WO2022161491A1 - 一种针对SARS-CoV-2病毒的中和性抗体 - Google Patents

一种针对SARS-CoV-2病毒的中和性抗体 Download PDF

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WO2022161491A1
WO2022161491A1 PCT/CN2022/074998 CN2022074998W WO2022161491A1 WO 2022161491 A1 WO2022161491 A1 WO 2022161491A1 CN 2022074998 W CN2022074998 W CN 2022074998W WO 2022161491 A1 WO2022161491 A1 WO 2022161491A1
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antibody
variable region
heavy chain
chain variable
light chain
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PCT/CN2022/074998
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English (en)
French (fr)
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唐宏
江陆斌
张晓明
孟璐
曹龙
李腾
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中国科学院上海巴斯德研究所
中科南京生命健康高等研究院
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Publication of WO2022161491A1 publication Critical patent/WO2022161491A1/zh

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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
    • A61K47/6839—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 material from viruses
    • A61K47/6841—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 material from viruses the antibody targeting a RNA virus
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—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
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    • C07—ORGANIC CHEMISTRY
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    • 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
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the invention belongs to the field of biotechnology or medicine, and in particular relates to the preparation and application of an anti-SARS-CoV-2 virus neutralizing monoclonal antibody.
  • SARS-CoV-2 virus One of the key steps for the SARS-CoV-2 virus to invade host cells is that the RBD region of its structural protein Spike binds to the receptor angiotensin-converting enzyme 2 (ACE2) on the surface of the host cell membrane to form an ACE2-RBD complex.
  • ACE2 angiotensin-converting enzyme 2
  • Zhou Qiang's team had analyzed the protein structure of Spike protein by cryo-electron microscopy.
  • the Wrapp team also pointed out that the SARS-CoV-2 S protein binds the host cell ACE2 receptor with an affinity more than 10 times higher than that of SARS-CoV.
  • designing a monoclonal antibody targeting the S protein can block the fusion of the virus and the host by competitively inhibiting the binding of the S protein to the receptor, thereby inhibiting the virus from infecting host cells, thereby preventing new crown infection or treating infected patients.
  • Antibody drugs will likely become a new treatment option for the new crown.
  • Convalescent plasma therapy as one of the currently approved treatments for COVID-19, although it can be used to fight viral infection to a certain extent, the plasma sources are different, the components are relatively complex and there are many adverse reactions, and the safety and effectiveness cannot be guaranteed.
  • the successful application of convalescent plasma still implies that neutralizing monoclonal antibodies can be used to treat new coronavirus infection.
  • the purpose of the present invention is to provide an effective method for diagnosing and treating SARS-CoV-2 coronavirus for diagnosing and treating pneumonia caused by novel coronavirus infection.
  • a heavy chain variable region of an antibody having a complementarity determining region CDR selected from the group consisting of:
  • VH-CDR1 shown in SEQ ID NO:3, VH-CDR2 shown in SEQ ID NO:4, and VH-CDR3 shown in SEQ ID NO:5;
  • any one of the amino acid sequences in the above-mentioned amino acid sequence also includes a derivative that optionally undergoes addition, deletion, modification and/or substitution of at least one amino acid and can retain the binding affinity with the RBD domain of the SARS-CoV-2 S protein sequence.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:2.
  • a heavy chain of an antibody wherein the heavy chain has the variable region of the heavy chain according to the first aspect of the present invention.
  • the heavy chain further includes a heavy chain constant region.
  • the heavy chain constant region is of human or murine origin.
  • the heavy chain constant region is a human antibody heavy chain IgG1 constant region.
  • a light chain variable region of an antibody having a complementarity determining region CDR selected from the group consisting of:
  • VL-CDR1 shown in SEQ ID NO:8, VL-CDR2 shown in SEQ ID NO:9, and VL-CDR3 shown in SEQ ID NO:10;
  • any one of the amino acid sequences in the above-mentioned amino acid sequence also includes a derivative that optionally undergoes addition, deletion, modification and/or substitution of at least one amino acid and can retain the binding affinity with the RBD domain of the SARS-CoV-2 S protein sequence.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO:7.
  • a light chain of an antibody the light chain has the light chain variable region according to the third aspect of the present invention.
  • the light chain further includes a light chain constant region.
  • the light chain constant region is of human or murine origin.
  • the light chain constant region is a human antibody light chain lambda constant region.
  • amino acid sequence of the light chain is shown in SEQ ID NO:7.
  • an antibody having a heavy chain variable region as described in the first aspect of the present invention, and/or a light chain variable region as described in the third aspect of the present invention ;
  • the antibody has a heavy chain as described in the second aspect of the invention, and/or a light chain as described in the fourth aspect of the invention;
  • any one of the amino acid sequences in the above-mentioned amino acid sequence also includes a derivative that optionally undergoes addition, deletion, modification and/or substitution of at least one amino acid and can retain the binding affinity with the RBD domain of the SARS-CoV-2 S protein sequence.
  • the number of added, deleted, modified and/or substituted amino acids is 1-5 (eg 1-3, preferably 1-2, more preferably 1).
  • the derivative sequence that has been added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity of the RBD domain of the SARS-CoV-2 S protein is homology or sequence An amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
  • the antibody further includes a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is of human origin, and/or the light chain constant region is of human origin.
  • the heavy chain constant region is a human antibody heavy chain IgG1 constant region
  • the light chain constant region is a human antibody light chain lambda constant region
  • variable region of the heavy chain of the antibody further includes a human framework region
  • variable region of the light chain of the antibody further includes a human framework region
  • the heavy chain variable region of the antibody further includes a murine framework region, and/or the light chain variable region of the antibody further includes a murine framework region.
  • the antibody is selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or a combination thereof.
  • the antibody is a partially or fully humanized or fully human monoclonal antibody.
  • the antibody is a diabody or a single-chain antibody.
  • the antibody is a full-length antibody protein, or an antigen-binding fragment.
  • the antibody is a bispecific antibody or a multispecific antibody.
  • the antibody is in the form of a drug conjugate.
  • the antibody has one or more properties selected from the group consisting of:
  • the antibody has the variable region of the heavy chain according to the first aspect of the present invention and the variable region of the light chain according to the third aspect of the present invention;
  • variable region of the heavy chain and the variable region of the light chain comprise CDRs selected from the group consisting of:
  • VH-CDR1 as shown in SEQ ID NO:3, VH-CDR2 as shown in SEQ ID NO:4, VH-CDR3 as shown in SEQ ID NO:5, as shown in SEQ ID NO:8 VL-CDR1, VL-CDR2 shown in SEQ ID NO:9, and VL-CDR3 shown in SEQ ID NO:10.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:2
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:7.
  • the amino acid sequence of the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, and 94% of the amino acid sequence shown in SEQ ID NO: 2. %, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • the amino acid sequence of the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94% with the amino acid sequence shown in SEQ ID NO:7 %, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • recombinant protein comprises:
  • variable region of the heavy chain according to the first aspect of the present invention the heavy chain according to the second aspect of the present invention, the variable region of the light chain according to the third aspect of the present invention, the variable region of the light chain according to the fourth aspect of the present invention
  • the tag sequence includes a 6His tag.
  • the recombinant protein includes fusion protein.
  • the recombinant protein is a monomer, a dimer, or a multimer.
  • a polynucleotide encoding a polypeptide selected from the group consisting of the heavy chain variable region according to the first aspect of the present invention, the heavy chain variable region according to the second aspect of the present invention.
  • the recombinant protein of the aspect is provided.
  • polynucleotide encoding the variable region of the heavy chain is shown in SEQ ID NO: 1; and/or the polynucleotide encoding the variable region of the light chain is shown in SEQ ID NO: 6 shown.
  • a vector comprising the polynucleotide according to the seventh aspect of the present invention.
  • the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • a genetically engineered host cell contains the vector according to the eighth aspect of the present invention or the polynucleotide according to the seventh aspect of the present invention is integrated into the genome .
  • an antibody conjugate is provided, and the antibody conjugate contains:
  • a conjugation moiety conjugated to the antibody moiety selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, Magnetic nanoparticles, viral coat proteins or VLPs, or combinations thereof.
  • the antibody moiety and the coupling moiety are coupled through chemical bonds or linkers.
  • the radionuclide includes:
  • a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and/or
  • a therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133Yb-169, Yb-177, or a combination thereof.
  • the coupling moiety is a drug or a toxin.
  • the drug is a cytotoxic drug.
  • the cytotoxic drugs are selected from the group consisting of anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy A sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
  • cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatins, camptothecins camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (eg DM1 and DM4), taxanes ( taxanes), benzodiazepines, or benzodiazepine-containing drugs (eg, pyrrolo[1,4]benzodiazepines (PBDs), indoline benzodiazepines) indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.
