting, zymogen activation, receptor-mediated endocytosis, and synaptic vesicle proton gradient generation.
  • the ATP6V1B2 protein can include a cytoplasmic V1 domain and a transmembrane V0 domain.
  • the accession number of human ATP6V1B2 in GenBank is 526.
  • the accession number of human ATP6V1B2 in UniProt can be P21281.
  • proton pump-associated protein generally refers to proteins that encode and/or express proton pumps.
  • the proton pumps can be proteins that actively transport hydrogen ions across a biological membrane against the electrochemical potential difference between hydrogen ions on either side of the membrane.
  • These proton pumps can include Na-K pumps, Ca2+ pumps, H+-ATP pumps, and H+ pyrophosphate pumps.
  • expression level generally refers to the protein, RNA or mRNA level of a specific related gene. Any method known in the art can be used to determine the expression level of a specific related gene (e.g., human ATP6V1B2 gene).
  • expression generally refers to the process of converting the information encoded by a gene into a structure that exists in and operates in a cell.
  • reverse transcription and amplification analysis e.g., PCR, linked RT-PCR or quantitative RT-PCR
  • hybridization analysis e.g., Northern blotting, dot blotting, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, protein blotting, immunohistochemistry, immunostaining or mass spectrometry
  • Analysis can be performed directly on a biological sample or on proteins/nucleic acids separated from a sample.
  • the term "activity" generally refers to any activity associated with a particular protein.
  • the activity may include, for example, any activity associated with the ATP6V1B2 protein.
  • the activity may include an enzymatic activity associated with a protease.
  • the activity may include a biological activity.
  • the activity may include binding of the protein to a receptor, for example, where the binding may produce a measurable downstream effect.
  • the activity may include any activity that would be attributed to the protein by one skilled in the art.
  • the term "variant" generally refers to a polypeptide comprising an amino acid sequence that differs from the amino acid sequence of a parent or reference polypeptide (e.g., a wild-type polypeptide) by at least one amino acid residue.
  • the variant may have a high (e.g., at least 80%) homology with the parent or reference polypeptide.
  • the homology may include sequence similarity or identity.
  • the homology may be determined using standard techniques known in the art (see, e.g., Smith and Waterman, Adv. Appl.
  • the percentage of identity shared by polynucleotide or polypeptide sequences is determined by direct comparison of sequence information between molecules, the comparison being performed by sequence alignment and determining identity using methods known in the art.
  • An example of an algorithm suitable for determining sequence similarity is the BLAST algorithm (see Altschul et al., J. Mol. Biol., 215:403-410 [1990]).
  • Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI).
  • the "variant" and/or “functional variant” can be, for example, a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein and/or the polypeptide (e.g., a binder that binds to ATP6V1B2 or a fragment thereof).
  • the variant may comprise a protein or polypeptide that has been subjected to amino acid changes by at least 1, for example, 1-30, 1-20 or 1-10, for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions and/or insertions.
  • the functional variant may substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition).
  • the "variant" may be a homolog.
  • the homolog may be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the protein and/or polypeptide (e.g., a binding agent that specifically binds to ATP6V1B2 or a fragment thereof).
  • amino acid generally refers to naturally occurring, synthetic, or non-natural amino acids, as well as amino acid analogs and amino acid mimetics that function in a similar manner to naturally occurring amino acids.
  • exemplary amino acids include naturally occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs with variant side chains; and stereoisomers of any of the foregoing.
  • the 20 commonly used amino acids and their abbreviations used in this application are in accordance with conventional usage. See Immunology-A Synthesis (2nd edition, E.S. Golub and D.R. Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), the contents of which are incorporated herein by reference.
  • This application uses commonly used amino acid single-letter abbreviations and three-letter abbreviations (Bruce Alberts et al., Molecular Biology of the Cell, Garland Publishing, Inc., New York (4th edition, 2002)).
  • Conservative amino acid substitution groups can be, for example, valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
  • non-conservative substitution also referred to as “radical replacement” or “radical substitution” generally refers to the substitution of an amino acid with another amino acid having different properties in a protein, such as charge, side chain size, hydrophobicity/hydrophilicity, main-chain conformation, and rigidity.
  • non-conservative substitutions can also involve the use of unnatural amino acids.
  • polar amino acid can include polar uncharged amino acids, basic (positively charged) amino acids, acidic (negatively charged) amino acids.
  • polar amino acids can include threonine, serine, cystine, tyrosine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid, glutamic acid.
  • fusion polypeptide generally refers to a polypeptide that comprises at least two discrete peptides or polypeptides (which do not exist together in this way in natural polypeptides, that is, these parts do not naturally exist in the same polypeptide or in the same order), or is composed of at least two discrete peptides or polypeptides (which do not exist together in this way in natural polypeptides, that is, these parts do not naturally exist in the same polypeptide or in the same order).
  • the discrete peptides or polypeptides can be linked together directly or indirectly to form a fusion polypeptide.
  • the discrete peptides or polypeptides can be linked to form a fusion polypeptide by peptide bonds.
  • the discrete peptides or polypeptides can be linked to form a fusion polypeptide by a linker.
  • the fusion polypeptide described in the present application may include a fusion polypeptide formed by connecting a polypeptide capable of binding to ATP6V1B2 and a polypeptide having a specific function.
  • the term "fusion protein" generally refers to a protein composed of two or more polypeptides.
  • endocytosis also known as “endocytosis” or “entry,” generally refers to the manner in which a substance enters a cell.
  • the endocytosis described herein may include various mechanisms by which a substance enters a cell.
  • the endocytosis may include phagocytosis, pinocytosis, and/or receptor-mediated endocytosis.
  • the "protein that mediates endocytosis” described herein generally refers to proteins and/or polypeptide molecules that participate in the endocytosis process.
  • membrane-penetrating peptide generally refers to a class of short peptides that can cross cell membranes or tissue barriers.
  • membrane-penetrating peptides can carry biomacromolecules such as proteins, RNA, and DNA into cells through mechanisms such as endocytosis and direct penetration to exert their effector functions.
  • Membrane-penetrating peptides related to the endocytic mechanism may involve multiple cellular endocytosis pathways.
  • the direct membrane-penetrating mechanism is usually because the membrane-penetrating peptide causes perturbation and instability of the cell membrane, thereby changing the permeability of the cell membrane.
  • membrane-penetrating peptides can be divided into three categories: cationic, amphiphilic, and hydrophobic.
  • Cationic cell-penetrating peptides generally have a high net positive charge.
  • Amphiphilic cell-penetrating peptides generally contain both polar and non-polar groups, thus possessing both hydrophilic and hydrophobic properties.
  • Hydrophobic cell-penetrating peptides generally contain only non-polar groups and therefore have a lower net charge.
  • isolated generally refers to a substance obtained artificially from its natural state.
  • a polynucleotide or polypeptide naturally present in a living animal, which has not been isolated, and a highly purified identical polynucleotide or polypeptide isolated from this natural state can be considered isolated.
  • isolated does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.
  • the term "cell” generally refers to a single cell, cell line or cell culture that may be or has been a recipient of a subject's plasmid or vector, including a nucleic acid molecule as described herein or a vector as described herein.
  • a cell may include the offspring of a single cell. Due to natural, accidental or intentional mutations, the offspring may not necessarily be identical to the original parent cell (in terms of the morphology of the total DNA complement or in the genome).
  • a cell may include a cell transfected in vitro with a vector as described herein.
  • immunoconjugate generally refers to a substance formed by linking a polypeptide with other active agents, which may be small molecule active agents, such as therapeutic agents, imaging probes, or spectroscopic probes.
  • pharmaceutically acceptable carrier generally refers to a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed thereto at the doses and concentrations employed.
  • Physiologically acceptable carriers may include suitable substances.
  • Pharmaceutically acceptable carriers are generally not the same substance as vectors used to insert nucleic acids in genetic engineering.
  • the term “specific binding” or “specific” generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules).
  • an antibody that specifically binds to a target (which may be an epitope) may be one that binds to that target with greater affinity, avidity, more readily, and/or for a greater duration than it binds to other targets.
  • an antibody specifically binds to an epitope on a protein that is conserved among proteins of different species.
  • specific binding may include, but does not require, exclusive binding.
  • the term "transmitter release” generally refers to neurotransmitter release, that is, the process by which a neuron releases neurotransmitters encapsulated in vesicles into the synaptic cleft, which acts on another neuron to transmit information.
  • the synapse the basic structure of the neural circuit, may be involved.
  • the transmitter release can be referred to as synaptic transmission.
  • the methods of transmitter release can include synchronous release, asynchronous release, and spontaneous release.
  • discharge frequency generally refers to the discharge frequency of action potentials.
  • Action potential can refer to the rapid and reversible reversal and recovery process of the potential on both sides of the membrane generated on the basis of the resting potential when an excitable cell is stimulated.
