WO2022083675A1 - 针对多种冠状病毒的单克隆抗体及应用 - Google Patents

针对多种冠状病毒的单克隆抗体及应用 Download PDF

Info

Publication number
WO2022083675A1
WO2022083675A1 PCT/CN2021/125173 CN2021125173W WO2022083675A1 WO 2022083675 A1 WO2022083675 A1 WO 2022083675A1 CN 2021125173 W CN2021125173 W CN 2021125173W WO 2022083675 A1 WO2022083675 A1 WO 2022083675A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
antigen
binding fragment
variable region
monoclonal antibody
Prior art date
Application number
PCT/CN2021/125173
Other languages
English (en)
French (fr)
Inventor
王春河
陈玉宁
陈艺丽
陈淦均
王桂凤
王琪
Original Assignee
中国科学院上海药物研究所
达石药业(广东)有限公司
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 中国科学院上海药物研究所, 达石药业(广东)有限公司 filed Critical 中国科学院上海药物研究所
Publication of WO2022083675A1 publication Critical patent/WO2022083675A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6871—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an enzyme
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to monoclonal antibodies against various coronaviruses, antigen-binding fragments thereof and applications thereof.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV-2 is a virus that causes a worldwide respiratory epidemic (COVID-19) and is currently mainly divided into two types: wild-type SARS- CoV-2, and the more infectious mutant SARS-CoV-2-G614 that is currently circulating abroad.
  • SARS-CoV-2 has infected more than 38.5 million people worldwide and caused more than 1.09 million deaths.
  • vaccines against SARS-CoV-2 have been developed in this field, the efficacy is still unknown, and there is no specific therapeutic drug.
  • SARS-CoV-2, SARS-CoV and HCoV-NL63 are all coronaviruses.
  • the object of the present invention is to provide monoclonal antibodies and applications for various coronaviruses.
  • the binding site is H34.
  • the antibody is a monoclonal antibody.
  • the monoclonal antibody is a humanized antibody or a fully human antibody.
  • the monoclonal antibody is an IgG.
  • the antigen-binding fragment is a scFv, Fv, Fab or F(ab) 2 .
  • the monoclonal antibody comprises a heavy chain variable region ( VH ) and a light chain variable region ( VL ), wherein the VH comprises a heavy chain variable region comprising the sequence SEQ ID NO: 1 Region CDR1, heavy chain variable region CDR2 comprising sequence SEQ ID NO:2, and heavy chain variable region CDR3 comprising sequence SEQ ID NO:3, and the VL comprises a light chain comprising sequence SEQ ID NO:5
  • the amino acid sequence of the heavy chain variable region is at least 90% identical to the VH
  • the amino acid sequence of the light chain variable region is at least 90% identical to the VL identity.
  • amino acid sequence of the heavy chain variable region is SEQ ID NO:4
  • amino acid sequence of the light chain variable region is SEQ ID NO:8.
  • a bispecific antibody comprising the aforementioned monoclonal antibody or an antigen-binding fragment thereof.
  • An aforementioned monoclonal antibody or antigen-binding fragment thereof, or a conjugate of the aforementioned bispecific antibody and an effector molecule is an aforementioned monoclonal antibody or antigen-binding fragment thereof, or a conjugate of the aforementioned bispecific antibody and an effector molecule.
  • the effector molecule is a detectable label.
  • the detectable label is a fluorescent label, a radiolabel, avidin, biotin, or an enzyme.
  • the effector molecule is a toxin or chemotherapeutic agent.
  • the toxin is Pseudomonas aeruginosa exotoxin.
  • the nucleic acid molecule is operably linked to a promoter.
  • a plasmid containing the aforementioned nucleic acid molecule A plasmid containing the aforementioned nucleic acid molecule.
  • a kit for detecting the level of ACE2 in a subject comprising: the aforementioned monoclonal antibody or antigen-binding fragment thereof, bispecific antibody or conjugate.
  • a pharmaceutical composition comprising a prophylactically effective amount of the aforementioned monoclonal antibody or antigen-binding fragment, the aforementioned bispecific antibody, the aforementioned conjugate, the aforementioned nucleic acid, and optionally, a pharmaceutically acceptable carrier.
  • the disease is selected from coronavirus infection or hypertension.
  • the coronavirus includes, but is not limited to, SARS-CoV-2 (D614 and G614), SARS-CoV or HCoV-NL63.
  • the present invention creatively proposes the application of the binding sites Q18, E23, Q24, F28, K31, H34 and Y83 of ACE2 in the preparation of ACE2-related diagnostic, preventive or therapeutic preparations.
  • the specific antibody 3E8 of the present invention shows neutralizing ability for SARS-CoV-2 (D614 and G614), SARS-CoV and HCoV-NL63, and at the same time it has no effect on the catalytic activity of ACE2, therefore, it can be It can be used to prevent or treat diseases related to the above viruses in clinic, and can also be used for the detection of ACE2 or the classification of related diseases.