  • PPDs pyrrolo[1,4]benzodiazepines
  • indoline benzodiazepines indolinobenzodiazepines and oxazolidinobenzodiazepines
  • vinca alkaloids or combinations thereof.
  • the toxin is selected from the following group:
  • Auristatins eg, auristatin E, auristatin F, MMAE, and MMAF
  • chlortetracycline maytansoid, gamatoxin, gamatoxin A-chain, combretastatin, docarmicin, Lastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, autumn Narcissin, Dihydroxyanthraxdione, Actinomycin, Diphtheria Toxin, Pseudomonas Exotoxin (PE) A, PE40, Acacia toxin, Acacia A chain, Capsule root toxin A chain, ⁇ - Sarcinus, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin,
  • the coupling moiety is a detectable label.
  • the conjugate is selected from: fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable Enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles, prodrugs Activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin).
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the immunoconjugate contains: multivalent (eg bivalent) (a).
  • the multivalent means that the amino acid sequence of the immunoconjugate contains multiple repeats of (a).
  • an immune cell expressing or exposing the antibody according to the fifth aspect of the present invention outside the cell membrane.
  • the immune cells include NK cells and T cells.
  • the immune cells are derived from human or non-human mammals (eg, mice).
  • a pharmaceutical composition contains:
  • an active ingredient selected from the group consisting of a heavy chain variable region as described in the first aspect of the present invention, a heavy chain as described in the second aspect of the present invention, and a heavy chain as described in the third aspect of the present invention
  • the light chain variable region of the present invention, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, or the tenth aspect of the present invention The antibody conjugate described above, the immune cell according to the eleventh aspect of the present invention, or a combination thereof; and
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition comprises 0.01-99.99% of the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, and the tenth aspect of the present invention.
  • an active ingredient selected from the group consisting of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain variable region as described in the second aspect of the present invention
  • the diagnostic reagent is a test piece or a test plate.
  • the diagnostic reagent or kit is used for: detecting SARS-CoV-2 S protein or a fragment thereof in a sample.
  • the antibody is in the form of a drug conjugate (ADC).
  • ADC drug conjugate
  • a method for detecting SARS-CoV-2 virus or SARS-CoV-2 S protein or a fragment thereof in a sample in vitro comprising the steps of:
  • the detection includes diagnostic or non-diagnostic.
  • a kit in the fifteenth aspect of the present invention, includes:
  • the second container contains a secondary antibody against the antibody of the fifth aspect of the present invention
  • the kit contains a detection plate, the detection plate includes: a substrate (support plate) and a test strip, the test strip contains the antibody according to the fifth aspect of the present invention, and the sixth aspect of the present invention.
  • the recombinant polypeptide is the antibody according to the fifth aspect of the present invention or the recombinant protein according to the sixth aspect of the present invention, the methods include:
  • a pharmaceutical combination comprising:
  • a first active ingredient comprises the antibody according to the fifth aspect of the present invention, or the recombinant protein according to the sixth aspect of the present invention, or the antibody according to the tenth aspect of the present invention
  • a second active ingredient comprising other drugs for treating SAR-CoV-2 virus infection.
  • the other drugs for treating SAR-CoV-2 virus infection include: other protective monoclonal antibodies, small molecule drugs such as Remdesivir, or other proprietary Chinese medicines.
  • a method for diagnosing SAR-CoV-2 virus infection comprising the steps of:
  • the sample is a blood sample or a throat swab sample, or a sample from other tissues and organs.
  • a method for treating SARS-CoV-2 virus-infected diseases comprising the steps of: administering an effective amount of the antibody according to the fifth aspect of the present invention, or The recombinant protein according to the sixth aspect of the present invention, or the antibody conjugate according to the tenth aspect of the present invention, or the immune cell according to the eleventh aspect of the present invention, or the twelfth aspect of the present invention the pharmaceutical composition, or a combination thereof.
  • Figure 1 shows the SDS-PAGE electropherogram of neutralizing antibody expression.
  • lane 1 is protein marker
  • lane 2 is control antibody IgG
  • lane 3 is monoclonal antibody XG-Ab008.
  • Figure 2 shows the results of the antibody neutralization effect test and the antigen-antibody affinity test.
  • Figures 2A and 2B show the neutralization test results of candidate antibodies Ab001-Ab010 against (Figure 2A) SARS-CoV-2 and (Figure 2B) SARS-CoV lentivirus infection system.
  • Figure 2C shows the neutralization effect of XG-Ab008 in the SARS-CoV-2 pseudovirus system at approximately 1 nM.
  • Figure 2D shows the neutralization effect of XG-Ab008 in the SARS-Cov-2 eukaryotic system, about 4.5 nM.
  • Figure 3 shows CD20-/CD38+/IgD-/IgM-/RBD+ B cells sorted by flow cytometry.
  • Figure 4 shows the light and heavy chains amplified from B cell reverse-transcribed cDNA as templates: upper heavy chain, middle light chain ⁇ , lower light chain ⁇ .
  • the successful amplification of double-stranded templates is: 3, 4, 6, 7 and 8.
  • Figure 5 shows the results of XG-Ab008 neutralizing antibody affinity detection, KD is about 1.39nM.
  • the inventors of the present invention developed for the first time an antibody specific for the 2019 novel coronavirus for the first time. Specifically, the inventors screened out a monoclonal antibody extracted from the blood samples of recovered patients with SARS-Cov-2, which has high binding force and neutralizing effect on the Spike protein on the surface of the SARS-Cov-2 virus. The experimental results show that the neutralizing antibody of the present invention can effectively inhibit the infection of pseudovirus at the cellular level, and it can play a neutralizing effect by inhibiting the binding of ACE2 receptor and RBD protein.
  • the inventors first isolated PBMCs from the blood samples of patients who had recovered from COVID-19, and obtained CD19+ B cells through magnetic bead enrichment. After incubation with fluorescein-labeled SARS-CoV-2 target antigen, single IgG+ B cells were flow-sorted into 96-well plate lysate. The heavy chain and light chain of each single cell were amplified by PCR, and the monoclonal antibody expression plasmid was recombinantly inserted; after the recombinant plasmid was transfected into CHO cells, the cell culture supernatant was collected and purified with protein G to obtain monoclonal antibody Antibody. Through the Vero-E6 cells expressing ACE2 in vitro and the pseudovirus system expressing SARS-Cov-2, the monoclonal antibody with high neutralizing effect and high binding ability to the virus S protein in the present invention was screened out.
  • antibody or "immunoglobulin” is a heterotetraglycan protein of about 150,000 Daltons having the same structural characteristics, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions.
  • VH variable region
  • Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is opposite the first constant domain of the heavy chain, and the variable domain of the light chain is opposite the variable domain of the heavy chain .
  • VL variable domain
  • Particular amino acid residues form the interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence that contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable regions are called the framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • the variable regions of native heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs that form linking loops, and in some cases can form part of a ⁇ -sheet structure.
  • the CDRs in each chain are tightly packed together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • immunoglobulins can be grouped into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be classified into different classes according to the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.
  • the heavy chain constant regions corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those in the art.
  • variable regions which are separated into four framework regions (FRs), four
  • FRs framework regions
  • the amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a circular structure, and the ⁇ -sheets formed by the FRs in between are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • Which amino acids make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • VH-CDR1 and “CDR-H1” can be used interchangeably, and both refer to the CDR1 of the heavy chain variable region;
  • VH-CDR2 and “CDR-H2” can be used interchangeably, and both refer to the heavy chain CDR2 of the variable region;
  • VH-CDR3 and “CDR-H3” are used interchangeably, and both refer to the CDR3 of the heavy chain variable region.
  • VL-CDR1" and CDR-L1 are used interchangeably, and both refer to the CDR1 of the light chain variable region;
  • CDR2; “VL-CDR3” and “CDR-L3” are used interchangeably, and both refer to the CDR3 of the light chain variable region.
  • the present invention includes not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of said antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, which can be obtained by standard recombinant DNA techniques, are useful antibodies.
  • a chimeric antibody is a molecule in which different parts are derived from different animal species, such as a chimeric antibody having variable regions from a murine monoclonal antibody, and constant regions from a human immunoglobulin (see, e.g., U.S. Patent No. 4,816,567 and US Patent 4,816,397, which is hereby incorporated by reference in its entirety).
  • a humanized antibody refers to an antibody molecule derived from a non-human species having one or more complementarity determining regions (CDRs) derived from the non-human species and framework regions derived from a human immunoglobulin molecule (see U.S. Patent No. 5,585,089, is hereby incorporated by reference in its entirety).
  • CDRs complementarity determining regions
  • These chimeric and humanized monoclonal antibodies can be prepared using recombinant DNA techniques well known in the art.
  • antibodies may be monospecific, bispecific, trispecific, or more multispecific.
  • the antibody of the present invention also includes its conservative variants, which means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, most preferably Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide.