  • the action potential can be composed of a peak potential and an afterpotential, corresponding to the depolarization (Depolarisation) and hyperpolarization (Hyperpolarization) processes respectively.
  • the discharge of action potentials can have the characteristics of pulses.
  • the discharge frequency of pulses is the ratio of the number of pulse discharges to the time.
  • the discharge frequency can include the discharge frequency of synaptic vesicles releasing neurotransmitters.
  • the discharge frequency can include the discharge frequency of excitatory postsynaptic currents.
  • neuron generally refers to a nerve cell, which is the main functional unit of the nervous system.
  • a neuron can be composed of a cell body and its protrusions, an axon, and one or more dendrites. Neurons can transmit information to other neurons or cells by releasing neurotransmitters at synapses.
  • learning ability generally refers to all abilities related to or required for the learning/cognitive process. Such learning ability may include the ability to acquire new information, knowledge, and/or skills through processes such as experience, learning, or training. Such learning ability may include imagination, attention, perceptual observation, reading ability, analytical ability, operational ability, adaptability, summarization, problem-solving ability, or a combination thereof.
  • athleteic ability generally refers to the ability to participate in sports and training.
  • Athletic ability can include aerobic fitness, muscle strength, body flexibility, balance, and reaction ability.
  • Athletic ability can be a comprehensive manifestation of multiple factors such as physical shape, quality, skills, techniques, and psychological ability.
  • spatial exploration ability generally refers to the ability to explore the shape and/or position of an object.
  • the spatial exploration ability includes observing, thinking, imagining, recognizing and/or exploring the shape and/or position of an object.
  • the term "assessment score of learning ability” generally refers to a quantitative assessment score of a subject's learning ability.
  • the assessment score of learning ability can be obtained by conducting an assessment including attention/executive function (e.g., Wechsler Memory Test), language ability assessment (e.g., the language screening test (LAST)), visual-spatial and structural ability assessment (e.g., visual-motor integration test, Hooper visual organization test, object assembly test, pattern arrangement test, clock drawing test), application ability assessment, daily function assessment (e.g., disability assessment for dementia (DAD)) and/or neuropsychological scale scores.
  • the assessment score of learning ability can be obtained by conducting the mini-mental state examination (MMSE), the Montreal cognitive assessment (MoCA), the Alzheimer disease assessment scale-cog (ADAS-cog) and the clinical dementia rating scale (CDR).
  • the term "novel object recognition test” generally refers to a test that detects the time required for an animal (such as a mouse) to learn to recognize a new object by having the animal (such as a mouse) recognize an object in a specific space.
  • the novel object recognition test can refer to the test method described in Ennaceur et al., Behav Brain Res 80 9-25, 1996.
  • the novel object recognition test may include the following steps: placing two identical objects in a container of fixed volume, placing a mouse in the container to recognize the two objects, and after a period of time, replacing one of the two objects in the same container with a new object with a different shape, and then measuring the mouse's search time for the new object.
  • water maze test generally refers to a test in which an animal (e.g., a mouse) is forced to swim, thereby learning to find a platform hidden in the water.
  • the water maze test e.g., Morris water maze
  • the water maze test can test a mouse's learning ability and/or memory ability for a sense of spatial position and direction.
  • the water maze test can also include acquisition training, exploratory training, counterpoint training, or counterpoint exploratory training. If the time required for an animal (e.g., a mouse) to find a platform from entering the water is shorter, and the distance moved during this period is approximately shorter, the assessment score for the animal's (e.g., mouse's) learning ability is correspondingly approximately higher.
  • the water maze evaluation test can be an important experiment for evaluating learning ability.
  • neurodegenerative disease generally refers to cognitive disorders such as dementia caused by the gradual loss of neuronal structure and function, including neuronal death and glial cell imbalance.
  • age e.g., Alzheimer's disease (AD), Parkinson's disease (PD)
  • genetic mutations that affect CNS cell function e.g., Huntington's disease, early-onset AD or PD, amyotrophic lateral sclerosis (ALS)
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • ALS amyotrophic lateral sclerosis
  • the neurodegenerative disease may have changes and/or conditions selected from the following: protein misfolding and aggregation; neuroinflammation (e.g., CNS inflammation that occurs under toxic stimulation (e.g., protein aggregation), infection, traumatic injury, or autoimmunity); changes in cell signal transduction; acquired aging/cell death (e.g., interrupted apoptosis signal transduction, mitochondrial dysfunction, impaired autophagy, and necrotic bodies activated by stress/inflammation); motor cell damage and epigenetic changes.
  • neuroinflammation e.g., CNS inflammation that occurs under toxic stimulation (e.g., protein aggregation), infection, traumatic injury, or autoimmunity)
  • changes in cell signal transduction e.g., acquired aging/cell death (e.g., interrupted apoptosis signal transduction, mitochondrial dysfunction, impaired autophagy, and necrotic bodies activated by stress/inflammation); motor cell damage and epigenetic changes.
  • Alzheimer's disease generally refers to precocious dementia, senile dementia, a neurodegenerative disease with a slow progression of disease that worsens over time.
  • the most common early symptom is loss of short-term memory (difficulty remembering recent events).
  • As the disease progresses at least one of the following symptoms may gradually appear: language disorders, disorientation (e.g., easily getting lost), emotional instability, loss of motivation, inability to take care of oneself, and behavioral problems.
  • the true cause of Alzheimer's disease is still unknown, and its progression may be related to the deposition of amyloid plaques in the brain and fibrillary tangles caused by hyperphosphorylation of Tau proteins.
  • There is currently no treatment that can prevent or reverse the course of the disease and only a few methods may temporarily relieve or improve symptoms.
  • the terms “early cognitive impairment (MCI)” and “mild cognitive impairment” are used interchangeably and generally refer to a clinical state intermediate between normal cognition and cognitive impairment.
  • the MCI may include cognitive impairment that meets the criteria for dementia but exceeds the degree of normal aging.
  • MCI varies in clinical manifestations, etiology, prognosis and prevalence.
  • MCI may be a pathological stage of Alzheimer's disease. Certain forms of cognitive impairment can be considered an early manifestation of neurodegenerative diseases that will eventually lead to dementia.
  • intermediate cognitive impairment and “moderate cognitive impairment” are used interchangeably and may include more severe memory impairment that affects the patient's ability to live independently and/or may be accompanied by sphincter disorders.
  • late cognitive impairment and “severe cognitive impairment” are used interchangeably and may include severe intellectual impairment, inability to take care of oneself, complete dependence on others for care, and/or obvious sphincter disorders.
  • the severity of cognitive impairment can be determined by clinical manifestations, impairment of daily activities or cognitive assessment. For example, the severity of cognitive impairment can be diagnosed using the activity of daily living scale (ADL), the clinical dementia rating (CDR), or the global deterioration scale (GDS).
  • ADL activity of daily living scale
  • CDR clinical dementia rating
  • GDS global deterioration scale
  • normal aging-related cognitive impairment generally refers to cognitive impairment due to normal aging.
  • normal aging-related cognitive impairment may manifest as memory loss, confusion about the location of familiar places, taking longer than usual to complete daily tasks, or changes in mood and personality.
  • Lewy body dementia generally refers to Lewy body dementia.
  • Lewy body dementia is characterized by abnormal buildup of proteins into lumps called Lewy bodies.
  • Lewy body dementia causes a gradual decline in mental abilities. People with Lewy body dementia may experience visual hallucinations and changes in alertness and attention. Other effects include muscle stiffness, slowed movements, difficulty walking, and tremors. People with Lewy bodies in their brains can also have plaques and tangles associated with Alzheimer's disease.
  • frontotemporal dementia generally refers to Pick's disease, a rare, progressive disorder in which the tau protein affects only the frontal and temporal lobes of the brain.
  • People with frontotemporal dementia have difficulty with higher-level reasoning, expressive language, speech perception, and memory formation. Over time, the frontal and temporal lobes of the brain can shrink in people with frontotemporal dementia.
  • vascular dementia generally refers to problems with reasoning, judgment, and memory caused by impaired blood flow to the brain.
  • vascular dementia can include dementia caused by factors that increase the risk of heart disease and stroke, such as high blood pressure and high cholesterol.
  • multi-infarct type generally refers to small, non-cortical infarcts caused by occlusion of a single perforating branch of a large cerebral artery.
  • the multi-infarct type can be a special type of cerebral infarction, also known as ischemic stroke.
  • the multi-infarct type can manifest as hemisensory disturbances, aphasia, dysarthria, slow movements, and clumsiness (particularly difficulty with fine motor skills such as handwriting).
  • Parkinson's disease generally refers to a progressive neurodegenerative disease.
  • the clinical features of Parkinson's disease (PD) can include motor symptoms (e.g., tremor, bradykinesia, muscle rigidity, and postural instability), as well as neuropsychiatric and other non-motor manifestations.