  • Figure 1 The binding ability of the specific antibody 3E8 of the present invention to ACE2 was detected by ELISA (A), biofilm interference technology (BLI) (B) and flow cytometry (FACS) (C).
  • A ELISA
  • B biofilm interference technology
  • FACS flow cytometry
  • Figure 2 Detecting the neutralizing ability of the specific antibody 3E8 of the present invention to SARS-CoV-2-S1 (D614 and G614), SARS-CoV-S1 and HCoV-NL63-S1 by ELISA (A: SARS-CoV- 2-S1 (D614 and G614), SARS-CoV-S1 and HCoV-NL63-S1 bind to ACE2, B: mAb 3E8 against SARS-CoV-2-S1 (D614 and G614), SARS-CoV-S1 and HCoV-NL63-S1 disrupts the aforementioned binding).
  • A SARS-CoV- 2-S1 (D614 and G614)
  • B mAb 3E8 against SARS-CoV-2-S1 (D614 and G614)
  • SARS-CoV-S1 and HCoV-NL63-S1 disrupts the aforementioned binding).
  • Figure 3 Detecting the neutralization of SARS-CoV-2 (D614(B) and G614(A)), SARS-CoV(C) and HCoV-NL63(D) pseudovirus by the specific antibody 3E8 of the present invention by ELISA ability.
  • Figure 4 The neutralization ability of the specific antibody 3E8 of the present invention to the live virus SARS-CoV-2 (D614) in vitro (A) and in vivo (B) was detected by qRT-PCR.
  • Figure 5 The effect of the specific antibody 3E8 of the present invention on the catalytic activity of ACE2 at the molecular level (A) and the cellular level (B).
  • Figure 6 Schematic representation of the site where the specific antibody 3E8 competes for binding to ACE2.
  • the DNA fragment encoding extracellular hACE2 (residues 19-740) from the plasmid encoding full-length human ACE2 was subcloned into mammalian expression vectors pTT5 (his-tag) and pINFUSE-hIgG1-Fc1 (Fc-tag), and the The purified plasmid was transfected into cells for expression. When the antibody is enriched to a certain extent, it is purified by protein A chromatography.
  • mice The immunized mice were selected from female balb/c mice, and the antigens and adjuvants required for immunization were prepared into a homogenate, and the toe injection and subcutaneous multi-point injection were used. After the initial immunization, multiple booster immunizations were performed. Prime and boost immunizations were with ACE2-Fc + CpG ODN + incomplete Freund's adjuvant. After immunization, the mouse B cells were extracted and fused with myeloma cells SP2/0 to prepare hybridoma cells.
  • Anti-monoclonal antibody its variable region is obtained from hybridoma screening, and its constant region is obtained from human antibody IgG.
  • Chimeric antibodies need to be humanized through complementarity determining region (CDR) grafting, specificity-determining region (SDR) grafting, superhumanization or framework shuffling ) and guided selection to effectively reduce the immunogenicity of the antibody in clinical use, and optimize the CDR region of the antibody molecule to improve the affinity of the antibody.
  • CDR complementarity determining region
  • SDR specificity-determining region
  • framework shuffling superhumanization or framework shuffling
  • the antibody 3E8 clone was selected according to the test results, specifically: the plasmids for expressing the heavy and light chains of 3E8 were transferred into HEK293F cells. After 5 days, it was purified by protein A chromatography and analyzed for protein purity by size exclusion chromatography.
  • the heavy chain and light chain variable region sequences of antibody 3E8 are respectively: SEQ ID NO:4 and SEQ ID NO:8, the heavy chain and light chain constant region sequences are respectively: SEQ ID NO:9 and SEQ ID NO:10,
  • the nucleotide sequences of the heavy chain and light chain constant regions are respectively: SEQ ID NO:11 and SEQ ID NO:12, and the full-length sequences of the heavy chain and light chain are respectively: SEQ ID NO:13 and SEQ ID NO:14,
  • the nucleotide sequences (original) of the variable regions of the heavy and light chains are: SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and the nucleotide sequences (optimized) of the constant regions of the heavy and light chains are: SEQ ID NO: 15 and SEQ ID NO: 16, respectively.
  • ID NO: 17 and SEQ ID NO: 18 were used to detect the binding ability of monoclonal antibody 3E8 to ACE2 by ELISA, FACS technology and biofilm interference technology (BL
  • 96-well plates were coated with purified recombinant hACE2-his protein, overnight at 4°C. After blocking with 1% casein protein for 1 hour at room temperature, the plates were washed with phosphate buffered saline (PBST) containing 0.05% Tween-20 and incubated with serial dilutions of 3E8 for one hour. After washing with PBST, HRP-labeled goat anti-human IgG was added and incubated for 1 hour. Wash with PBST, add TMB substrate, stop with 2M H2SO4 , and measure OD450 with a SpectraMax M5e microplate reader.