  • conservatively variant polypeptides are best produced by amino acid substitutions according to Table A.
  • the antibody is an antibody that specifically binds to the RBD domain of the SARS-CoV-2 S protein.
  • the present invention provides a high-specificity and high-affinity antibody against the RBD domain of the SARS-CoV-2 S protein, comprising a heavy chain and a light chain, the heavy chain containing the heavy chain variable region (VH) amino acid sequence, The light chain contains the light chain variable region (VL) amino acid sequence.
  • the heavy chain variable region (VH) has a complementarity determining region CDR selected from the group consisting of:
  • VH-CDR1 shown in SEQ ID NO:3,
  • VH-CDR2 shown in SEQ ID NO:4, and
  • VH-CDR3 shown in SEQ ID NO:5;
  • the light chain variable region (VL) has a complementarity determining region CDR selected from the group consisting of:
  • VL-CDR1 shown in SEQ ID NO:8,
  • VL-CDR2 shown in SEQ ID NO:9
  • VL-CDR3 shown in SEQ ID NO: 10;
  • any one of the amino acid sequences in the above-mentioned amino acid sequence also includes a derivative that optionally undergoes addition, deletion, modification and/or substitution of at least one amino acid and can retain the binding affinity with the RBD domain of the SARS-CoV-2 S protein sequence.
  • sequence formed by adding, deleting, modifying and/or replacing at least one amino acid sequence is preferably homology or sequence identity of at least 80%, preferably at least 85%, more preferably Preferably at least 90%, and optimally at least 95% of the amino acid sequence.
  • the preferred method of determining identity is to obtain the greatest match between the sequences tested.
  • Methods for determining identity are codified in publicly available computer programs.
  • Preferred computer program methods for determining the identity between two sequences include, but are not limited to: the GCG package (Devereux, J. et al, 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. et al, 1990).
  • the BLASTX program is publicly available from NCBI and other sources (BLAST Handbook, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990).
  • the well-known Smith Waterman algorithm can also be used to determine identity.
  • the antibody described herein is an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody fragment (scFv), a single domain antibody (single domain antibody). , sdAb) and one or more of single-domain antibodies (Signle-domain antibodies), as well as monoclonal or polyclonal antibodies prepared from the above-mentioned antibodies.
  • the monoclonal antibody can be developed by various methods and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.
  • the full-length antibody protein is a conventional full-length antibody protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region.
  • the heavy chain variable region and light chain variable region of the protein together with the human heavy chain constant region and the human light chain constant region constitute a fully human antibody full-length protein.
  • the antibody full-length protein is IgG1, IgG2, IgG3 or IgG4; more preferably IgG1.
  • the antibodies of the present invention may be double-chain or single-chain antibodies, and may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully human Antibody.
  • the antibody derivatives of the present invention can be single-chain antibodies, and/or antibody fragments, such as: Fab, Fab', (Fab')2 or other known antibody derivatives in the field, etc., as well as IgA, IgD, IgE , IgG and IgM antibodies or any one or more of other subtypes of antibodies.
  • the single-chain antibody is a conventional single-chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.
  • the animal is preferably a mammal, such as a mouse.
  • the antibodies of the present invention may be chimeric antibodies, humanized antibodies, CDR-grafted and/or modified antibodies targeting the SARS-CoV-2 Spike protein.
  • the number of added, deleted, modified and/or substituted amino acids is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33% , more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.
  • the number of amino acids added, deleted, modified and/or substituted may be 1-7, more preferably 1-5, more preferably 1-3, more preferably for 1-2.
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:2.
  • the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:7.
  • the antibody targeting the RBD domain of the SARS-CoV-2 S protein is XG-Ab008.
  • each antibody of the present invention specifically includes each of the following VL and VH sequences, as well as Fc, CL and CH1 sequences.
  • the present invention also provides a polynucleotide encoding the above-mentioned antibody or a recombinant protein comprising the same, or a heavy chain variable region or a light chain variable region thereof.
  • nucleotide sequence of the nucleic acid encoding the variable region of the heavy chain is shown in SEQ ID NO: 1 in the Sequence Listing; and/or, the nucleotide sequence of the nucleic acid encoding the variable region of the light chain is as follows: The sequence listing is shown in SEQ ID NO: 6.
  • nucleotide sequence of the nucleic acid encoding the variable region of the heavy chain is shown in SEQ ID NO: 1 in the Sequence Listing; and the nucleotide sequence of the nucleic acid encoding the variable region of the light chain is shown in the SEQ ID NO: 1 in the Sequence Listing ID NO: 6.
  • the preparation method of the nucleic acid is a conventional preparation method in the field, preferably, comprising the following steps: obtaining a nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above-mentioned protein by artificial full sequence synthesis .
  • homologs of the polynucleotides of the present invention can be prepared by substituting, deleting or adding one or more bases of the gene encoding the protein sequence within the range that maintains the activity of the antibody.
  • the present invention also provides a recombinant expression vector comprising the nucleic acid.
  • the recombinant expression vector can be obtained by conventional methods in the art, that is, constructed by linking the nucleic acid molecule described in the present invention to various expression vectors.
  • the expression vector is any conventional vector in the art, as long as it can accommodate the aforementioned nucleic acid molecule.
  • the vectors preferably include: various plasmids, cosmids, phage or viral vectors and the like.
  • the present invention also provides a recombinant expression transformant comprising the above recombinant expression vector.
  • the preparation method of the recombinant expression transformant is a conventional preparation method in the art, preferably by transforming the above recombinant expression vector into a host cell.
  • the host cells are various conventional host cells in the field, as long as the above-mentioned recombinant expression vector can stably replicate by itself, and the nucleic acid carried by it can be effectively expressed.
  • the host cells are E.coli TG1 or E.coli BL21 cells (expressing single chain antibody or Fab antibody), or HEK293 or CHO cells (expressing full-length IgG antibody).
  • the preferred recombinant expression transformants of the present invention can be obtained by transforming the aforementioned recombinant expression plasmids into host cells.
  • the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a thermal shock method or an electrotransformation method.
  • sequences of the DNA molecules of the antibodies or fragments thereof of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
  • the coding sequences for the light and heavy chains can be fused together to form single chain antibodies.
  • recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
  • synthetic methods can also be used to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • the DNA sequences encoding the antibodies of the present invention can be obtained completely by chemical synthesis.
  • This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • Preferred animal cells include, but are not limited to: CHO-S, HEK-293 cells.
  • the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography and other techniques in the art
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography and other techniques in the art
  • the antibody of the present invention can be obtained by conventional separation and purification methods well known to those skilled in the art.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • the antibody of the present invention can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • ADCs Antibody-Drug Conjugates
  • the present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.
  • ADC antibody-drug conjugate
  • the antibody-drug conjugate includes the antibody, and an effector molecule, and the antibody is conjugated to the effector molecule, preferably chemically conjugated.
  • the effector molecule is preferably a drug with therapeutic activity.
  • the effector molecule can be one or more of a toxin, a chemotherapeutic drug, a small molecule drug, or a radionuclide.
  • the antibody of the present invention and the effector molecule can be coupled through a coupling agent.
  • the coupling agent may be any one or more of non-selective coupling agents, coupling agents utilizing carboxyl groups, peptide chains, and coupling agents utilizing disulfide bonds.
  • the non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde and the like.
  • the coupling agent utilizing the carboxyl group may be any one or more of a cis-aconitic anhydride type coupling agent (such as cis-aconitic anhydride) and an acyl hydrazone type coupling agent (the coupling site is an acyl hydrazone).
  • antibodies are used to link with various functional groups, including imaging reagents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes) , stabilizers (eg, ethylene glycol polymers) and therapeutic agents.
  • imaging reagents such as chromophores and fluorophores
  • diagnostic reagents such as MRI contrast agents and radioisotopes
  • stabilizers eg, ethylene glycol polymers
  • therapeutic agents eg, ethylene glycol polymers
  • Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates.
  • Functional agents eg, drugs, detection reagents, stabilizers
  • the functional agent can be attached to the antibody either directly or indirectly through a linker.
  • Antibodies can be conjugated to drugs to form antibody drug conjugates (ADCs).
  • ADC antibody drug conjugates
  • the ADC contains a linker between the drug and the antibody.
  • Linkers can be degradable or non-degradable linkers. Degradable linkers are typically susceptible to degradation in the intracellular environment, eg, at the target site, where the linker is degraded, thereby releasing the drug from the antibody.
  • Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (eg, lysosomal or endosomal proteases), or sugar linkers that, for example, can be degraded by glucuronides Enzymatically degraded glucuronide-containing linkers.
  • Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine.
  • degradable linkers include, for example, pH sensitive linkers (eg, linkers that hydrolyze at pH less than 5.5, eg, hydrazone linkers) and linkers that degrade under reducing conditions (eg, disulfide linkers).
  • Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.
  • the linker Before being attached to the antibody, the linker has a reactive reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive reactive group.
  • Sulfhydryl-specific reactive groups are preferred and include, for example, maleimides, haloamides (eg, iodo, bromo, or chloro); haloesters (eg, iodo, bromo, or chloro) ); halogenated methyl ketones (eg iodo, bromo or chloro), benzyl halides (eg iodo, bromo or chloro); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6- bis-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate.
  • Linkers can include, for example, maleimide
  • the drug can be any cytotoxic, cytostatic or immunosuppressive drug.
  • the linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker.
  • the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker.
  • the drug is directly attached to the linker, the drug has a reactive reactive group prior to attachment to the antibody.
  • drug-linkers can be used to form ADCs in one simple step.
  • bifunctional linker compounds can be used to form ADCs in a two- or multi-step process. For example, a cysteine residue reacts with the reactive moiety of the linker in the first step, and in a subsequent step, the functional group on the linker reacts with the drug to form the ADC.
  • functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety.
  • azide-based moieties can be used to specifically react with reactive alkynyl groups on drug moieties.
  • the drug is covalently bound to the linker through a 1,3-dipolar cycloaddition between the azide and the alkynyl group.
  • Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines).
  • the present invention also provides a method for preparing an ADC, which may further include: combining the antibody with the drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • the methods of the invention comprise binding the antibody to a bifunctional linker compound under conditions sufficient to form the antibody-linker conjugate. In these embodiments, the methods of the invention further comprise: conjugating the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.
  • the antibody drug conjugate ADC is represented by the following molecular formula:
  • Ab is an antibody
  • D is a drug
  • the present invention also provides the use of the antibodies, antibody conjugate ADCs, recombinant proteins, and/or immune cells of the present invention, for example, for the preparation of diagnostic preparations or the preparation of medicaments.
  • the medicament is a medicament for preventing and/or treating SARS-CoV-2 virus infection.
  • Antibodies or ADCs thereof of the invention can be used in detection applications, eg, in detection of samples, to provide diagnostic information.
  • the sample (sample) used includes cells, tissue samples and biopsy specimens.
  • the sample is a blood sample or a throat swab sample from the test subject.
  • Biopsy shall include all kinds of biopsies known to those skilled in the art. Biopsies used in the present invention may thus include, for example, excised samples of tumors, tissue samples prepared by endoscopic methods or puncture or needle biopsy of organs.
  • Samples used in the present invention include fixed or preserved cell or tissue samples.
  • the present invention also provides a kit containing the antibody (or its fragment) of the present invention.
  • the kit further includes a container, instructions for use, buffers, and the like.
  • the antibody of the present invention can be immobilized on a detection plate.
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding immune cells, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the route of administration of the pharmaceutical composition of the present invention is preferably injection administration or oral administration.
  • the injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.
  • the pharmaceutical composition is in various conventional dosage forms in the field, preferably in the form of solid, semi-solid or liquid, can be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, capsule, granule , injection or infusion, etc.
  • the antibody of the present invention can also be expressed in a cell by a nucleotide sequence for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing and/or treating SARS-CoV-2 virus-infected diseases.
  • the pharmaceutical composition of the present invention can be directly used to bind SARS-CoV-2 S protein molecule or its fragment, thus can be used to prevent and treat diseases caused by viral infection.
  • the pharmaceutical composition of the present invention contains the above-mentioned monoclonal antibody (or its conjugate) of the present invention in a safe and effective amount (eg, 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) and a pharmaceutical an acceptable carrier or excipient.
  • a pharmaceutical an acceptable carrier or excipient include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, about 1 microgram/kg body weight to about 5 mg/kg body weight per day.
  • the polypeptides of the present invention may also be used with other therapeutic agents.
  • the pharmaceutical composition of the present invention further comprises one or more pharmaceutically acceptable carriers.
  • the pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the art, preferably including pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above protein and 0.01-99.99% of a pharmaceutical carrier, and the percentage is the mass percentage of the pharmaceutical composition.
  • the administration amount of the pharmaceutical composition is an effective amount
  • the effective amount is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition.
  • the effective amount can be determined on an individual basis and will be based in part on consideration of the symptoms to be treated and the results sought. An effective amount can be determined by one skilled in the art using the above-mentioned factors on an individual basis and the like and using no more than routine experimentation.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 20 mg/kg body weight.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the present invention provides the application of the above-mentioned pharmaceutical composition in the preparation of medicines for preventing and/or treating SARS-CoV-2 virus-infected diseases.
  • the SARS-CoV-2 virus-infected disease is pneumonia.
  • novel coronavirus As used herein, the terms "novel coronavirus”, “2019-nCov” or “SARS-CoV-2” are used interchangeably, which is the seventh coronavirus known to infect humans and cause COVID-19 (COVID-19), is one of the serious infectious diseases threatening human health worldwide.
  • Coronavirus belongs to the family Coronaviridae of the order Nidovirales. It is an enveloped positive-strand RNA virus.
  • HCoV-229E and HCoV-NL63 belong to alpha coronaviruses, and HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and SARS-CoV-2 are all beta coronaviruses Virus.
  • SARS-CoV-2 is also known as 2019-nCov.
  • SARS-CoV highly pathogenic coronaviruses
  • MERS-CoV Middle East Respiratory Syndrome
  • SARS-CoV-2 The new coronavirus (SARS-CoV-2) is about 80% similar to SARS-CoV and 40% similar to MERS-CoV, and also belongs to the beta coronavirus.
  • the genome of this virus is a single-stranded positive-stranded RNA, which is one of the largest RNA viruses in the genome, encoding replicase, spike protein, envelope protein, envelope protein and nucleocapsid protein.
  • the genome is translated into two peptide chains of several thousand amino acids, the precursor polyprotein (Polyprotein), and then the precursor protein is cleaved by proteases to generate non-structural proteins (such as RNA polymerase and unwinding). enzymes) and structural proteins (such as spike proteins) and accessory proteins.
  • the antibody preparation process of the present invention is simple.
  • the present invention uses the CHO expression system, is simple to operate, can express a large amount of antibodies sustainably, and meets the requirements of downstream experiments and detection.
  • the monoclonal antibody provided by the present invention is a fully human antibody and does not have the antigenicity of other heterologous proteins.
  • the selected single clones are cultured in small volumes and the expression vector is purified
  • the monoclonal neutralizing antibody XG Ab-008 can be obtained by collecting the CHO cell expression supernatant solution and purifying it.
  • the SDS-PAGE results show ( Figure 1) that the purity of the purified candidate antibody can meet the requirements of follow-up test requirements.
  • Example 1 Obtaining mononuclear cells (PBMCs) from the blood of recovered patients with COVID-19
  • Collect 12-15ml of heparin anticoagulated blood from patients who have recovered from the new crown centrifuge at 500g for 10 minutes, and absorb the upper plasma. After that, add normal saline to dilute the lower blood cells by 1:1 volume, and mix well. The mixture was slowly stacked into a centrifuge tube containing an equal volume of Lymphoprep solution and mononuclear cells (PBMC) were obtained by density gradient centrifugation.
  • PBMC mononuclear cells
  • Physiological saline 0.9% sodium chloride, ultrapure water, filtered with 0.22um membrane before use.
  • Lymphoprep solution LymphoprepTM, 1 ⁇ D-PBS, 0.9% sodium chloride, 100 ⁇ penicillin-streptomycin double antibody, 0.5M EDTA, adjust pH to 8.0.
  • antibody dilution mixture Prepare "antibody dilution mixture” and dilute 0.6ul each of biotin-labeled anti-CD3, anti-CD11b, anti-CD14, anti-CD16, anti-CD56, and anti-CD235a antibodies in 150ul of antibody dilution.
  • incubate the PBMC suspension prepared in Example 1 with the antibody dilution mixture wash off the excess antibody
  • incubate the treated PBMC suspension with the anti-biotin magnetic bead suspension and collect the enriched PBMC suspension with LS colum collection of B cells.
  • MACS solution 10ml fetal bovine serum (FBS), 5mM EDTA, 500ml 1 ⁇ DPBS, adjust pH to 8.0, filter with 0.22um membrane and store at 4°C.
  • Example 2 Using the enriched B cells in Example 2, add 100ul Mouse FC dilution, and incubate at room temperature for 15 minutes in the dark. Wash off excess antibody with MACS, then add 100ul flow-through fluorescent antibody diluent, and incubate at room temperature for 15 minutes in the dark. After washing away excess antibody with MACS, flow cytometry Melody was used to sort single B cells that specifically bound S-RBD (Figure 3). The sorted B cells were collected in a 96-well PCR plate containing 4ul of cell lysate, one cell per well, sealed with membrane immediately after sorting and snap-frozen with dry ice.