  • the non-motor manifestations can include cognitive dysfunction and dementia, mood disorders (e.g., depression, anxiety, apathy), and sleep disorders.
  • AIDS generally refers to acquired immunodeficiency syndrome (AIDS).
  • Clinical manifestations of AIDS include changes in memory, concentration, attention, and motor skills.
  • AIDS patients may develop cognitive impairment. For example, approximately 50% of infected individuals may further develop HIV-associated neurocognitive disorders (HAND).
  • HAND HIV-associated neurocognitive disorders
  • CJD generally refers to a transmissible spongiform encephalopathy that occurs in humans.
  • CJD is caused by infection with a prion virus.
  • CJD patients may experience paranoid behavior, confusion, loss of appetite and weight, depression, and, in a minority of cases, visual or auditory abnormalities.
  • symptoms include progressive neurological deterioration (e.g., paresthesias, speech disorders, and aphasia).
  • MS multiple sclerosis
  • the term “multiple sclerosis (MS)” generally refers to a demyelinating neuropathy.
  • the insulating material i.e., myelin sheath
  • myelin sheath the insulating material on the surface of nerve cells in the brain or spinal cord is damaged, impairing signal transduction in the nervous system. This can lead to a range of symptoms that can affect the patient's mobility, mental state, and even mental state. These symptoms can include double vision, unilateral vision loss, muscle weakness, sensory impairment, or coordination problems.
  • ALS myotrophic lateral sclerosis
  • Lou Gehrig's disease also known as motor neuron disease, a progressive and fatal neurodegenerative disease.
  • a small number of ALS patients may develop frontotemporal dementia.
  • Some ALS patients experience degeneration of their senses of hearing, vision, touch, smell, and taste, and a very small number of ALS patients may also develop dementia.
  • HD Huntington's disease
  • the term "senile stage” generally refers to the aging stage of a subject.
  • the aging stage may be over 60 years old, over 70 years old, or over 75 years old; for mice, the aging stage may be over 10 months old, for example, over 13 months old or over 18 months old.
  • the aging stage of the subject may have one or more symptoms of learning impairment, memory impairment, memory loss, and/or brain dysfunction.
  • stroke generally refers to a condition caused by cell death due to blockage or bleeding of one or more blood vessels supplying blood to the brain, including stroke and damage caused by stroke.
  • stroke can be an acute cerebrovascular disease.
  • ischemic stroke generally refers to a stroke caused by blockage of one or more blood vessels supplying blood to the brain.
  • Ischemic stroke can be caused by thrombosis, embolism and/or hypotension.
  • Thrombosis can be caused by atherosclerosis, aneurysm, vascular malformation, arteritis and/or vasospasm.
  • Ischemic stroke includes cerebral infarction, which can include lacunar infarction, ischemic cerebral infarction and/or hemorrhagic infarction.
  • the types of ischemic stroke can include, for example, embolic stroke, cardioembolic stroke, thrombotic stroke, large vessel stroke, lacunar infarction, artery-artery stroke and cryptic stroke.
  • "hemorrhagic stroke” generally refers to a stroke caused by bleeding from one or more blood vessels supplying blood to the brain. Hemorrhagic stroke can include intraparenchymal hemorrhage, intraventricular hemorrhage and/or subarachnoid hemorrhage.
  • Hemorrhagic stroke can include primary cerebral hemorrhage and/or secondary cerebral hemorrhage.
  • Hemorrhagic stroke can include aneurysmal subarachnoid hemorrhage.
  • Hemorrhagic stroke can be caused by vascular malformations, aneurysms, blood diseases, cerebral amyloid angiopathy, abnormal vascular network at the base of the brain, cerebral arteritis, anticoagulation or thrombolytic therapy and/or aneurysmal stroke.
  • damage caused by stroke generally refers to a condition caused directly or indirectly by a stroke.
  • Damage caused by a stroke may include damage that occurs simultaneously with the stroke and damage caused after the stroke occurs.
  • Damage caused by a stroke may include damage in the brain.
  • the damage caused by a stroke may not be limited to the location where the stroke occurs, and may include damage to the entire body of the patient who has suffered a stroke, as long as it is related to the stroke or the occurrence of a stroke.
  • the damage caused by a stroke may manifest as damage to the learning ability and/or behavioral ability of the patient who has suffered a stroke.
  • the damage caused by a stroke may manifest as damage to the cognitive ability, motor ability and/or spatial exploration ability of the patient who has suffered a stroke.
  • the damage caused by a stroke can be detected by known means, including but not limited to imaging observations, novel object recognition tests and water maze tests.
  • nanoparticle generally refers to a microscopic particle having at least one dimension less than 100 nm.
  • nanoparticles typically have a diameter in the range of 50 nm to 500 nm (i.e., 0.05 ⁇ m to 0.5 ⁇ m); are structurally stable in physiological environments; and are capable of accommodating smaller molecules (such as molecules that bind to ATP6V1B2 and/or functionally active fragments thereof), which can then be delivered to the desired site.
  • the nanoparticles described herein include particles of different structures, such as nanospheres and nanocapsules. In this application, the nanoparticles include liposomes.
  • the term "liposome” generally refers to a vesicle with an internal space that is separated from the external medium by one or more bilayer membranes.
  • the bilayer membrane can be formed by amphiphilic molecules, such as synthetic or naturally derived lipids containing spatially separated hydrophilic and hydrophobic domains; for another example, the bilayer membrane can be formed by amphiphilic polymers and surfactants.
  • the internal space of the liposome can be loaded with hydrophilic drugs, while the space between its bilayer membranes can be loaded with lipophilic drugs.
  • the term "subject” generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.
  • the ATP6V1B2 may be derived from any organism.
  • the ATP6V1B2 may be derived from humans or mice.
  • the ATP6V1B2 and/or its functionally active fragment may comprise the amino acid sequence shown in any one of SEQ ID NO: 8, 10-11, 16.
  • nucleic acid sequence encoding the ATP6V1B2 and/or its functionally active fragment may include the nucleic acid sequence shown in SEQ ID NO:9 or 17.
  • the activity of ATP6V1B2 can include the biological activity of ATP6V1B2 protein and/or its functionally active fragment (e.g., can include a measurable downstream effect caused by it).
  • the activity of ATP6V1B2 can include increasing the expression level and/or activity of the proton pump-related protein.
  • the increase can include increasing the activity of the proton pump-related protein by at least about 10% compared to the activity of the original proton pump-related protein in the subject.
  • it can increase by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 500% or more.
  • the present application provides a binding agent comprising a molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof.
  • the binding agent may include a small molecule compound, a polymer and/or a biomacromolecule.
  • the binding agent may include a protein and/or a polypeptide.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment comprises at least one mutated amino acid residue compared to the amino acid sequence shown in SEQ ID NO: 85.
  • the mutated amino acid residues are 1 to 8.
  • the mutated amino acid residues are 1, 2, 3, 4, 5, 6, 7 or 8.
  • the at least one mutated amino acid residue is at least two mutated amino acid residues.
  • the mutated amino acid residues are 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment comprises 1, 2, 3, 4 or 5 mutated amino acid residues.
  • the mutation site of the at least one mutated amino acid residue is located at position 1, 2, 5, 6 and/or 8 of the amino acid sequence shown in SEQ ID NO: 85.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof comprises an amino acid mutation at position 1 of the amino acid sequence set forth in SEQ ID NO: 85.
  • the amino acid mutation at position 1 can be mutated to any amino acid.
  • the amino acid mutation at position 1 can be mutated to arginine or threonine.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof comprises an amino acid mutation at position 2 of the amino acid sequence set forth in SEQ ID NO: 85.
  • the amino acid mutation at position 2 can be mutated to any amino acid.
  • the amino acid mutation at position 2 can be mutated to tryptophan or aspartic acid.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof comprises an amino acid mutation at position 6 of the amino acid sequence set forth in SEQ ID NO: 85.
  • the amino acid mutation at position 6 can be mutated to any amino acid.