  • PBST phosphate buffered saline
  • FACS method to detect the binding ability of monoclonal antibody 3E8 to cell surface ACE2 Take 5 ⁇ 10 5 HEK293F cells stably transfected with ACE2 or Vero E6 cells endogenously expressing ACE2 in a 96-well plate, wash with PBS, and place the serially diluted 3E8 antibody was added to the plate, and PE-labeled goat anti-human IgG was added, incubated at 4°C for 40 minutes, washed with PBS, and detected on a flow cytometer.
  • Biofilm Layer Optical Interferometry (BLI) Technique Binding capacity was measured by BLI technique using Octet Red96. For 3E8, 15 ⁇ g/ml ACE2-his protein was captured by Ni-NTA biosensor and combined with different gradients of 3E8. For various S1 proteins bound to ACE2, 15 ⁇ g/ml of S1-his protein was immobilized on the Ni-NTA biosensor and bound to different gradients of ACE2-Fc. Equilibrate with PBS containing 0.05% Tween 20 for 60 s. The association and dissociation times were both 400 s. Average Kon, Koff and apparent KD values for binding affinity were calculated based on the overall fit (1:1) of all binding curves.
  • SARS-CoV-2 (D614 and G614), SARS-CoV and HCoV-NL63: HEK293T cells were co-transfected with two plasmids: one of the plasmids expressed Env-deficient HIV-1 (pNL4-3.luc.RE) (with a luciferase reporter gene), another plasmid expresses the full-length S protein of SARS-CoV-2 (D614 and G614), SARS-CoV or HCoV-NL63. The supernatant containing viral particles was collected 48 hours after transfection and filtered through a 0.45 ⁇ m filter.
  • 1.2 x 104 HEK293F /hACE2/EGFP cells were seeded per well.
  • Cells were incubated with 50 ⁇ l of serially diluted antibodies or ACE2-Fc, B38 (as a positive control), Isotype (isotype control) for 1 hour at 37°C, and various equal volumes of pseudoviruses were added.
  • 100 ⁇ l of DMEM containing 10% FBS was used as a negative control, and 50 ⁇ l of pseudovirus and the same volume of DMEM were used as a positive control. After 24 hours, the medium was replaced with fresh medium and incubated for an additional 48 hours.
  • Relative fluorescence units were measured using a firefly luciferase reporter gene assay kit. Neutralizing activity (%) [1-(RFU sample -RFU negative control )/(RFU positive control -RFU negative control )] ⁇ 100%. IC50 values were calculated by nonlinear regression in GraphPad Prism 8.
  • Vero E6 cells were seeded per well in a 24-well plate, and 24 hours later, serially diluted antibodies and SARS-CoV-2 (D614) were added to the plate. After 24 hours, the virus supernatant was collected, RNA was extracted, the level of viral RNA was detected by qRT-PCR, and the inhibition rate of each antibody against the virus was calculated.
  • the primer sequences are: RBD-qF1: 5'-caatggtttaacaggcacagg-3' (SEQ ID NO: 20), RBD-qR1: 5'-ctcaagtgtctgtggatcacg-3' (SEQ ID NO: 21), Probe: acagcatcagtagtgtcagcaatgtctc (SEQ ID NO: twenty two).
  • ACE2 enzymatic activity at the molecular level After incubating the diluted antibody (AF933/3E8/isotype) with 2 ⁇ g/ml ACE2-his on a shaker for 1 hour at room temperature, dilute it 5-fold with activity buffer, and add it to containing 50 ⁇ l of 200mM base in the plate of the material. Incubate at 37°C for 20 minutes and read at 325nm/393nm.
  • Vero E6 cells were plated in 96-well plates at 1 ⁇ 10 5 cells per well, overnight at 37°C. Discard the supernatant, wash the cells with PBS, then add 20 ⁇ g/ml AF933/3E8/isotype, 100 ⁇ l per well, incubate at 37°C for 1 hour; discard the liquid in the plate, add 50 ⁇ l activity buffer and 50 ⁇ l 200mM substrate, Incubate at 37°C for 20 minutes and read at 325nm/393nm.
  • Dilution buffer and activation buffer were prepared as follows: Dilution buffer: 100 mM Glycine, 50 ⁇ M ZnCl 2 , 150 mM NaCl, 1% BSA; Activation buffer: 75 mM Tris, 10 ⁇ M ZnCl 2 , 150 mM NaCl, 0.01% TritonX-100, pH 7. 2.
  • the binding site of 3E8 and ACE2 was analyzed and data processed by cryo-electron microscopy, and finally it was found that the CDR2 of 3E8 interacted with H34, K31, E23 and Q18 of ACE2, and the CDR3 of 3E8 interacted with Q24, Y83 and F28 of ACE2, thereby inhibiting the interaction between ACE2 and ACE2.
  • the binding of SARS-CoV-2, SARS-CoV-2 and HCoV-NL63 to ACE2 was interrupted (see Figure 6), in which the H34 site on ACE2 plays a key role, which can simultaneously inhibit the binding of multiple coronaviruses to ACE2.
  • the human ACE2 amino acid sequence is SEQ ID NO:19.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Pathology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Virology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pulmonology (AREA)
  • Plant Pathology (AREA)