  • Lysis solution 300ul RNasin, 40U/ul RNase inhibitor, 200ul 1*DPBS, 400ul DL-DTT (100mM), 3100ul nuclease-free water.
  • Example 3 The single B cells sorted in Example 3 were reversed with SuperScript III RT reverse transcriptase (Invitrogen, 18080-044) to obtain cDNA.
  • the paired light and heavy chain sequences obtained in Example 4 were inserted into the light chain expression plasmid and the heavy chain expression plasmid, respectively, according to the method described in the ExpiCHO expression system commercial manual (ExpiCHO TM Expression System Kit, A29133 ), transfected and expressed CHO cells. 500g, 10min centrifugation to collect the cell suspension, and follow the commercial instructions of Protein G Sepharose (Protein G Sepharose). 4 Fast Flow, 17-0618-01) method, purification of IgG antibodies
  • SARS-Cov-2 pseudovirus construct expression vector, including: mouse leukemia virus (MLV) gag/pol, retrovirus EGFP, full-length sequence of SARS-CoV-2 S protein (GenBank: QHD43419.1) or SARS - CoV S protein full-length sequence (AY569693).
  • the present invention provides a strategy based on single B cell expansion and expression of monoclonal antibodies, and a screened high-efficiency neutralizing monoclonal antibody targeting S protein.

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Abstract

本发明提供了针对SARS-CoV-2病毒具有中和效果的单克隆抗体。同时，本发明提供了对所述抗体的中和能力与结合能力的检测方法。本发明抗体的制备过程简单、产物纯度高且为全人IgG抗体，并且本发明的抗体与SARS-CoV-2病毒具有较强的中和效果。

Description

一种针对SARS-CoV-2病毒的中和性抗体 技术领域
本发明属于生物技术或医药领域，具体涉及一种抗SARS-CoV-2病毒中和性单克隆抗体的制备和应用。
背景技术
在急性传染病中，绝大部分都是病毒性传染病，病毒性传染病的发病率高，死亡率也很高。由于检测和诊断手段有限，导致新病毒引发的新疫情爆发往往具有突发性、随机性和不可预测性等特点，一旦爆发，如无有效的防治手段，极易造成大规模流行，严重威胁人民健康生命安全。
SARS-CoV-2病毒入侵宿主细胞的关键步骤之一是其结构蛋白Spike的RBD区域与宿主细胞膜表面受体血管紧张素转化酶2(ACE2)结合实现，形成ACE2-RBD复合物。早在2020年2月，周强团队已通过冷冻电镜的方法解析了Spike蛋白的蛋白质结构。Wrapp团队更指出SARS-CoV-2 S蛋白结合宿主细胞ACE2受体的亲和力比SARS-CoV高10倍以上。基于这些研究，设计靶向S蛋白的单抗，通过竞争性抑制S蛋白与受体结合，可以阻断病毒与宿主融合，进而抑制病毒感染宿主细胞，由此预防新冠感染或治疗已感染患者。抗体类药物将很有可能成为一种新的新冠治疗方案。
在全球范围内，抗体药物研发早已成为研究热点，其中绝大部分以S蛋白为药物靶点。目前，至少有26个国家超过200家机构参与抗体药物研发。截至2020年11月6日，在COVID-19 Antibody Therapeutics Tracker上可搜索到全球范围内针对S蛋白设计抗体、多肽或重组蛋白研发方案共105种(大部分为单抗)，其中临床一期10项(JS016、TY027、CT-P59、BRII-196、BRII-198、SCTA01、MW33、STI-1499/COVI-SHIELD、SAB-185、IgY-110/anti-SARS-CoV-2IgY)，二期2项(DXP-593、APN01)，三期4项(REGN-COV2、LY3819253、VIR-7831、AZD7442)。
然而，目前暂无针对S蛋白的中和抗体被批准上市，而全球抗体疗法研究中也只有两款：俄罗斯联邦卫生部今年6月批准了Levilimab(BCD-089，商品名Ilsira)和印度批在7月批准的Itolizumab(EQ001，H-T1，T1-h)，都用于控制细胞因子风暴以减少新冠重症患者死亡率
目前，国内已批准的抗病毒药物均有着一定程度的毒副作用，需要在医师监管下使用。恢复期血浆疗法作为目前已被批准的新冠疗法之一，虽然可以一定程度上用来对抗病毒感染，但是血浆来源不一，成分相对复杂不良反应较多， 安全性和有效性无法得到保证。但是恢复性血浆的成功应用依然暗示着中和性单克隆抗体可以用于治疗新冠病毒感染。
综上所述，本领域迫切需要开发针对SARS-CoV-2冠状病毒有效诊断和治疗方法，以用于诊断和治疗新型冠状病毒感染引起的肺炎。
发明内容
本发明的目的就是提供一种针对SARS-CoV-2冠状病毒有效诊断和治疗方法，以用于诊断和治疗新型冠状病毒感染引起的肺炎。
在本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区具有选自下组的互补决定区CDR：
SEQ ID NO:3所示的VH-CDR1、SEQ ID NO:4所示的VH-CDR2，和SEQ ID NO:5所示的VH-CDR3；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留与SARS-CoV-2 S蛋白RBD结构域的结合亲和力的衍生序列。
在另一优选例中，所述重链可变区具有如SEQ ID NO:2所示的氨基酸序列。
在本发明的第二方面，提供了一种抗体的重链，所述的重链具有如本发明第一方面所述的重链可变区。
在另一优选例中，所述重链还包括重链恒定区。
在另一优选例中，所述重链恒定区为人源或鼠源的。
在另一优选例中，所述重链恒定区为人源抗体重链IgG1恒定区。
在本发明的第三方面，提供了一种抗体的轻链可变区，所述的轻链可变区具有选自下组的互补决定区CDR：
SEQ ID NO:8所示的VL-CDR1、SEQ ID NO:9所示的VL-CDR2，和SEQ ID NO:10所示的VL-CDR3；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留与SARS-CoV-2 S蛋白RBD结构域的结合亲和力的衍生序列。
在另一优选例中，所述轻链可变区具有如SEQ ID NO:7所示的氨基酸序列。
在本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有如本发明第三方面所述的轻链可变区。
在另一优选例中，所述轻链还包括轻链恒定区。
在另一优选例中，所述轻链恒定区为人源或鼠源的。
在另一优选例中，所述轻链恒定区为人源抗体轻链λ恒定区。
在另一优选例中，所述轻链的氨基酸序列如SEQ ID NO:7所示。
在本发明的第五方面，提供了一种抗体，所述抗体具有如本发明第一方面所述的重链可变区，和/或如本发明第三方面所述的轻链可变区；
或者，所述抗体具有如本发明第二方面所述的重链，和/或如本发明第四方面所述的轻链；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留与SARS-CoV-2 S蛋白RBD结构域的结合亲和力的衍生序列。
在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量为1-5个(如1-3个，较佳地1-2个，更佳地1个)。
在另一优选例中，所述的经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留SARS-CoV-2 S蛋白RBD结构域的结合亲和力的衍生序列为同源性或序列相同性为至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的氨基酸序列。
在另一优选例中，所述的抗体还包括重链恒定区和/或轻链恒定区。
在另一优选例中，所述的重链恒定区为人源的，和/或所述的轻链恒定区为人源的。
在另一优选例中，所述重链恒定区为人源抗体重链IgG1恒定区，且所述轻链恒定区为人源抗体轻链λ恒定区。
在另一优选例中，所述抗体的重链可变区还包括人源的框架区，和/或所述抗体的轻链可变区还包括人源的框架区。
在另一优选例中，所述抗体的重链可变区还包括鼠源的框架区，和/或所述抗体的轻链可变区还包括鼠源的框架区。
在另一优选例中，所述抗体选自下组：动物源抗体、嵌合抗体、人源化抗体、全人抗体、或其组合。
在另一优选例中，所述的抗体是部分或全人源化、或全人的单克隆抗体。
在另一优选例中，所述的抗体为双链抗体、或单链抗体。
在另一优选例中，所述抗体为抗体全长蛋白、或抗原结合片段。
在另一优选例中，所述抗体为双特异性抗体、或多特异性抗体。
在另一优选例中，所述的抗体为药物偶联物形式。
在另一优选例中，所述抗体具有选自下组的一个或多个特性：
(a)特异性结合SARS-CoV-2 S蛋白RBD结构域；
(b)阻断SARS-CoV-2病毒与人血管紧张素转化酶2(ACE2)的结合；和
(c)有效中和SARS-CoV-2病毒感染。
在另一优选例中，所述的所述抗体具有如本本发明第一方面所述的重链可变区和如本发明第三方面所述的轻链可变区；
其中，所述的重链可变区和所述的轻链可变区包括选自下组的CDR：
(1)如SEQ ID NO:3所示的VH-CDR1、如SEQ ID NO:4所示的VH-CDR2、如SEQ ID NO:5所示的VH-CDR3、如SEQ ID NO:8所示的VL-CDR1、如SEQ ID NO:9所示的VL-CDR2，和如SEQ ID NO:10所示的VL-CDR3。
在另一优选例中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:7所示。