  • the amino acid mutation at position 6 can be mutated to threonine, phenylalanine, or tyrosine.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment contains a mutated amino acid combination compared to the amino acid sequence shown in SEQ ID NO: 85.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment comprises an amino acid mutation at position 1, an amino acid mutation at position 2, an amino acid mutation at position 5, and/or an amino acid mutation at position 6 of the amino acid sequence as shown in SEQ ID NO: 85.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment comprises an amino acid mutation at position 2 to aspartic acid, an amino acid mutation at position 5 to tyrosine, and/or an amino acid mutation at position 6 to tyrosine, compared to the amino acid sequence as shown in SEQ ID NO: 85.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment comprises an amino acid mutation at position 2, an amino acid mutation at position 5, an amino acid mutation at position 6, and/or an amino acid mutation at position 8 of the amino acid sequence as shown in SEQ ID NO: 85.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment comprises an amino acid mutation at position 2 to aspartic acid, an amino acid mutation at position 5 to phenylalanine, an amino acid mutation at position 6 to threonine, and/or an amino acid mutation at position 8 to asparagine, compared to the amino acid sequence as shown in SEQ ID NO: 85.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment comprises an amino acid mutation at position 1, an amino acid mutation at position 2, an amino acid mutation at position 5, and/or an amino acid mutation at position 6 of the amino acid sequence as shown in SEQ ID NO: 85.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment comprises an amino acid mutation at position 1 to threonine, an amino acid mutation at position 2 to tryptophan, an amino acid mutation at position 5 to phenylalanine, and/or an amino acid mutation at position 6 to threonine, compared to the amino acid sequence as shown in SEQ ID NO: 85.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment comprises an amino acid mutation at position 1 to arginine, an amino acid mutation at position 2 to tryptophan, an amino acid mutation at position 5 to phenylalanine, and/or an amino acid mutation at position 6 to tyrosine, compared to the amino acid sequence as shown in SEQ ID NO: 85.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment comprises an amino acid mutation at position 1, an amino acid mutation at position 2, an amino acid mutation at position 5, an amino acid mutation at position 6, and/or an amino acid mutation at position 8 of the amino acid sequence as shown in SEQ ID NO: 85.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment comprises, compared to the amino acid sequence as shown in SEQ ID NO: 85, an amino acid mutation at position 1 to threonine, an amino acid mutation at position 2 to tryptophan, an amino acid mutation at position 5 to phenylalanine, an amino acid mutation at position 6 to threonine, and/or an asparagine mutation at position 8.
  • the molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof comprises the amino acid sequence of SEQ ID NO: 19 and/or a variant thereof, wherein X is any amino acid.
  • the amino acid sequence represented by SEQ ID NO: 19 is: XXVDXXC[X-], where X is any amino acid, and amino acid position 8 may not be present.
  • the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NOs: 20-24 and/or its variants, wherein X is any amino acid.
  • the molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof comprises an amino acid sequence and/or variant thereof as set forth in any one of SEQ ID NOs: 25-30, wherein X is any amino acid.
  • the amino acid sequence set forth in SEQ ID NO: 26 is: XXVDXXC[-S], wherein X can be any amino acid, and [-S] indicates that position 8 can be serine or not present.
  • the amino acid sequence set forth in SEQ ID NO: 28 is: XXVDXXCX, wherein X can be any amino acid.
  • the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NOs: 31-35 and/or its variants, wherein X is any amino acid.
  • the molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NOs: 36-41 and/or a variant thereof, wherein X is any amino acid.
  • amino acid sequence set forth in SEQ ID NO: 37 is: [RTS][PWD]VD[GFYV][VTY]C[-S], wherein position 1 can be arginine, threonine, or serine, position 2 can be proline, tryptophan, or aspartic acid, position 5 can be glycine, phenylalanine, tyrosine, or valine, position 6 can be valine, threonine, or tyrosine, and position 8 can be serine or absent.
  • the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NOs: 42-48 and/or its variants, wherein X is any amino acid.
  • the molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof does not comprise the amino acid sequence set forth in SEQ ID NO: 85.
  • the amino acid sequence of the molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof is not completely identical to the amino acid sequence set forth in SEQ ID NO: 85.
  • the variant may include an amino terminal deletion, a carboxyl terminal deletion, an internal deletion and/or an amino acid substitution, while the remaining amino acid sequence generally has the same amino acids as the corresponding positions of the amino acid sequence from which it is derived.
  • the variant generally has at least 60%, 70%, 80%, 90%, or 100% of the biological activity of the amino acid sequence from which it is derived.
  • the binding agent or molecule capable of binding to ATP6V1B2 and/or its functionally active fragment comprises a protein and/or a polypeptide.
  • the molecule capable of binding to ATP6V1B2 and/or its functionally active fragment can be a polypeptide, which can contain cysteine residues.
  • the cysteine residues in the amino acid sequence of the polypeptide may not be modified to block sulfhydryl groups. For example, if the cysteine residues in the amino acid sequence of the polypeptide are blocked by sulfhydryl groups, the polypeptide may lose its ability to bind to the ATP6V1B2 protein.
  • the molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof may be a polypeptide, which may contain a serine residue, and the serine residue in the amino acid sequence of the polypeptide is not phosphorylated.
  • the polypeptide may lose the ability to bind to the ATP6V1B2 protein.
  • the molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof may be a multimer.
  • the multimer may include a homodimer.
  • the binding agent may comprise a single molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof as a first portion. In certain embodiments, the binding agent may optionally further comprise a second portion.
  • the binding agent or molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof can be a fusion protein and/or fusion polypeptide.
  • the fusion protein and/or fusion polypeptide can include a first portion and a second portion.
  • the fusion protein and/or polypeptide can include a binding agent for ATP6V1B2 described herein and/or a molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof described herein as a first portion.
  • the fusion protein and/or polypeptide can include a portion that is identical, different, or not identical to the first portion as a second portion.
  • the fusion protein and/or polypeptide may include a binder to ATP6V1B2 as described herein and/or a molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof as described herein as a first portion, and a cell-penetrating peptide as a second portion.
  • the molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof and the cell-penetrating peptide may be linked by a peptide bond to form a fusion polypeptide and/or fusion protein.
  • the binding agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 49-71 and/or a variant thereof, wherein X is any amino acid. In certain embodiments, the binding agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 72-78 and/or a variant thereof.
  • the present application provides a binding agent, which may comprise two parts, the first part comprising a molecule that binds to ATP6V1B2 and/or a functionally active fragment thereof, and the second part comprising a molecule that delivers the first part.
  • the form of the second part is not limited, and all forms of molecules that can achieve delivery of the first part are included.
  • the second part can include a modifying group, a small molecule, a nanoparticle, an exosome, a virus, a nucleic acid, a protein and/or a polypeptide.
  • the second part has the function of delivering the first part into cells and/or transporting the first part across the blood-brain barrier to the brain.
  • the second part is capable of delivering the first part into cells expressing ATP6V1B2.
  • the second portion includes a modification or transformation of the molecule of the first portion so that it can be delivered to a location where it can function.
  • the second portion may include a group that is rationally designed and modified to modify the molecule of the first portion, changing its properties to make it easier to cross the blood-brain barrier and/or enter cells.
  • the second portion may include a group for modifying the first portion.
  • the second portion may include a group for PEGylation, lipidation, and/or glycosylation of the first portion.
  • the first part can be wrapped inside the second part so that it can be delivered to the position where it can play a role.
  • the second part can include nanoparticles.
  • the second part can include liposomes.
  • the nanoparticles and/or liposomes can be further modified or transformed to improve the delivery effect of the first part.
  • the second part can include liposomes, and the liposomes can include modified liposomes, and the modifications include polypeptide modification, antibody modification, glycosyl modification, ligand modification, nucleic acid aptamer and/or multi-target modification.
  • the second portion may include a molecule that interferes with the physiological barrier function of the blood-brain barrier and/or a binder to a transporter protein on the blood-brain barrier.
  • the interference with the physiological barrier function of the blood-brain barrier may include increasing the permeability of the blood-brain barrier.
  • the interference with the physiological barrier function of the blood-brain barrier may include inhibiting the efflux system of the blood-brain barrier.
  • the molecule that interferes with the physiological barrier function of the blood-brain barrier includes an efflux inhibitor.
  • the binding agent for the transporter on the blood-brain barrier may include a transporter binding protein on the blood-brain barrier.
  • the transporter binding protein on the blood-brain barrier may include an antibody and/or an antigen-binding fragment thereof that targets the transporter on the blood-brain barrier.
  • the transporter binding protein on the blood-brain barrier may include an antibody and/or an antigen-binding fragment thereof that targets the transferrin receptor.
  • the second portion may include a cell-penetrating peptide.
  • the cell-penetrating peptide may include a cell-penetrating peptide that enters the cell through an endocytic pathway and/or a direct penetration pathway.
  • the cell-penetrating peptide may include: a cationic cell-penetrating peptide, an amphipathic cell-penetrating peptide and/or a hydrophobic cell-penetrating peptide.
  • the cell-penetrating peptide may include a linear peptide and/or a cyclic peptide.
  • the second portion may include a cell-penetrating peptide and/or a variant thereof selected from the following table:
  • the second portion may include a cell-penetrating peptide derived from TAT protein.
  • the second portion may include a variant of a cell-penetrating peptide derived from TAT protein, and the variant may include amino terminal deletion, carboxyl terminal deletion, internal deletion and/or amino acid replacement, and the remaining amino acid sequence generally has the same amino acid as the corresponding position of the amino acid sequence from which it is derived.