Abstract

本发明涉及针对多种冠状病毒的单克隆抗体及应用。所述单克隆抗体与氨基酸序列为SEQ ID NO:19的人ACE2在结合位点结合，所述结合位点选自Q18，E23，Q24，F28，K31，H34和Y83位中的任一个或两种以上的组合。所述单克隆抗体或其抗原结合片段可在临床上预防或治疗多种冠状病毒(包括但不限于SARS-CoV-2(D614和G614)、SARS-CoV和HCoV-NL63)相关疾病，亦可用于ACE2的检测或相关疾病分型。

Description

针对多种冠状病毒的单克隆抗体及应用 技术领域
本发明属于生物技术领域，具体涉及针对多种冠状病毒的单克隆抗体、其抗原结合片段及其应用。
背景技术
现有技术公开了严重急性呼吸系统综合征冠状病毒2(SARS-CoV-2)是一种引起世界范围内呼吸道流行病(COVID-19)的病毒，目前主要分为两种：野生型SARS-CoV-2，以及目前国外流行的感染力更强的变异株SARS-CoV-2-G614。近一年来，SARS-CoV-2已感染全球3850余万人，并导致109余万人死亡。目前本领域虽已研制出针对SARS-CoV-2的疫苗，但药效尚不可知，也无特效的治疗性药物。SARS-CoV-2、SARS-CoV和HCoV-NL63同属于冠状病毒。研究显示，这三种病毒都能通过各自的病毒刺突蛋白S1亚基与宿主受体人血管紧张素转化酶2(ACE2)结合，介导病毒的入侵，所以ACE2是该类病毒最主要的受体，而S1-ACE2结合被认为是有效的阻断靶标。抗体在抗感染治疗及紧急被动免疫预防方面扮演着重要的角色。因此，研制特异性SARS-CoV-2(D614和G614)、SARS-CoV和HCoV-NL63的抗体，用于紧急预防和治疗是十分必要和可行的。
发明内容
本发明的目的在于提供针对多种冠状病毒的单克隆抗体及应用。
一种疫苗、抗体或其抗原结合片段，其与氨基酸序列为SEQ ID NO:19的人ACE2在结合位点结合，所述结合位点选自SEQ ID NO:19中的Q18， E23，Q24，F28，K31，H34和Y83位中的任一个或两种以上的组合。
在具体实施方式中，所述结合位点为H34。
在具体实施方式中，所述抗体为单克隆抗体。
在具体实施方式中，所述单克隆抗体是人源化抗体或全人源抗体。
在具体实施方式中，所述单克隆抗体为IgG。
在具体实施方式中，所述抗原结合片段是scFv、Fv、Fab或F(ab) 2。
在具体实施方式中，所述单克隆抗体包括重链可变区(V H)和轻链可变区(V L)，其中所述V H包括含序列SEQ ID NO:1的重链可变区CDR1，含序列SEQ ID NO:2的重链可变区CDR2，以及含序列SEQ ID NO:3的重链可变区CDR3，并且所述V L包括含序列SEQ ID NO:5的轻链可变区CDR1，含序列SEQ ID NO:6的轻链可变区CDR2，以及含序列SEQ ID NO:7的轻链可变区CDR3。
在具体实施方式中，所述重链可变区的氨基酸序列与所述V H具有至少90％的同一性，且所述轻链可变区的氨基酸序列与所述V L具有至少90％的同一性。
在具体实施方式中，所述重链可变区的氨基酸序列为SEQ ID NO:4，且所述轻链可变区的氨基酸序列为SEQ ID NO:8。
一种双特异性抗体，其含有前述单克隆抗体或其抗原结合片段。
一种前述单克隆抗体或其抗原结合片段，或前述双特异性抗体与一种效应分子的偶联物。
在具体实施方式中，所述效应分子是可检测标记。
在具体实施方式中，所述可检测标记是荧光标记、放射性标记、亲和素、生物素，或酶。
在具体实施方式中，所述效应分子是毒素或化疗剂。
在具体实施方式中，所述毒素是绿脓杆菌外毒素。
一种核酸分子，其编码前述单克隆抗体或抗原结合片段，前述双特异性抗体。
在具体实施方式中，所述核酸分子可操作地连接至启动子。
一种质粒，其含有前述核酸分子。
一种用于检测受试者的ACE2水平的试剂盒，其包括：前述单克隆抗体或其抗原结合片段、双特异性抗体或偶联物。
一种药用组合物，其含有有效预防剂量的前述单克隆抗体或抗原结合片段，前述双特异性抗体，前述偶联物，前述核酸，和任选地，药学上可接受载体。
前述单克隆抗体或其抗原结合片段，双特异性抗体，或偶联物，或前述药用组合物在制备用于预防或治疗或诊断与ACE2相关疾病的药物中的用途。
在具体实施方式中，所述疾病选自冠状病毒感染或高血压。
在具体实施方式中，所述冠状病毒包括但不限于SARS-CoV-2(D614和G614)、SARS-CoV或HCoV-NL63。
有益效果
本发明创造性地提出了ACE2的结合位点Q18，E23，Q24，F28，K31，H34和Y83位在制备ACE2相关的诊断、预防或治疗制剂中的应用。
进一步地，本发明的特异性抗体3E8显示出对于SARS-CoV-2(D614和G614)、SARS-CoV和HCoV-NL63的中和能力，同时其对于ACE2的催化活性没有影响，因此，其可在临床上预防或治疗与上述病毒相关的疾病，亦可用于ACE2的检测或相关疾病的分型。
附图说明
图1：利用ELISA(A)、生物膜干涉技术(BLI)(B)和流式细胞术(FACS)(C)法检测本发明的特异性抗体3E8与ACE2的结合能力。
图2：利用ELISA法检测本发明的特异性抗体3E8对SARS-CoV-2-S1(D614和G614)、SARS-CoV-S1和HCoV-NL63-S1的中和能力(A：SARS-CoV-2-S1(D614和G614)、SARS-CoV-S1和HCoV-NL63-S1与ACE2结合，B：单克隆抗体3E8对SARS-CoV-2-S1(D614和G614)、SARS-CoV-S1和HCoV-NL63-S1破坏前述结合)。