在另一优选例中，所述重链可变区的氨基酸序列与如SEQ ID NO:2所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
在另一优选例中，所述轻链可变区的氨基酸序列与如SEQ ID NO:7所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
在本发明的第六方面，提供了一种重组蛋白，所述的重组蛋白包括：
(i)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或如本发明第五方面所述的抗体；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。
在本发明的第七方面，提供了一种多核苷酸，所述多核苷酸编码选自下组的多肽：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、如本发明第五方面所述的抗体，或如本发明第六方面所述的重组蛋白。
在另一优选例中，编码所述重链可变区的多核苷酸如SEQ ID NO:1所示；和/或，编码所述轻链可变区的多核苷酸如SEQ ID NO:6所示。
在本发明的第八方面，提供了一种载体，所述载体含有如本发明第七方面所 述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
在本发明的第九方面，提供了一种遗传工程化的宿主细胞，所述宿主细胞含有如本发明第八方面所述的载体或基因组中整合有如本发明第七方面所述的多核苷酸。
在本发明的第十方面，提供了一种抗体偶联物，抗体偶联物含有：
(a)抗体部分，所述抗体部分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白，或其组合；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP，或其组合。
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。
在另一优选例中，所述的放射性核素包括：
(i)诊断用同位素，所述的诊断用同位素选自下组：Tc-99m、Ga-68、F-18、I-123、I-125、I-131、In-111、Ga-67、Cu-64、Zr-89、C-11、Lu-177、Re-188、或其组合；和/或
(ii)治疗用同位素，所述的治疗用同位素选自下组：Lu-177、Y-90、Ac-225、As-211、Bi-212、Bi-213、Cs-137、Cr-51、Co-60、Dy-165、Er-169、Fm-255、Au-198、Ho-166、I-125、I-131、Ir-192、Fe-59、Pb-212、Mo-99、Pd-103、P-32、K-42、Re-186、Re-188、Sm-153、Ra223、Ru-106、Na24、Sr89、Tb-149、Th-227、Xe-133Yb-169、Yb-177、或其组合。
在另一优选例中，所述偶联部分为药物或毒素。
在另一优选例中，所述的药物为细胞毒性药物。
在另一优选例中，所述的细胞毒性药物选自下组：抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱、或其组合。
特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、 依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))、长春花生物碱(vinca alkaloids)、或其组合。
在另一优选例中，所述的毒素选自下组：
耳他汀类(例如，耳他汀E、耳他汀F、MMAE和MMAF)、金霉素、类美坦西醇、篦麻毒素、篦麻毒素A-链、考布他汀、多卡米星、多拉司他汀、阿霉素、柔红霉素、紫杉醇、顺铂、cc1065、溴化乙锭、丝裂霉素、依托泊甙、替诺泊甙(tenoposide)、长春新碱、长春碱、秋水仙素、二羟基炭疽菌素二酮、放线菌素、白喉毒素、假单胞菌外毒素(PE)A、PE40、相思豆毒素、相思豆毒素A链、蒴莲根毒素A链、α-八叠球菌、白树毒素、迈托毒素(mitogellin)、局限曲菌素(retstrictocin)、酚霉素、依诺霉素、麻疯树毒蛋白(curicin)、巴豆毒素、卡奇霉素、肥皂草(Sapaonaria officinalis)抑制剂、糖皮质激素、或其组合。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联物选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(如DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(如顺铂)。
在另一优选例中，所述免疫偶联物含有：多价(如二价)的(a)。
在另一优选例中，所述多价是指在所述免疫偶联物的氨基酸序列中包含多个重复的(a)。
在本发明的第十一方面，提供了一种免疫细胞，所述免疫细胞表达或在细胞膜外暴露有如本发明第五方面所述的抗体。
在另一优选例中，所述的免疫细胞包括NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
在本发明的第十二方面，提供了一种药物组合物，所述药物组合物含有：
(i)活性成分，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第十方面所述的抗体偶联物、如本发明第十一方面所述的免 疫细胞、或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
在另一优选例中，所述的药物组合物包括0.01～99.99％的如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第十方面所述的抗体偶联物、如本发明第十一方面所述的免疫细胞、或其组合和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
在本发明的第十三方面，提供了一种活性成分的用途，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第十方面所述的抗体偶联物、如本发明第十一方面所述的免疫细胞、或其组合，其中所述活性成分被用于(a)制备SARS-CoV-2病毒感染的诊断试剂或试剂盒；和/或(b)制备预防和/或治疗SARS-CoV-2病毒感染的药物。
在另一优选例中，所述的诊断试剂为检测片或检测板。
在另一优选例中，所述诊断试剂或试剂盒用于：检测样品中的SARS-CoV-2 S蛋白或其片段。
在另一优选例中，所述的抗体为药物偶联物(ADC)形式。
在本发明的第十四方面，提供了一种体外检测样品中SARS-CoV-2病毒或SARS-CoV-2 S蛋白或其片段的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在SARS-CoV-2病毒或SARS-CoV-2 S蛋白或其片段。
在另一优选例中，所述的检测包括诊断性的或非诊断性的。
在本发明的第十五方面，提供了一种试剂盒，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有如本发明第五方面的抗体；和/或
(2)第二容器，所述第二容器中含有抗本发明第五方面所述抗体的二抗；
或者，
所述试剂盒含有一检测板，所述检测板包括：基片(支撑板)和测试条，所述的测试条含有如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第十方面所述的抗体偶联物、如本发明第十一方面所述的免疫细胞、或其 组合。
在本发明的第十六方面，提供了一种重组多肽的制备方法，所述的重组多肽是如本发明第五方面所述的抗体或如本发明第六方面所述的重组蛋白，所述的方法包括：
(a)在适合表达的条件下，培养如本发明第九方面所述的宿主细胞；和
(b)从培养物中分离出所述的重组多肽。
在本发明的第十七方面，提供了一种药物组合，包括：
(i)第一活性成分，所述第一活性成分包括如本发明第五方面所述的抗体、或如本发明第六方面所述的重组蛋白、或如本发明第十方面所述的抗体偶联物、或如本发明第十一方面所述的免疫细胞、或如本发明第十二方面所述的药物组合物、或其组合；
(ii)第二活性成分，所述第二活性成分包括其他治疗SAR-CoV-2病毒感染的药物。
在另一优选例中，所述其他治疗SAR-CoV-2病毒感染的药物包括：其他保护性单抗或瑞德西韦等小分子化药或其他中成药。
在本发明的第十八方面，提供了一种SAR-CoV-2病毒感染的诊断方法，包括步骤：
(i)从诊断对象获取一样品，将所述的样品与如本发明第五方面所述的抗体接触；和
(ii)检测是否形成抗原-抗体复合物，其中形成复合物就表示所述的对象为SAR-CoV-2病毒确诊患者。
在另一优选例中，所述的样品为血液样品或咽拭子样品，或其他组织器官中的样品。
在本发明的第十九方面，提供了一种治疗SARS-CoV-2病毒感染的疾病的方法，包括步骤：向有需要的对象施用有效量的如本发明第五方面所述的抗体、或如本发明第六方面所述的重组蛋白、或如本发明第十方面所述的抗体偶联物、或如本发明第十一方面所述的免疫细胞、如本发明第十二方面所述的药物组合物，或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。 限于篇幅，在此不再一一累述。
附图说明
图1显示了中和抗体表达的SDS-PAGE电泳图。
其中，1道是蛋白marker；2道是对照抗体IgG；3道是单克隆抗体XG-Ab008。
图2显示了抗体中和效果测试与抗原抗体亲和力测试结果。
其中，图2A和2B显示了候选抗体Ab001-Ab010对(图2A)SARS-CoV-2以及(图2B)SARS-CoV慢病毒感染体系中和试验结果。图2C显示了XG-Ab008在SARS-CoV-2假病毒体系中的中和效果，约为1nM。图2D显示了XG-Ab008在SARS-Cov-2真病毒体系中的中和效果，约为4.5nM。
图3显示了通过流式细胞仪分选得CD20-/CD38+/IgD-/IgM-/RBD+的B细胞。
图4显示了以B细胞反转录cDNA为模板扩增出的轻重链：上重链，中轻链κ，下轻链λ，成功扩增双链模板为：3、4、6、7和8。
图5显示了XG-Ab008中和抗体亲和力检测结果，KD约为1.39nM。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次开发了一种高效地特异性针对2019新型冠状病毒的抗体。具体地，本发明人筛选出了一种从新型冠状病毒痊愈者血液样本中提取的对SARS-Cov-2病毒表面Spike蛋白有高结合力和中和效果的单克隆抗体。实验结果表明，本发明的中和性抗体能够在细胞水平上有效地抑制假病毒的感染，其可以通过抑制ACE2受体和RBD蛋白的结合来发挥中和作用。
具体地，本发明人从COVID-19痊愈者血样中首先分离PBMC，通过磁珠富集得到CD19+B细胞。加入荧光素标记的SARS-CoV-2靶抗原孵育后，流式分选单个IgG+B细胞至96孔板裂解液中。分别对每个单个细胞的重链和轻链进行PCR扩增，重组插入单克隆抗体表达质粒；将重组质粒转染CHO细胞后，收集细胞培养上清液并使用protein G进行纯化，获得单克隆抗体。经过体外表达ACE2的Vero-E6细胞及表达SARS-Cov-2假病毒体系，筛选出了本发明中对病毒S蛋白具有高中和效果、高结合能力的单克隆抗体。