  • the variant generally has at least 60%, 70%, 80%, 90%, or 100% biological activity of the amino acid sequence from which it is derived.
  • the variant of the cell-penetrating peptide derived from TAT protein has the function of delivering the first portion to intracellular and/or allowing it to be transported to the brain across the blood-brain barrier.
  • the second part may comprise the amino acid sequence shown in SEQ ID NO: 79 and/or its variants.
  • the second portion may comprise the amino acid sequence shown in any one of SEQ ID NOs: 80-84 and/or its variants.
  • the first portion may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 19-30, 93-99, and/or variants thereof, wherein X is any amino acid.
  • the first portion may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 31-48, 86-92, and/or variants thereof.
  • the first portion may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 85, and/or variants thereof.
  • the first portion may be a polypeptide, which may contain a serine residue, and the serine residue in the amino acid sequence of the polypeptide is not phosphorylated.
  • the serine residue in the amino acid sequence of the polypeptide may lose the ability to bind to the ATP6V1B2 protein.
  • the binding agent may be a fusion protein and/or a fusion polypeptide.
  • the fusion protein and/or fusion polypeptide may include the first part and the second part.
  • the first part includes a molecule that binds to ATP6V1B2 and/or a functionally active fragment thereof
  • the second part includes a molecule that delivers the first part
  • the first part and the second part form a fusion polypeptide.
  • the binding agent comprises the amino acid sequence shown in any one of SEQ ID NOs: 100-104 and/or a variant thereof.
  • the binding agent may comprise the amino acid sequence shown in SEQ ID NO: 1 and/or a variant thereof.
  • the binding agent may comprise the amino acid sequence shown in SEQ ID NO: 5 and/or a variant thereof.
  • the binding agent can regulate the expression level and/or activity of a proton pump-related protein.
  • the binding agent can increase the expression level and/or activity of a proton pump-related protein.
  • the expression level of the proton pump-related protein includes the expression level of a gene encoding a proton pump-related protein, the transcription level of a gene encoding a proton pump-related protein, and/or the expression level of a proton pump-related protein.
  • the increase includes increasing the expression level and/or activity of the proton pump-related protein by at least about 10% compared to the expression level and/or activity of the original proton pump-related protein in the subject.
  • the increase may include increasing the expression level of the proton pump-related protein by at least about 10% compared to the expression level of the original proton pump-related protein in the subject.
  • the improvement can be at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 500%, or more.
  • the expression level of a proton pump-related protein can be measured by conventional techniques in the art. For example, including but not limited to measuring the expression level of a proton pump-related protein by performing an assay selected from the group consisting of: qPCR, qRT-PCR, hybridization analysis, Northern blotting, dot blotting, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, Western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • an assay selected from the group consisting of: qPCR, qRT-PCR, hybridization analysis, Northern blotting, dot blotting, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, Western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • the expression level of a proton pump-related protein can be measured, but not limited to, by utilizing a substance selected from the group consisting of: a primer capable of specifically amplifying a gene encoding a proton pump-related protein, a nucleic acid molecule that specifically binds to a gene encoding a proton pump-related protein, a nucleic acid molecule that specifically binds to a proton pump-related protein, a small molecule that specifically binds to a proton pump-related protein, a probe that specifically binds to a proton pump-related protein, and a polypeptide that specifically binds to a proton pump-related protein.
  • a primer capable of specifically amplifying a gene encoding a proton pump-related protein a nucleic acid molecule that specifically binds to a gene encoding a proton pump-related protein
  • a nucleic acid molecule that specifically binds to a proton pump-related protein a small molecule that specifically
  • the isolated polypeptide can be used as a binder for ATP6V1B2. In the present application, the isolated polypeptide can be used to prepare a binder for ATP6V1B2. In the present application, the isolated polypeptide can have one or more functions of an ATP6V1B2 binder.
  • the polypeptide can regulate the expression level and/or activity of a proton pump-related protein.
  • the polypeptide can regulate the expression level and/or biological activity of ATP6V1B2 and/or its functionally active fragments.
  • the polypeptide can increase the expression level and/or biological activity of ATP6V1B2 and/or its functionally active fragments.
  • the polypeptide can improve cognitive ability.
  • the fusion protein and/or fusion polypeptide may include a first portion and a second portion.
  • the fusion protein and/or polypeptide may include, as the first portion, a binder to ATP6V1B2, a molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof, and/or a polypeptide as described herein.
  • the fusion protein and/or polypeptide may include, as the second portion, a portion that is identical, different, or not identical to the first portion.
  • the second part may have a specific function.
  • the second part can be transported across the blood-brain barrier to the brain and/or cross the cell membrane.
  • the fusion protein and/or fusion polypeptide also comprises a molecule capable of being transported across the blood-brain barrier to the brain and/or a molecule capable of crossing the cell membrane.
  • the molecule capable of being transported across the blood-brain barrier to the brain and/or the molecule capable of crossing the cell membrane comprises a polypeptide.
  • the molecule capable of being transported across the blood-brain barrier to the brain and/or the molecule capable of crossing the cell membrane comprises a transmembrane peptide.
  • the transmembrane peptide comprises an amino acid sequence as shown in any one of SEQ ID NOs: 79-84 or a variant thereof.
  • the first part and the second part can be linked by a peptide bond to form a fusion polypeptide and/or fusion protein.
  • a fusion polypeptide or protein comprising the first portion and the second portion may bind to ATP6V1B2 more strongly than the binding of the first portion to ATP6V1B2 by at least about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more.
  • the second portion can weaken the binding ability of the first portion to ATP6V1B2.
  • the binding ability of a fusion polypeptide or protein comprising the first and second portions to ATP6V1B2 is weaker than the binding ability of the first portion to ATP6V1B2.
  • the binding ability of a fusion polypeptide or protein comprising the first and second portions to ATP6V1B2 is at least about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more weaker than the binding ability of the first portion to ATP6V1B2.
  • the binding ability can be measured using techniques commonly used in the art.
  • the binding ability can be represented by the equilibrium dissociation constant, Kd.
  • the binding ability can be determined by co-immunoprecipitation.
  • the polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 1 and/or a variant thereof. In some embodiments, the polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 5 and/or a variant thereof.
  • the fusion protein and/or polypeptide may comprise an amino acid sequence and/or variant thereof as shown in any one of SEQ ID NOs: 49-71, wherein X is any amino acid.
  • the fusion protein and/or polypeptide may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 72-78 and/or variants thereof.
  • the fusion protein and/or polypeptide may include a binding agent for ATP6V1B2 as described herein, a molecule capable of binding to ATP6V1B2 and/or a functionally active fragment thereof as described herein, and/or a polypeptide as described herein as a first portion and a molecule that delivers the first portion as a second portion.
  • the binding agent described herein may be a fusion protein and/or polypeptide, the binding agent consisting of two portions, the first portion including a molecule that binds to ATP6V1B2 and/or a functionally active fragment thereof, and the second portion including a molecule that delivers the first portion.
  • the fusion protein and/or fusion polypeptide may have one or more functions of an ATP6V1B2 binder.
  • the fusion protein and/or fusion polypeptide may regulate the expression level and/or activity of a proton pump-related protein.
  • the fusion protein and/or fusion polypeptide may regulate the expression level and/or biological activity of ATP6V1B2 and/or its functionally active fragments.
  • the fusion protein and/or fusion polypeptide may increase the expression level and/or biological activity of ATP6V1B2 and/or its functionally active fragments.
  • the fusion protein and/or fusion polypeptide may improve cognitive ability.
  • the fusion protein and/or fusion polypeptide may increase neuronal synaptic transmitter release.
  • the fusion protein and/or fusion polypeptide may increase the frequency of excitatory postsynaptic currents.
  • the fusion protein and/or fusion polypeptide may improve learning ability, treat cognitive impairment, prevent and/or treat neurodegenerative diseases, and/or prevent and/or treat stroke.
  • the fusion polypeptide may prevent and/or treat lysosome-related diseases.
  • the lysosome-related disease may include a disease associated with lysosome abnormality.
  • the lysosome-related disease may include a disease associated with abnormal lysosomal acidity.
  • the present application provides an immunoconjugate comprising the binding agent described herein, the polypeptide described herein, and/or the fusion polypeptide described herein.
  • the present application provides one or more vectors comprising the nucleic acid molecules.
  • the vector may comprise one or more nucleic acid molecules.
  • the vector may further comprise other genes, such as marker genes that allow the vector to be selected in an appropriate host cell and under appropriate conditions.
  • the vector may further comprise expression control elements that allow the coding region to be correctly expressed in an appropriate host.
  • control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the one or more nucleic acid molecules described herein may be operably linked to the expression control elements.
  • the vector may include, for example, a plasmid, a cosmid, a virus, a phage or other vectors commonly used in, for example, genetic engineering.
  • the vector is an expression vector.