图3：利用ELISA法检测本发明的特异性抗体3E8对SARS-CoV-2(D614(B)和G614(A))、SARS-CoV(C)和HCoV-NL63(D)假病毒的中和能力。
图4：qRT-PCR法检测本发明的特异性抗体3E8在体外(A)和体内(B)对活病毒SARS-CoV-2(D614)的中和能力。
图5：本发明的特异性抗体3E8在分子水平(A)和细胞水平(B)对ACE2的催化活性的影响。
图6：示意性示出特异性抗体3E8竞争性结合ACE2的位点。
具体实施方式
以下通过具体实施例详细描述本发明，然而下述实施例并非用于限制本发明的范围。
本发明实例中未明确具体条件的实验方法，均为常规方法或按厂商推荐条件。未注明具体来源的试剂，为市场中的常规试剂。
实施例1 ACE2蛋白的产生
将编码全长人ACE2的质粒中编码胞外hACE2(19-740位残基)的DNA片段亚克隆到哺乳动物表达载体pTT5(his-tag)和pINFUSE-hIgG1-Fc1(Fc-tag)，将纯化的质粒转染细胞进行表达。当抗体富集到一定程度后，通过protein A色谱法纯化。
实施例2免疫小鼠获得杂交瘤细胞
免疫小鼠选用雌性balb/c小鼠，将免疫所需抗原及佐剂制备匀浆，采用脚趾注射和皮下多点注射法。初次免疫后，再多次加强免疫。初次免疫和加强免疫用ACE2-Fc+CpG ODN+弗氏不完全佐剂。免疫完成后，提取小鼠B细胞，将其与骨髓瘤细胞SP2/0融合，制备杂交瘤细胞。
实施例3抗体分子的人源化
抗单克隆抗体，其可变区来自于杂交瘤筛选获得，恒定区来自于人源抗体IgG。嵌合抗体需要进行人源化，通过互补决定区(complementarity determining region，CDR)移植、特异性决定区(specificity-determining region，SDR)移植、超级人源化(superhumanization)或框架区轮换(framework shuffling)、导向性选择三种策略进行，以有效降低抗体在临床使用中的免疫源性，并对抗体分子的CDR区进行优化，提高抗体的亲和力。
实验实施例1单克隆抗体3E8与靶点ACE2的结合。
根据测试结果选择抗体3E8克隆，具体为：将用于表达3E8的重链和轻链的质粒转入HEK293F细胞。5天后，用protein A色谱法纯化，用分子排阻色谱分析蛋白纯度。
抗体3E8的重链和轻链可变区序列分别为：SEQ ID NO:4和SEQ ID NO:8，重链和轻链恒定区序列分别为：SEQ ID NO:9和SEQ ID NO:10，重链和轻链恒定区的核苷酸序列分别为：SEQ ID NO:11和SEQ ID NO:12，重链和轻链全长序列分别为：SEQ ID NO:13和SEQ ID NO:14，重链和轻链可变区的核苷酸序列(原始)分别为：SEQ ID NO:15和SEQ ID NO:16，重链和轻链恒定区的核苷酸序列(优化)分别为：SEQ ID NO:17和SEQ ID NO:18，分别用ELISA法、FACS技术和生物膜干涉技术(BLI)检测单克隆抗体3E8与ACE2的结合能力。
ELISA法检测单克隆抗体3E8与细胞表面ACE2的结合能力：将96孔板包被纯化的重组hACE2-his蛋白，4℃过夜。在室温下用1％casein蛋白封闭1小时后，将板用含0.05％Tween-20的磷酸盐缓冲液(PBST)洗涤，并加入梯度稀释的3E8孵育一小时。PBST洗涤后，加入HRP标记的山羊抗人IgG，孵育1小时。PBST洗涤，加入TMB底物，并用2M的H 2SO 4终止，再用SpectraMax M5e酶标仪检测OD 450。为了测量S1蛋白与hACE2的结合能力，将2μg/ml的各种S1蛋白包被到平板中，再加入梯度稀释的ACE2-Fc，利用辣根过氧化物酶(HRP)标记的山羊抗人IgG检测结合的ACE2-Fc。
FACS法检测单克隆抗体3E8与细胞表面ACE2的结合能力：取5×10 5个稳定转染ACE2的HEK293F细胞或内源表达ACE2的Vero E6细胞于96孔板中，PBS清洗，将梯度稀释的3E8抗体加入到板中，加入PE标记的山羊抗人IgG，4℃孵育40分钟，PBS洗，流式细胞仪上机检测。
生物膜层光学干涉(BLI)技术：使用Octet Red96通过BLI技术测量结合能力。针对3E8，利用Ni-NTA生物传感器捕获15μg/ml ACE2-his蛋白，并与不同梯度的3E8进行结合。针对与ACE2结合的各种S1蛋白，将15μg/ml的S1-his蛋白固定在Ni-NTA生物传感器上，并与不同梯度的ACE2-Fc进行结合。用含0.05％Tween 20的PBS持续60s进行平衡。结合和解离时间均为400s。根据所有结合曲线的整体拟合度(1：1)，计算结合亲和力的平均Kon，Koff和表观KD值。
实验结果如图1所示，从图1可以看出，在分子和细胞水平上，通过不同方法都证明了3E8能特异性地结合ACE2，且结合能力较强。
实验实施例2单克隆抗体3E8对SARS-CoV-2-S1(D614和G614)、SARS-CoV-S1和HCoV-NL63-S1的中和能力。
具体为：将2μg/ml的各种S1蛋白在4℃下包被过夜。将梯度稀释的3E8或同型对照与5μg/ml ACE2-Fc在室温下预孵育30分钟，再将混合物加入包被的板中孵育1小时。利用HRP标记的山羊抗人IgG检测结合的ACE2-Fc。