在此基础上，完成了本发明。
抗体
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的 重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明中，“VH-CDR1”与“CDR-H1”可互换使用，均指重链可变区的CDR1；“VH-CDR2”与“CDR-H2”可互换使用，均指重链可变区的CDR2；“VH-CDR3”与“CDR-H3”可互换使用，均指重链可变区的CDR3。“VL-CDR1”与“CDR-L1”可互换使用，均指轻链可变区的CDR1；“VL-CDR2”与“CDR-L2”可互换使用，均指轻链可变区的CDR2；“VL-CDR3”与“CDR-L3”可互换使用，均指轻链可变区的CDR3。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以通过标准的DNA重组技术获得，它们都是有用的抗体。嵌合抗体是一个分子，其中不同的部分来自不同的动物种，例如具有来自鼠的单克隆抗体的可变区，和来自人免疫球蛋白的恒定区的嵌合抗体(见例如美国专利4,816,567和美国专利4,816,397,在此通过引用方式整体引入本文)。人源化的抗体是指来源于非人物种的抗体分子，具有一个或多个来源于非人物种的互补决定区(CDRs)和来源于人免疫球蛋白分子的框架区域(见美国专利5,585,089，在此通过引用方式整体引入本文)。这些嵌合和人源化的单克隆抗体可以采用本领域熟知的DNA重组技术制备。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本发明中，本发明的抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明中，所述抗体为特异性结合SARS-CoV-2 S蛋白RBD结构域的抗体。本发明提供一种针对SARS-CoV-2 S蛋白RBD结构域的高特异性和高亲和力的抗体，其包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。
优选地，
所述的重链可变区(VH)具有选自下组的互补决定区CDR：
SEQ ID NO:3所示的VH-CDR1，
SEQ ID NO:4所示的VH-CDR2，和
SEQ ID NO:5所示的VH-CDR3；
所述的轻链可变区(VL)具有选自下组的互补决定区CDR：
SEQ ID NO:8所示的VL-CDR1，
SEQ ID NO:9所示的VL-CDR2，和
SEQ ID NO:10所示的VL-CDR3；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留与SARS-CoV-2 S蛋白RBD结构域的结合亲和力的衍生序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性或序列相同性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
本领域普通技术人员公知的测定序列同源性或相同性的方法包括但不限于：计算机分子生物学(Computational Molecular Biology)，Lesk，A.M.编，牛津大学出版社，纽约，1988；生物计算：信息学和基因组项目(Biocomputing:Informatics and Genome Projects)，Smith，D.W.编，学术出版社，纽约，1993；序列数据的计算机分析(Computer Analysis of Sequence Data)，第一部分，Griffin，A.M.和Griffin，H.G.编，Humana Press，新泽西，1994；分子生物学中的序列分析(Sequence Analysis in Molecular Biology)，von Heinje，G.，学术出版社，1987和序列分析引物(Sequence Analysis Primer)，Gribskov，M.与Devereux，J.编M Stockton Press，纽约，1991和Carillo，H.与Lipman，D.，SIAM J.Applied Math.，48:1073(1988)。测定相同性的优选方法要在测试的序列之间得到最大的匹配。测定相同性的方法编译在公众可获得的计算机程序中。优选的测定两条序列之间相同性的计算机程序方法包括但不限于：GCG程序包(Devereux，J.等，1984)、BLASTP、BLASTN和FASTA(Altschul，S，F.等，1990)。公众可从NCBI和其它来源得到BLASTX程序(BLAST手册，Altschul，S.等，NCBI NLM NIH Bethesda，Md.20894；Altschul，S.等，1990)。熟知的Smith Waterman算法也可用于测定相同性。
较佳地，本文所述抗体为抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(single chain antibody fragment，scFv)、单域抗体(single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。所述的蛋白质的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成全人源抗体全长蛋白。较佳地，所述的抗体全长蛋白为IgG1、IgG2、IgG3或IgG4；更佳地为IgG1。
本发明的抗体可以是双链或单链抗体，并且可以是选自动物源抗体、嵌合抗体、人源化抗体，更优选为人源化抗体、人-动物嵌合抗体，更优选为全人源化抗体。
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab’、(Fab’)2或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
所述的单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。
其中，所述动物优选为哺乳动物，如鼠。
本发明抗体可以是靶向SARS-CoV-2 Spike蛋白的嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
本发明上述内容中，更优选地，所述添加、缺失、修饰和/或取代的氨基酸数量，可以是1-7个，更优选为1-5个，更优选为1-3个，更优选为1-2个。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO:2所示的氨基酸序列。
在另一优选例中，所述抗体的轻链可变区含有SEQ ID NO:7所示的氨基酸序列。
在本发明的实施方式中，所述靶向SARS-CoV-2 S蛋白RBD结构域的抗体为XG-Ab008。
在一个更加优选的实施方式中，本发明各抗体具体地包括以下各VL和VH序列，以及Fc、CL和CH1序列。
表B XG-Ab008抗体序列总结
Figure PCTCN2022074998-appb-000001
编码的多核苷酸
本发明还提供一种多核苷酸，其编码上述的抗体或包含其的重组蛋白或其重链可变区或轻链可变区。
较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID NO:1所示；和/或，编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID NO:6所示。
更佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID NO:1所示；且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID NO:6所示。
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述蛋白质的核酸分子，或者通过人工全序列合成的方法得到编码上述蛋白质的核酸分子。
本领域技术人员知晓，编码上述蛋白质的氨基酸序列的碱基序列可以适当引 入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该蛋白序列基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
载体
本发明还提供一种包含所述核酸的重组表达载体。
其中所述重组表达载体可通过本领域常规方法获得，即：将本发明所述的核酸分子连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能够容载前述核酸分子即可。所述载体较佳地包括：各种质粒、粘粒、噬菌体或病毒载体等。
本发明还提供一种包含上述重组表达载体的重组表达转化体。
其中，所述重组表达转化体的制备方法为本领域常规的制备方法，较佳地为：将上述重组表达载体转化至宿主细胞中制得。所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。较佳地，所述宿主细胞为E.coli TG1或E.coli BL21细胞(表达单链抗体或Fab抗体)，或者HEK293或CHO细胞(表达全长IgG抗体)。将前述重组表达质粒转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
抗体的制备
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码所述的本发明的抗体(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。优选的动物细胞包括(但并不限于)：CHO-S、HEK-293细胞。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。单克隆抗体的结合亲和力例如可用Munson等,Anal.Biochem.,107:220(1980)的Scatchard分析来测定。
本发明的抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
抗体-药物偶联物(ADC)
本发明还提供了基于本发明抗体的抗体偶联药物(antibody-drug conjugate，ADC)。
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。
本发明抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至 抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，并包括：例如马来酰亚胺类化合物，卤代酰胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