  • the present application provides a pharmaceutical composition, which may include the binding agent described herein, the polypeptide described herein, the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein and/or the cell described herein, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can include a pharmaceutical product suitable for pharmaceutical uses (e.g., improving learning ability, treating cognitive impairment and/or treating neurodegenerative diseases (e.g., Alzheimer's disease)).
  • the pharmaceutical composition can be used to prevent and/or treat neurodegenerative diseases and/or prevent and/or treat stroke.
  • the pharmaceutical composition can be used to prevent and/or treat a disease associated with a lysosome.
  • the disease associated with a lysosome can include a disease associated with a lysosomal abnormality.
  • the disease associated with a lysosome can include a disease associated with an abnormal lysosomal acidity.
  • the pharmaceutical composition can be a composition comprising one or more active ingredients (such as the binding agents described herein) and one or more inert ingredients; as well as any product directly or indirectly obtained by the combination, complex or aggregation of any two or more ingredients, or by the dissociation of one or more ingredients, or by other types of reactions or interactions of one or more ingredients.
  • active ingredients such as the binding agents described herein
  • inert ingredients such as the binding agents described herein
  • the pharmaceutically acceptable carrier may include sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions immediately before use.
  • suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles may include water, ethanol, polyols (e.g., propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil) and injectable organic esters such as ethyl oleate.
  • the present application provides a kit comprising the binding agent described herein, the polypeptide described herein, the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein and/or the cell described herein; and one or more additional components selected from the following group: analytical buffer, controls, substrates, standards, detection materials, laboratory supplies, equipment, instruments, cells, organs, tissues and user manuals or instructions.
  • the kit can detect ATP6V1B2 in a sample.
  • the kit can detect the expression level and/or biological activity of ATP6V1B2 and/or its functional fragments.
  • the kit can include instructions describing specific steps for using the kit to detect the expression level and/or biological activity of ATP6V1B2 and/or its functional fragments, and/or specific steps for using the test results to determine whether the candidate drug can prevent and/or treat cognitive impairment and/or neurodegenerative disease in a subject.
  • ATP6V1B2 when the expression level and/or activity of ATP6V1B2 in the subject is increased, the learning ability of the subject can be significantly improved (for example, the cognitive ability, motor ability, memory ability and/or spatial exploration ability can be significantly improved compared to before the expression level and/or activity of ATP6V1B2 in the subject is increased). Therefore, ATP6V1B2 can be used as a potential target for improving learning ability. For example, ATP6V1B2 can be used as a potential target for treating neurodegenerative diseases (such as Alzheimer's disease) and/or cognitive disorders.
  • neurodegenerative diseases such as Alzheimer's disease
  • the learning ability may include all abilities related to or required for the learning/cognitive process.
  • the learning ability may include cognitive ability, motor ability, memory ability and/or spatial exploration ability.
  • the present application provides uses of the binding agent described herein, the polypeptide described herein, the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, the drug combination described herein and/or the pharmaceutical composition described herein in improving learning ability.
  • the present application provides the use of the binding agent described herein, the polypeptide described herein, the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, the drug combination described herein and/or the pharmaceutical composition described herein in the preparation of an agent for preventing and/or treating a disease.
  • the present application provides a method for preventing and/or treating a disease, which comprises administering to a subject in need thereof the binding agent described herein, the polypeptide described herein, the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, the drug combination described herein, the pharmaceutical composition described herein and/or the reagent for preventing and/or treating a disease described herein.
  • the agents described herein can be administered in any manner.
  • the agents described herein can be administered orally and/or by injection.
  • the agents described herein can be formulated into a form suitable for their mode of use.
  • the agents described herein can be formulated into a form suitable for oral administration and/or by injection.
  • the prevention and/or treatment not only includes preventing and/or treating the disease, but also generally includes preventing the onset of the disease, slowing or reversing the progression of the disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or duration of the disease and/or any symptoms associated therewith and/or preventing further increase in the severity of the disease and/or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease, and generally any pharmacological effect that is beneficial to the patient being treated.
  • the present application provides a method for detecting ATP6V1B2 in a sample, which comprises administering the binding agent described herein, the polypeptide described herein, the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, the pharmaceutical composition described herein and/or the kit described herein.
  • detecting ATP6V1B2 in a sample as described herein may include detecting the presence of ATP6V1B2 in the sample.
  • detecting ATP6V1B2 in a sample as described herein may include detecting the amount of ATP6V1B2 in the sample.
  • the ATP6V1B2 may include ATP6V1B2 and/or a nucleic acid encoding ATP6V1B2.
  • the methods provided herein may directly test a sample.
  • the methods provided herein may simply involve contacting a sample with a binding agent.
  • the methods provided herein may simply involve contacting a sample with a binding agent and detecting binding.
  • the methods provided herein may involve contacting a sample with a binding agent, removing non-specifically bound substances, and detecting binding.
  • the binding agents described herein, the polypeptides described herein, the fusion polypeptides described herein, the immunoconjugates described herein, the nucleic acid molecules described herein, the vectors described herein, the cells described herein, the pharmaceutical compositions described herein, and/or the kits described herein can be attached to a carrier.
  • the binding agent is directly or indirectly attached to a carrier such as a magnetic bead or resin, or a mixture thereof.
  • the binding agent can also be directly or indirectly attached to a solid surface or substrate.
  • the binding agent can also be attached to particles, such as beads or microspheres.
  • the binding agent can also be labeled with substances including, but not limited to, magnetic labels, fluorescent moieties, enzymes, chemiluminescent probes, metal particles, non-metallic colloidal particles, polymeric dye particles, pigment molecules, pigment particles, electrochemically active substances, semiconductor nanocrystals or other nanoparticles (including quantum dots or gold particles).
  • substances including, but not limited to, magnetic labels, fluorescent moieties, enzymes, chemiluminescent probes, metal particles, non-metallic colloidal particles, polymeric dye particles, pigment molecules, pigment particles, electrochemically active substances, semiconductor nanocrystals or other nanoparticles (including quantum dots or gold particles).
  • the neurodegenerative disease may include acute neurodegenerative diseases and chronic neurodegenerative diseases.
  • the neurodegenerative disease may include neurodegenerative diseases caused by neuronal death and glial cell homeostasis, neurodegenerative diseases caused by aging, neurodegenerative diseases caused by affected CNS cell function, neurodegenerative diseases caused by abnormal intercellular communication and/or neurodegenerative diseases caused by impaired cell motility.
  • the cognitive impairment may include mild cognitive impairment (MCI), moderate cognitive impairment and severe cognitive impairment.
  • MCI mild cognitive impairment
  • the cognitive impairment may include cognitive impairment caused by normal aging, Lewis's body dementia (LBD), frontotemporal dementia and/or vascular dementia.
  • LBD Lewis's body dementia
  • the inducing disease of the cognitive impairment may include Alzheimer's disease, multi-infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).
  • the stroke may include ischemic stroke and/or hemorrhagic stroke.
  • the ischemic stroke may include cerebral infarction.
  • the cerebral infarction may include lacunar infarction, ischemic cerebral infarction and/or hemorrhagic infarction.
  • the ischemic stroke may be caused by factors including: thrombosis, embolism and/or hypotension.
  • the thrombosis may be caused by factors including: atherosclerosis, aneurysm, vascular malformation, arteritis and/or vasospasm.
  • the hemorrhagic stroke may include intracerebral hemorrhage, intraventricular hemorrhage and/or subarachnoid hemorrhage.
  • the hemorrhagic stroke may include primary cerebral hemorrhage and/or secondary cerebral hemorrhage.
  • the hemorrhagic stroke may include aneurysmal subarachnoid hemorrhage.
  • the hemorrhagic stroke may be caused by factors including: vascular malformation, aneurysm, blood disease, cerebral amyloid angiopathy, abnormal vascular network at the base of the brain, cerebral arteritis, anticoagulation or thrombolytic therapy and/or tumor stroke.
  • the stroke may also include damage caused by the stroke.
  • the damage may include damage observed by imaging.
  • the damage observed by imaging may include intracerebral hematoma, intraventricular hemorrhage and/or subarachnoid hemorrhage.
  • the damage observed by imaging may include edema, hematoma and/or space-occupying effect.
  • the damage observed by imaging may include dense artery sign, island band sign, blurred outline of lenticular nucleus or reduced density, occlusion of cerebral perforator arterioles, edema and/or space-occupying effect.
  • the imaging observation may include conventional imaging observation means in this area.
  • the imaging observation may include but is not limited to ultrasound, CT, cranial angiography (CTA), CT perfusion scan (CTP), nuclear magnetic resonance (MRI) and digital subtraction angiography (DSA).
  • the impairment may include an impairment in learning abilities, including cognitive abilities, motor abilities, memory abilities, and/or spatial exploration abilities.