实验结果如图2所示，从图2可以看出，SARS-CoV-2-S1(D614和G614)、SARS-CoV-S1和HCoV-NL63-S1都能和ACE2结合(图2A和下表1)，而这种结合能够被3E8竞争破坏，即单克隆抗体3E8对SARS-CoV-2-S1(D614和G614)、SARS-CoV-S1和HCoV-NL63-S1具有很好的中和能力。
表1
Figure PCTCN2021125173-appb-000001
实验实施例3单克隆抗体3E8对SARS-CoV-2(D614和G614)、SARS-CoV和HCoV-NL63假病毒的中和能力。
构建假病毒SARS-CoV-2(D614和G614)、SARS-CoV和HCoV-NL63：将两个质粒共转HEK293T细胞：其中一个质粒表达Env缺陷型HIV-1(pNL4-3.luc.RE)(带有荧光素酶报告基因)，另一个质粒表达SARS-CoV-2(D614和G614)、SARS-CoV或HCoV-NL63的全长S蛋白。转染48小时后收集含有病毒颗粒的上清液，0.45μm过滤膜过滤。
在96孔板中，每孔接种1.2×10 4个HEK293F/hACE2/EGFP细胞。将细胞与50μl梯度稀释的抗体或ACE2-Fc、B38(作为阳性对照)、Isotype(同型对照)在37℃温育1小时，加入各种相同体积的假病毒。100μl含10％FBS的DMEM作为阴性对照，50μl假病毒和同体积的DMEM设为阳性对照。24小时后，更换新鲜培养基并再温育48小时。利用萤火虫荧光素酶报告基因检测试剂盒测量相对荧光单位(RLU)。中和活性(％)[1-(RFU 样品–RFU 阴 性对照)/(RFU 阳性对照–RFU 阴性对照)]×100％。通过GraphPad Prism 8中的非线性回归计算IC 50值。
实验结果如图3和表2所示，从图3可以看出，单克隆抗体3E8能够浓度依赖性地竞争SARS-CoV-2(D614和G614)、SARS-CoV和HCoV-NL63假病毒与ACE2的结合，即3E8对各种假病毒具有很好的中和能力。
表2
Figure PCTCN2021125173-appb-000002
实验实施例4特异性抗体3E8在体内外对活病毒SARS-CoV-2(D614)的中和能力。
细胞水平：在24孔板中，每孔接种1×10 5个Vero E6细胞，24小时后，将梯度稀释的抗体和SARS-CoV-2(D614)加入板中。24小时后，收集病毒上清，提取RNA，用qRT-PCR法检测病毒RNA水平，计算各抗体对病毒的抑制率。
动物水平：6-8周龄的Balb/c雌性小鼠(n＝5)用avertin(250mg/ml)麻醉后，首先以10 6FFU VRP-hACE2感染每只小鼠。12小时后，腹腔注射抗体(3E8/B38/Isotype)，再12小时后将10 5PFU SARS-CoV-2通过滴鼻的方式感染小鼠。感染3天后，取每只小鼠的肺组织，检测各组SARS-CoV-2的RNA水平。引物序列为：RBD-qF1：5’-caatggtttaacaggcacagg-3’(SEQ ID NO:20),RBD-qR1：5’-ctcaagtgtctgtggatcacg-3’(SEQ ID NO:21)，Probe：acagcatcagtagtgtcagcaatgtctc(SEQ ID NO:22)。
实验结果如图4，表3和表4所示，特异性抗体3E8能够在细胞和动物水平显著降低活SARS-CoV-2-D614的RNA水平，并优于阳性对照B38。
表3细胞水平的抗新冠病毒活性
Figure PCTCN2021125173-appb-000003
表4动物水平的抗新冠病毒活性
Figure PCTCN2021125173-appb-000004
实验实施例5特异性抗体3E8对于ACE2的催化活性的影响。
分子水平ACE2酶活性检测：将稀释的抗体(AF933/3E8/isotype)与2μg/ml ACE2-his在振荡器上室温孵育1小时后，用活性缓冲液稀释5倍，再加到含有50μl 200mM底物的平板中。37℃孵育20分钟，读取325nm/393nm数值。
细胞水平ACE2酶活性检测：将Vero E6细胞以每孔1×10 5个细胞铺在96孔板中，37℃过夜。弃上清，用PBS洗细胞，再加入浓度为20μg/ml的AF933/3E8/isotype，每孔100μl，37℃孵育1小时；弃去板中液体，加50μl活性缓冲液和50μl 200mM底物，于37℃孵育20分钟，读取325nm/393nm数值。
稀释缓冲液和活性缓冲液的配制如下：稀释缓冲液：100mM甘氨酸，50μMZnCl 2、150mM NaCl，1％BSA；活性缓冲液：75mM Tris，10μM ZnCl 2、150mM NaCl，0.01％TritonX-100，pH7.2。
实验结果如图5所示，从图5可以看出，3E8在高浓度下(分子水平100μg/ml；细胞水平20μg/ml)，均未对ACE2的酶催化活性产生影响，安全性很好。其中AF933作为阳性对照，能够显著抑制ACE2的酶活。
实验实施例6特异性抗体3E8结合ACE2的结构基础。
利用冷冻电镜法对3E8与ACE2的结合位点进行分析和数据处理，最终发现3E8的CDR2与ACE2的H34、K31、E23和Q18作用，3E8的CDR3 与ACE2的Q24、Y83和F28作用，从而阻断了SARS-CoV-2、SARS-CoV-2和HCoV-NL63与ACE2的结合(参见图6)，其中ACE2上H34位点起着关键作用，能够同时抑制多种冠状病毒与ACE2的结合。
人ACE2氨基酸序列为SEQ ID NO:19。