  • the damage of described learning ability can comprise and compare with the assessment score of learning ability before the generation cerebral stroke of experimenter, and the assessment score of the learning ability of described experimenter after the damage caused by cerebral stroke improves and reduces about 10%.For example, can reduce at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 500% or more.
  • the subject may include a mammal.
  • the subject may include a rodent and/or a primate, for example, the subject may include a human.
  • the subject may include a non-cognitive disorder patient and/or a non-neurodegenerative disease patient.
  • the subject may be a normal person and/or a healthy person.
  • the subject may have a need and/or desire to further improve their learning ability.
  • the subject may include a patient with a neurodegenerative disease.
  • the subject may include a patient with Alzheimer's disease.
  • the patient with Alzheimer's disease may be in the early, early, middle, or late stages of Alzheimer's disease.
  • the subject may be elderly.
  • the subject may have exhibited cognitive impairment due to normal aging.
  • the subject may have exhibited symptoms of early cognitive impairment (MCI).
  • MCI early cognitive impairment
  • the subject may have exhibited symptoms of a neurodegenerative disease (e.g., Alzheimer's disease).
  • the subject may include a patient with ischemic stroke and/or hemorrhagic stroke.
  • the subject may include a patient with cerebral infarction.
  • the patient may include a patient with lacunar infarction, ischemic infarction and/or hemorrhagic infarction.
  • the patient may include a patient with thrombosis, embolism and/or hypotension.
  • the patient may include a patient with atherosclerosis, aneurysm, vascular malformation, arteritis and/or vasospasm.
  • the subject may include patients with intracerebral hemorrhage, intraventricular hemorrhage and/or subarachnoid hemorrhage.
  • the subject may include patients with primary cerebral hemorrhage and/or secondary cerebral hemorrhage.
  • the subject may include patients with aneurysmal subarachnoid hemorrhage.
  • the patient may include patients with vascular malformations, aneurysms, hematological diseases, cerebral amyloid angiopathy, abnormal vascular network at the base of the brain, cerebral arteritis, anticoagulation or thrombolytic therapy and/or tumor stroke.
  • the subject may also include patients with damage caused by stroke.
  • the patient may include patients with observed intracerebral hematoma, intraventricular hemorrhage and/or subarachnoid hemorrhage.
  • the patient may include patients with observed edema, hematoma and/or mass effect.
  • the patient may include patients with observed dense artery sign, island band sign, blurred outline or decreased density of the lenticular nucleus, occlusion of cerebral perforator arterioles, edema and/or mass effect.
  • This example demonstrates that the ATP6V1B2 binder described in this application can bind to the ATP6V1B2 protein through immunoprecipitation experiments with the ATP6V1B2 binder and the ATP6V1B2 protein.
  • hATP6V1B2-myc expression plasmid was constructed and transfected into HEK 293 cells.
  • Cell protein was extracted 48 hours after transfection. Protein extraction was performed using Pierce RIPA lysis buffer.
  • 500 ⁇ L of Pierce RIPA lysis buffer containing protease inhibitors was added.
  • the cells were harvested and transferred to a clean 1.5 mL EP tube. After mixing on a rotary mixer at 4°C for 1 hour, the cells were centrifuged at 13,000 rpm for 10 minutes at 4°C to remove cellular debris.
  • hATP6V1B2 was aliquoted into seven portions.
  • Protein G agarose beads (Roche, 11243233001) were placed in an EP tube and washed three times with Pierce RIPA lysis buffer to remove interference from the original storage solution. The protein incubated overnight with the antibody was added to the agarose beads and incubated at 4°C for 2 hours to allow for full binding of the antibody to the protein. Centrifuge at 4000 rpm for 1 minute, remove the supernatant, and add 600 ⁇ L Pierce RIPA lysis buffer. Gently invert and centrifuge to remove the supernatant. Repeat the above steps three times to wash nonspecifically bound proteins. Finally, add 30 ⁇ L 1 ⁇ loading buffer, mix well, and heat at 60°C for 20 minutes to denature the protein and store at -20°C. Proteins were detected by Western blot (Myc-Tag (9B11) Mouse mAb, CST, 2276S; Anti-QD202, GL Biochem).
  • This example demonstrates the therapeutic effects of ATP6V1B2 binders by examining changes in the frequency and amplitude of spontaneous excitatory postsynaptic currents in mouse hippocampal slices following treatment with ATP6V1B2 binders.
  • Spontaneous excitatory postsynaptic currents are closely related to synaptic transmission, neuronal plasticity, and overall neural network activity. Effects on spontaneous excitatory postsynaptic currents suggest a therapeutic effect of ATP6V1B2 binders.
  • mice Two- to three-month-old C57 mice were anesthetized with an intraperitoneal injection of 0.1-0.15 ml of 20% ulose. The brain tissue was then rapidly decapitated and removed. The mice were then placed in an ice-cold mixture of artificial cerebrospinal fluid (ACSF) pre-gassed with 95% O2 and 5% CO2 for 2 minutes.
  • the ACSF composition was as follows: 11.7 mM NaCl , 0.36 mM KCl, 0.12 mM NaH2PO4 , 0.25 mM CaCl2, 0.12 mM MgCl2, 25 mM NaHCO3, and 11 mM glucose. 350 mm coronal hippocampal slices were cut using a vibratome.
  • Hippocampal slices were placed in room temperature ACSF for at least 30 minutes to recover. The slices were then placed in a recording tank and continuously perfused with mixed ACSF at a rate of 2-2.5 ml/min. Under an Olympus BX51 upright microscope, hippocampal CA1 pyramidal neurons were blind-ligated and whole-cell patch clamp recordings were performed using an Axon 700B amplifier and a 1550B digital-to-analog converter. Spontaneous excitatory postsynaptic currents (sEPSCs) of pyramidal neurons were recorded with voltage clamp at a clamp voltage of -70 mV. After 5 minutes of sEPSC recording, the ATP6V1B2 binder described herein was administered for 5 minutes, followed by a 10-minute perfusion with ACSF. The recorded data were analyzed using Minianalysis software.
  • sEPSCs post-to-analog converter
  • LysoSensor TM Green DND-189 lysosomal green fluorescent probe staining is acidophilic and can accumulate in acidic organelles through protonation, and its fluorescence intensity is pH-dependent (acidophilic) as the degree of acidification of the organelles increases.
  • the specific steps are as follows: HEK293 cells are seeded in a 35mm glass-bottomed confocal culture dish, cultured in DMEM high-glucose medium, and grown to a density of 70-80% at 37°C and 5% carbon dioxide content.
  • a drug-containing cell culture medium with a final DMSO concentration of 0.1% is prepared, and the drug concentrations are 1nM V-ATPase inhibitor Bafilomycin A1, 5 ⁇ M ATP6V1B2 binder described in this application, and 20 ⁇ M ATP6V1B2 binder described in this application.
  • the culture medium was discarded and the cells were incubated with drug-containing medium or blank control medium at 37°C and 5% CO2 for 3 hours.
  • the culture medium was aspirated, the cells were washed once with PBS, and the cells were incubated with 37°C preheated culture medium containing the probe.
  • the cells were incubated in a growth state for 30 minutes.
  • the staining solution was replaced with fresh culture medium and the cells were observed under a fluorescence microscope. Five to six images were collected for each dish of cells, and the mean fluorescence intensity of the images was analyzed using ImageJ software. Three parallel experiments were performed.
  • the experimental results are shown in Figure 3.
  • the fluorescence intensity results show that the fluorescence intensity of the lysosomes in the group treated with the vesicular proton pump inhibitor Bafilomycin A1 was significantly weaker than that in the untreated group. This suggests that inhibiting the vesicular proton pump can reduce lysosomal acidity.
  • the fluorescence intensity of the lysosomes in the group treated with 20M QD202 was stronger than that in the untreated group, indicating that the acidity of the lysosomes was increased.
  • the ATP6V1B2 binder described in this application has the same function as QD202, it suggests that the ATP6V1B2 binder described in this application may activate the function of the lysosomal vesicular proton pump by binding to the ATP6V1B2 protein, thereby increasing lysosomal acidity.
  • an ATP6V1B2 binder was administered to mice, and their novel object recognition ability and water maze behavior were tested to evaluate the improvement effect of the ATP6V1B2 binder on learning ability.
  • Novel object recognition was tested using an open field box (40 ⁇ 40 ⁇ 35 cm, made of blue opaque plastic) with reference to the behavioral test protocol described in Leger, M., et al. Object recognition test in mice. Nat Protoc. 8, 2531-2537 (2013).
  • mice On the first day, mice were placed in the open field box for 10 min of acclimatization.
  • each mouse On the second day, each mouse was gently placed in the center of the box, and two similar objects (No. 1 batteries) were placed in the central area. The mice were allowed to explore freely for 10 min, and then returned to their home cages. 3 h later, the mice were placed back in the box (one battery was replaced with a 10 cm tall human toy) for 10 min for a memory retention test.