Claims (22)

  1. 一种抗体或其抗原结合片段，其与氨基酸序列为SEQ ID NO:19的人ACE2在结合位点结合，所述结合位点选自SEQ ID NO:19中的Q18，E23，Q24，F28，K31，H34和Y83位中的任一个或两种以上的组合。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其中，所述单克隆抗体包括重链可变区(V H)和轻链可变区(V L)，其中所述V H包括含序列SEQ ID NO:1的重链可变区CDR1，含序列SEQ ID NO:2的重链可变区CDR2，以及含序列SEQ ID NO:3的重链可变区CDR3，并且所述V L包括含序列SEQ ID NO:5的轻链可变区CDR1，含序列SEQ ID NO:6的轻链可变区CDR2，以及含序列SEQ ID NO:7的轻链可变区CDR3。
  3. 根据权利要求2所述的抗体或其抗原结合片段，其中，所述重链可变区的CDR区的氨基酸序列与所述V H的CDR区具有至少90％的同一性，且所述轻链可变区的CDR区氨基酸序列与所述V L的CDR区具有至少90％的同一性。
  4. 根据权利要求2所述的抗体或其抗原结合片段，其中，所述重链可变区的氨基酸序列为SEQ ID NO:4，且所述轻链可变区的氨基酸序列为SEQ ID NO:8。
  5. 根据权利要求1-4中任一项所述的抗体或其抗原结合片段，其中，所述结合位点为H34。
  6. 根据权利要求1-4中任一项所述的抗体或其抗原结合片段，其中，所述抗体为单克隆抗体。
  7. 根据权利要求6所述的抗体或其抗原结合片段，其中，所述单克隆抗 体是人源化抗体或全人源抗体。
  8. 根据权利要求6所述的抗体或其抗原结合片段，其中，所述单克隆抗体为IgG。
  9. 根据权利要求1-4中任一项所述的抗体或其抗原结合片段，其中，所述抗原结合片段是scFv、Fv、Fab或F(ab) 2。
  10. 一种双特异性抗体，其含有如权利要求1至9任一项所述的单克隆抗体或其抗原结合片段。
  11. 一种如权利要求1至9任一项所述的单克隆抗体或其抗原结合片段，或如权利要求10所述的双特异性抗体与一种效应分子的偶联物。
  12. 根据权利要求11所述的偶联物，其中，所述效应分子是可检测标记。
  13. 根据权利要求12所述的偶联物，其中，所述可检测标记是荧光标记、放射性标记、亲和素、生物素，或酶。
  14. 根据权利要求11所述的偶联物，其中，所述效应分子是毒素或化疗剂。
  15. 根据权利要求14所述的偶联物，其中，所述毒素是绿脓杆菌外毒素。
  16. 一种核酸分子，其编码如权利要求1至9任一项所述的单克隆抗体或抗原结合片段，或如权利要求10所述的双特异性抗体。
  17. 一种用于检测受试者的ACE2水平的试剂盒，其包括：如权利要求1至9任一项所述的单克隆抗体或其抗原结合片段、如权利要求10所述的双特异性抗体，或如权利要求11-15任一项所述的偶联物。
  18. 一种药用组合物，其含有有效预防剂量的如权利要求1至9任一项所述的单克隆抗体或抗原结合片段，如权利要求10所述的双特异性抗体，如权利要求11-15任一项所述的偶联物或如权利要求16所述的核酸分子，和任选地，药学上可接受载体。
  19. 如权利要求1至9任一项所述的单克隆抗体或其抗原结合片段，如权利要求10所述的双特异性抗体，如权利要求11-15任一项所述的偶联物，或如权利要求18所述的药用组合物在制备用于预防或治疗或诊断与ACE2相关疾病的药物中的用途。
  20. 根据权利要求19所述的用途，其中，所述疾病选自冠状病毒感染或高血压。
  21. 根据权利要求20所述的用途，其中，所述冠状病毒包括SARS-CoV-2、SARS-CoV-2-G614、SARS-CoV或HCoV-NL63。
  22. 一种疫苗，其至少包含如权利要求1-9任一项所述的抗体或其抗原结合片段或如权利要求10所述的双特异性抗体。
PCT/CN2021/125173 2020-10-23 2021-10-21 针对多种冠状病毒的单克隆抗体及应用 WO2022083675A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011143522.9A CN112409488B (zh) 2020-10-23 2020-10-23 针对多种冠状病毒的单克隆抗体及应用
CN202011143522.9 2020-10-23

Publications (1)

Publication Number Publication Date
WO2022083675A1 true WO2022083675A1 (zh) 2022-04-28

Family

ID=74840136

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/125173 WO2022083675A1 (zh) 2020-10-23 2021-10-21 针对多种冠状病毒的单克隆抗体及应用

Country Status (2)

Country Link
CN (1) CN112409488B (zh)
WO (1) WO2022083675A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024020646A1 (en) * 2022-07-27 2024-02-01 The Council Of The Queensland Institute Of Medical Research Methylation biomarker for infection resistance

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2021004130A (es) 2020-04-02 2021-06-15 Regeneron Pharma Anticuerpos contra glicoproteína de espícula anti-sars-cov-2 y fragmentos de unión al antígeno.
CR20220660A (es) 2020-06-03 2023-02-17 Regeneron Pharma Métodos para tratar o prevenir infecciones por sars-cov2 y covid-19 con anticuerpos de glicoproteína espícular anti-sars-cov-2
CN112409488B (zh) * 2020-10-23 2022-07-01 中国科学院上海药物研究所 针对多种冠状病毒的单克隆抗体及应用
WO2022191162A1 (ja) * 2021-03-09 2022-09-15 株式会社オーダーメードメディカルリサーチ 抗ａｃｅ２モノクローナル抗体