  • the video recordings were analyzed using Etho Vision XT 14 software, and the time the mice spent exploring the new/old objects was recorded.
  • the discrimination index was calculated as (Tnovel-Tfamliar)/(Tnovel+Tfamiliar).
  • mice were subjected to the above-mentioned behavioral tests.
  • mice Ten-month-old APP/PS1 transgenic mice (purchased from Shanghai Model Organisms) were selected and divided equally into two groups. These mice were intravenously injected with PBS buffer (Group 2) and different ATP6V1B2 binders described in this application (Group 3, with a dose of 1 mg/kg), respectively. Ten-month-old C57 BL/6 mice that were not intravenously injected with any reagent served as the control group (Group 1).
  • mice were subjected to the above-mentioned behavioral tests.
  • MCAO Middle cerebral artery occlusion
  • Edaravone is a drug that has been widely used to treat acute ischemic stroke.
  • the common carotid artery is clamped with an artery clamp, and a 5-0 suture is tied at the side of the external carotid artery near the brain, and a slipknot is tied at the external carotid artery near the common carotid bifurcation; (4)
  • the internal carotid artery is clamped with an artery clamp, and the external carotid artery is cut between the two knots with microscissors.
  • the suture is inserted into the internal carotid artery from the cut, and the internal carotid artery clamp is loosened and inserted into the middle cerebral artery until the suture bends.
  • the slipknot at the common carotid bifurcation is tightened, and the embolization time is 60 minutes; (5) After the embolization is completed, the slipknot is slightly loosened, and after the suture is pulled out, the external carotid artery is immediately tied with a slipknot, the common carotid artery clamp is loosened to suture the neck wound, and iodine is used for disinfection; (6) An appropriate amount of glucose solution is injected after the operation to ensure the basic survival status of the animal.
  • Neurobehavioral score results Compared with the Model + placebo group, the neurobehavioral score results of the Sham + placebo group were lower than those of the Model + placebo group, with a statistically significant difference (p ⁇ 0.001). Compared with the Model + placebo group, the neurobehavioral score results of the positive control group Edaravone-5mpk group were lower than those of the Model + placebo group, with a statistically significant difference (p ⁇ 0.01). The neurobehavioral score results of the ATP6V1B2 binder-1mpk group described in this application were lower than those of the Model + placebo group. The neurobehavioral score results of the ATP6V1B2 binder-3mpk group described in this application and the ATP6V1B2 binder-5mpk group described in this application were both lower than those of the Model + placebo group.
  • This example illustrates that the ATP6V1B2 binders described herein (eg, mutant 1, GS18, RS17, KS16, KS15, and RS14) have therapeutic effects on stroke.
  • the ATP6V1B2 binders described herein eg, mutant 1, GS18, RS17, KS16, KS15, and RS14
  • This example illustrates that the ATP6V1B2 binder described in the present application has free radical scavenging and antioxidant effects, thereby having neuroprotective effects and therapeutic effects on stroke, and the effect is better than that of edaravone.
  • the most widely used in vitro model is the neuronal oxygen-glucose deprivation model (OGD), which mainly simulates the occurrence process of ischemic damage in vivo.
  • OGD neuronal oxygen-glucose deprivation model
  • Mouse hippocampal HT22 cells were cultured in DMEM and 10% serum in a 5% CO2 incubator at 37°C.
  • Oxygen-Glucose Deprivation (OGD) Cell Model HT22 cells were seeded into 96-well plates at a density of approximately 1 ⁇ 105 and cultured in DMEM with 10% serum in a 37°C incubator containing 5% CO2 /95% air for 24 hours. HT22 cells were placed in a glucose-free medium and then transferred to a sealed container containing a 5% CO2 /95% N2 mixture. The sealed container was then placed in a 37°C incubator for 16 hours. Subsequently, the glucose-free medium was replaced with normal medium containing glucose and cultured in a 37°C incubator containing 5% CO2 /95% air for 24 hours, followed by reoxygenation.
  • OGD Oxygen-Glucose Deprivation
  • HT22 cells Normally cultured HT22 cells served as the control group. OGD-treated HT22 cells were reoxygenated in normal culture medium and given solvent, the ATP6V1B2 binder described in this application (0.1 ⁇ M, 1 ⁇ M, 5 ⁇ M), and the ROS scavenger edaravone (200 ⁇ M), respectively. After reoxygenation for 24 hours, ROS was quantitatively detected.
  • ROS Reactive oxygen species
  • DCFH-DA was diluted 1:1000 in serum-free culture medium to a final concentration of 10 ⁇ M. The culture medium was removed and the diluted DCFH-DA was added. The cells were incubated in a 37°C cell culture incubator for 20 minutes. The cells were washed three times with serum-free culture medium to fully remove the DCFH-DA that had not entered the cells. Observation was performed using a fluorescence microscope, and the mean fluorescence intensity was calculated using Image J software.
  • Example 7 ATP6V1B2 binders regulate the production of sleep-related substances in the body
  • This example illustrates that the ATP6V1B2 binders described in the present application can regulate sleep-related substances in the body, thereby being used to prevent and/or treat sleep disorders.
  • the ATP6V1B2 binder was administered orally by gavage at approximately 10:00 AM daily. Samples were collected 1 hour after the single-dose group and 1 hour after the last oral administration in the multiple-dose group, once daily for 3 consecutive days. Samples were collected 1 hour after the last oral administration in the PBS group, after a single oral administration of the corresponding volume of PBS.
  • Plasma Blood was collected from the heart of anesthetized mice, placed in EDTA anticoagulant tubes, stored on ice, and centrifuged at 4000 ⁇ g for 10 minutes at 4°C. The supernatant was collected for analysis.
  • 5-HT Serotonin/5-hydroxytryptamine
  • E-EL-0033c Norepinephrine ELISA Kit
  • E-EL-M0788c Mouse Melatonin ELISA Kit
  • Avidin labeled with horseradish peroxidase is added, and biotin specifically binds to the avidin to form an immune complex, and free components are washed away.
  • a chromogenic substrate (TMB) is added, which develops a blue color under the catalysis of horseradish peroxidase and turns yellow upon addition of the stop solution.
  • the OD value was measured at a wavelength of 450 nm using an enzyme-labeled instrument.
  • the antigen concentration was inversely proportional to the OD 450 value.
  • the concentration of the antigen in the sample was calculated by drawing a standard curve.
  • ELISA kit operation steps refer to the kit instructions, the specific method is as follows:
  • TMB substrate solution

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Abstract

L'invention concerne un agent de liaison à la pompe à protons et son utilisation dans la préparation d'un réactif. L'agent de liaison comprend une molécule capable de se lier à ATP6V1B2 et/ou un fragment fonctionnellement actif de celui-ci.
PCT/CN2025/076106 2024-02-07 2025-02-07 Agent de liaison à la pompe à protons et son utilisation dans la préparation d'un réactif Pending WO2025168025A1 (fr)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
US20070059681A1 (en) * 2003-09-22 2007-03-15 Biomerieux Method for production of bioresorable microparticles, microparticles thus obtained and use thereof
CN103571846A (zh) * 2012-07-18 2014-02-12 中国人民解放军总医院 Atp6v1b2基因突变体及其应用
CN114423413A (zh) * 2019-07-02 2022-04-29 俄亥俄州国家创新基金会 利用皮肤-脑轴的神经退行性疾病疗法
WO2023107893A2 (fr) * 2021-12-06 2023-06-15 The Board Of Trustees Of The Leland Stanford Junior University Traitement de maladies neurodégénératives par l'inhibition de l'ataxine-2
WO2023125744A1 (fr) * 2021-12-29 2023-07-06 上海魁特迪生物科技有限公司 Utilisation d'un modulateur de la pompe à protons dans la préparation d'un réactif

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Publication number Priority date Publication date Assignee Title
US20070059681A1 (en) * 2003-09-22 2007-03-15 Biomerieux Method for production of bioresorable microparticles, microparticles thus obtained and use thereof
CN103571846A (zh) * 2012-07-18 2014-02-12 中国人民解放军总医院 Atp6v1b2基因突变体及其应用
CN114423413A (zh) * 2019-07-02 2022-04-29 俄亥俄州国家创新基金会 利用皮肤-脑轴的神经退行性疾病疗法
US20220244275A1 (en) * 2019-07-02 2022-08-04 Ohio State Innovation Foundation Neurodegenerative disease therapies utilizing the skin-brain axis
WO2023107893A2 (fr) * 2021-12-06 2023-06-15 The Board Of Trustees Of The Leland Stanford Junior University Traitement de maladies neurodégénératives par l'inhibition de l'ataxine-2
WO2023125744A1 (fr) * 2021-12-29 2023-07-06 上海魁特迪生物科技有限公司 Utilisation d'un modulateur de la pompe à protons dans la préparation d'un réactif

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