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1914226A (zh) * 2003-11-25 2007-02-14 达纳-法伯癌症研究院有限公司 SARS－CoV抗体及其使用方法
CN111273016A (zh) * 2020-02-26 2020-06-12 浙江诺迦生物科技有限公司 一种基于s蛋白配体与ace2受体竞争法层析的冠状病毒快速检测的试剂盒
CN111592595A (zh) * 2020-04-27 2020-08-28 南京医科大学 抗新型冠状病毒SARS-Cov-2的中和性抗体及其应用
CN111647076A (zh) * 2020-04-27 2020-09-11 南京医科大学 抗新型冠状病毒SARS-Cov-2的中和性单域抗体及其应用
CN111647077A (zh) * 2020-06-02 2020-09-11 深圳市因诺赛生物科技有限公司 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用
CN112409488A (zh) * 2020-10-23 2021-02-26 中国科学院上海药物研究所 针对多种冠状病毒的单克隆抗体及应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007044695A2 (en) * 2005-10-07 2007-04-19 Dana-Farber Cancer Institute ANTIBODIES AGAINST SARS-CoV AND METHODS OF USE THEREOF
CN103864924B (zh) * 2014-02-14 2016-09-07 中国科学院微生物研究所 一种中东呼吸综合征冠状病毒抗体及其制备方法
MX2021004130A (es) * 2020-04-02 2021-06-15 Regeneron Pharma Anticuerpos contra glicoproteína de espícula anti-sars-cov-2 y fragmentos de unión al antígeno.
CN111793129B (zh) * 2020-07-28 2021-09-24 上海市公共卫生临床中心 一种特异性结合冠状病毒的抗体或其抗原结合片段

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1914226A (zh) * 2003-11-25 2007-02-14 达纳-法伯癌症研究院有限公司 SARS－CoV抗体及其使用方法
CN111273016A (zh) * 2020-02-26 2020-06-12 浙江诺迦生物科技有限公司 一种基于s蛋白配体与ace2受体竞争法层析的冠状病毒快速检测的试剂盒
CN111592595A (zh) * 2020-04-27 2020-08-28 南京医科大学 抗新型冠状病毒SARS-Cov-2的中和性抗体及其应用
CN111647076A (zh) * 2020-04-27 2020-09-11 南京医科大学 抗新型冠状病毒SARS-Cov-2的中和性单域抗体及其应用
CN111647077A (zh) * 2020-06-02 2020-09-11 深圳市因诺赛生物科技有限公司 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用
CN112409488A (zh) * 2020-10-23 2021-02-26 中国科学院上海药物研究所 针对多种冠状病毒的单克隆抗体及应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SEDOKANI AMIN, FEIZOLLAHZADEH SADEGH: "Plasmapheresis, Anti-ACE2 and Anti-FcγRII Monoclonal Antibodies: A Possible Treatment for Severe Cases of COVID-19", DRUG DESIGN, DEVELOPMENT AND THERAPY, vol. Volume 14, 6 July 2020 (2020-07-06), pages 2607 - 2611, XP055907088, DOI: 10.2147/DDDT.S262491 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024020646A1 (en) * 2022-07-27 2024-02-01 The Council Of The Queensland Institute Of Medical Research Methylation biomarker for infection resistance

Also Published As

Publication number Publication date
CN112409488B (zh) 2022-07-01
CN112409488A (zh) 2021-02-26

Similar Documents

Publication Publication Date Title
WO2022083675A1 (zh) 针对多种冠状病毒的单克隆抗体及应用
JP5575923B2 (ja) 抗凝固薬の解毒剤
JP7235256B2 (ja) 重症熱性血小板減少症候群ウイルスの外膜糖タンパク質に結合する抗体及びその用途
TW202146443A (zh) 一種抗新型冠狀病毒的單克隆抗體及其應用
AU2011208719B2 (en) Anticoagulant antidotes
US20200347130A1 (en) CD96 Antibody, Antigen-Binding Fragment and Pharmaceutical use Thereof
CN111333723B (zh) 针对狂犬病病毒g蛋白的单克隆抗体及其用途
CN110903394B (zh) 可结合cd4的多肽及其应用
EP3970798A1 (en) Sars-cov-2-nanobodies
US20220017604A1 (en) ANTI-SARS-CoV-2 MONOCLONAL ANTIBODIES
TW201302796A (zh) 抗凝血劑解毒劑
JP2023542107A (ja) ＳＡＲＳ－ＣｏＶ－２を標的とするシングルドメイン抗体
US20180016322A1 (en) Antibodies specific to glycoprotein (gp) of ebolavirus and uses for the treatment and diagnosis of ebola virus infection
CA3090327A1 (en) Binding molecule having neutralizing activity against middle east respiratory syndrome-coronavirus
WO2021254403A1 (en) Methods and compositions related to neutralizing antibodies against human coronavirus
WO2022037528A1 (zh) 结合bcma的单可变结构域及抗原结合分子
WO2021065846A1 (ja) 抗ＨＩＶ－１ Ｖ３抗体１Ｃ１０（０．５γ）に結合する抗体及びその抗原結合断片並びにその応用
TW202219063A (zh) Sars-cov(-2)棘糖蛋白結合域及包含其之多肽及其用途
KR20220034733A (ko) 타우의 입체형태-특이적 에피토프, 이에 대한 항체 및 이와 관련된 방법
US10435463B2 (en) Hepatitis C virus specific antibody
WO2022191162A1 (ja) 抗ａｃｅ２モノクローナル抗体
WO2023148641A1 (en) Monoclonal antibodies specific to receptor binding domain of sars-cov2 and uses thereof
KR20160093502A (ko) 항 eprs 모노클로날 항체 및 이의 용도
NZ788350A (en) Binding molecule specific for LIF and use thereof
KR20240008997A (ko) 사스-코로나 바이러스 2 중화 항체

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21882090

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21882090

Country of ref document: EP

Kind code of ref document: A1