在本发明中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier)中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的 反应活性功能基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
本发明还提供了制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。
在某些实施方式中，本发明方法包括：在足以形成抗体-接头偶联物的条件下，将抗体与双功能接头化合物进行结合。在这些实施方式中，本发明方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：
Figure PCTCN2022074998-appb-000002
其中：
Ab是抗体，
LU是接头；
D是药物；
而且下标p是选自1到8的值。
应用
本发明还提供了本发明抗体、抗体偶联物ADC、重组蛋白、和/或免疫细胞的用途，例如用于制备诊断制剂或制备药物。
较佳地，所述的药物是用于预防和/或治疗SARS-CoV-2病毒感染的疾病的药物。
检测用途和试剂盒
本发明的抗体或其ADC可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。
较佳地，所述的样本是来自于检测对象的血液样本或咽拭子样本。
本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如肿瘤的切除样本、通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的抗体可以固定于检测板。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白或其ADC或相应的免疫细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。
配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。典型地，本发明所述的药物组合物的给药途径较佳地为注射给药或口服给药。所述注射给药较佳地包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，更佳地为片剂、胶囊、颗粒剂、注射剂或输注剂等。
本发明所述抗体也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如，所述抗体用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。
本发明所述的药物组合物是用于预防和/或治疗SARS-CoV-2病毒感染的疾病的药物组合物。
本发明的药物组合物可直接用于结合SARS-CoV-2 S蛋白分子或其片段，因而可用于预防和治疗病毒感染所引发的疾病。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
本发明中，较佳地，本发明所述的药物组合物还包括一种或多种药用载体。所述的药用载体为本领域常规药用载体，所述的药用载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳的包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述蛋白质和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
本发明中，较佳地，所述的药物组合物的施用量为有效量，所述有效量为能够缓解或延迟疾病、退化性或损伤性病症进展的量。所述有效量可以以个体 基础来测定，并将部分基于待治疗症状和所寻求结果的考虑。本领域技术人员可以通过使用个体基础等上述因素和使用不超过常规的实验来确定有效量。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明提供上述药物组合物在制备预防和/或治疗SARS-CoV-2病毒感染的疾病的药物中的应用。较佳地，所述SARS-CoV-2病毒感染的疾病为肺炎。
冠状病毒
如本文所用，术语“新型冠状病毒”、“2019-nCov”或“SARS-CoV-2”可互换使用，该2019新型冠状病毒是已知感染人的第7种冠状病毒，并且造成新冠肺炎(COVID-19)，是威胁全球人类健康的严重传染性疾病之一。
冠状病毒(Coronavirus，CoV)属于套式病毒目(Nidovirales)冠状病毒科(Coronaviridae)，是一种有包膜的正链RNA病毒，其亚科包含α、β、δ及γ四属。
目前已知的感染人的冠状病毒中，HCoV-229E和HCoV-NL63属于α属冠状病毒，HCoV-OC43、SARS-CoV、HCoV-HKU1、MERS-CoV和SARS-CoV-2均为β属冠状病毒。SARS-CoV-2也被称为2019-nCov。
高致病性冠状病毒“非典”SARS-CoV和“中东呼吸综合征”MERS-CoV均属于β属冠状病毒。新型冠状病毒(SARS-CoV-2)与SARS-CoV有约80％相似性、与MERS-CoV有40％的相似性，也属于β属冠状病毒。
该类病毒的基因组是一条单股正链RNA，是基因组最大的RNA病毒之一，编码包括复制酶、刺突蛋白、囊膜蛋白、包膜蛋白和核壳蛋白等。在病毒复制的初始阶段，基因组被翻译成两条长达几千个氨基酸的肽链即前体多聚蛋白(Polyprotein)，随后前体蛋白被蛋白酶切割生成非结构蛋白(如RNA聚合酶和解旋酶)和结构蛋白(如刺突蛋白)及辅助蛋白。
本发明的主要优点包括：
(1)本发明的抗体制备过程简单。本发明使用CHO表达系统，操作简单，可持续地表达大量抗体，满足下游的实验和检测需求。
(2)本发明提供的单抗为全人抗体，不存在其他异种蛋白的抗原性。
(3)通过体外S蛋白慢病毒感染体系验证，本发明抗体对SARS-CoV-2病毒具有较强的中和效果(4.5nM)。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
以下为获得发明目的所述的单抗所需的基本实验方案。
1.收集新冠病毒感染痊愈者静脉采血12-15毫升，肝素抗凝。
2.外周肝素抗凝血使用Lymphoprep密度梯度离心法获得单个核细胞(PBMC)
3.使用磁珠负选富集外周血单个核细胞(PBMC)中的B细胞
4.再加入荧光抗体及荧光素标记的SARS-CoV-2靶抗原孵育，流式分选单个IgG+B细胞至96孔板中，理论上每孔收集一个细胞，每个孔预先加入含RNA酶抑制剂的细胞裂解液
5.随后提取单个B细胞RNA，逆转录成cDNA
6.以cDNA为模板，进行First PCR，再以first pcr产物为模板用seq-PCR方法扩增轻重链、筛选只能扩增出正确大小单一条带模板并测序。
7.用筛选后的模板扩总轻重链基因
8.重组轻重链基因，获得表达载体
9.将表达载体转染进DH5α感受态细胞中，并用含有氨苄西林的琼脂糖平板筛选挑选单克隆。
10.挑选出的单克隆进行小体积培养并纯化表达载体
11.用PCR方法扩增轻重链并测序，比较步骤6和步骤11获得的轻重链序列，筛选出结果一致的轻重链载体。
12.转染表达载体进CHO细胞，表达并纯化获得全人源抗体。
13.在BSL-2实验室中，用已建立的S蛋白慢病毒感染体系中，筛选获得有中和效果的候选抗体10株：Ab001、002、003至010。通过比较中和效果，获得3.1中目标特异性中和抗体：XG-Ab008。(简称为Ab008)
14.另外，使用Octet RED 96蛋白质相互作用工作站拟合得到XG-Ab008对S蛋白RBD的结合系数。
由上述步骤1-12，通过收集CHO细胞表达上清溶液，纯化可得所述单克隆中和抗体XG Ab-008，经SDS-PAGE结果显示(图1)，纯化后的候选抗体纯度可满足后续试验需求。
由上述步骤13-14，通过过表达ACE2的Vero-E6细胞作为应答细胞，使用 分别表达SARS-CoV-2、SARS-CoV S蛋白的假病毒体系，获得候选抗体对SARS-CoV-2慢病毒(图2A)和SARS-CoV慢病毒的抑制效果(图2B)，证明候选抗体Ab008对SARS-CoV-2病毒有特异性中和能力。其中，Ab008抗体特异的对SARS-CoV-2具有良好的中和效果1nM(图2C)。在真病毒感染体系中对SARS-CoV-2中和效果能达到4.5nM(图2D)通过链霉亲和素传感器固化S-RBD结构域后(RBD检测梯度浓度范围1.56-50nM)，Octet仪器检测候选抗体Ab008的亲和力，此抗体具有非常强的S-RBD结合能力(KD～1.39nM)(图5)
实施例1：新冠痊愈患者血液中获得单个核细胞(PBMC)
集新冠痊愈患者肝素抗凝血液12-15ml，500g离心10分钟，吸去上层血浆。之后，按1：1体积加入生理盐水稀释下层血细胞，混合均匀。混合物缓慢叠加到含有等体积Lymphoprep溶液的离心管中，用密度梯度离心法获得单个核细胞(PBMC)。
以上实施例中所需溶液配方如下所示：
生理盐水：0.9％氯化钠，超纯水，0.22um滤膜过滤后使用。
Lymphoprep溶液：LymphoprepTM、1×D-PBS、0.9％氯化钠、100×青链霉素双抗、0.5M EDTA，调整pH至8.0。
实施例2：B细胞富集
按照商业说明书方法准备LS column。
准备“抗体稀释混合液”，在150ul抗体稀释液稀释生物素标记的抗CD3、抗CD11b、抗CD14、抗CD16、抗CD56、抗CD235a抗体各0.6ul。
准备“抗生物素磁珠悬液”，在200ul抗体稀释液中加入20ul抗生物素磁珠。
按商业说明书方法，用抗体稀释混合液孵育实施例1中制得的PBMC悬液，洗去多余抗体后，再用抗生物素磁珠悬液孵育处理后的PBMC悬液，用LS colum收集富集的B细胞。
以上实施例中所需溶液配方如下所示：
MACS溶液：10ml胎牛血清(FBS)、5mM EDTA、500ml 1×DPBS，调整pH至8.0，0.22um滤膜过滤后使用4℃保存。
实施例3：B细胞分选
准备Mouse FC稀释液：抗体稀释液100ul中加入4ul mouse FC
准备流式荧光抗体稀释混合：抗体稀释液100ul中加入下表抗体
抗体/蛋白名称 荧光素 用量
S-RBD FITC 5ug/ml
S-RBD PE 5ug/ml
IgA VioBlue(PB channel) 1/25
IgM A647 1/100
IgD BV510 1/100
CD3 PerCPCy5.5 1/250
CD20 BV785 1/100
CD38 PECy7 1/250
使用实施例2中富集的B细胞，加入100ul Mouse FC稀释液，室温避光孵育15分钟。用MACS洗去多余抗体，再加入100ul流式荧光抗体稀释液，室温避光孵育15分钟。用MACS洗去多余抗体后，用流式细胞仪Melody分选特异性结合S-RBD的单个B细胞(图3)。分选出的B细胞用含有4ul细胞裂解液的96孔PCR板收集，每孔一个细胞，分选结束后立即用封膜并用干冰速冻。
以上实施例中所需溶液配方如下所示：
裂解液：300ul RNasin、40U/ul RNase抑制剂、200ul 1*DPBS、400ul DL-DTT(100mM)、3100ul无核酸酶水。
实施例4：筛选备选抗体表达载体模板
在实施例3中分选的到的单个B细胞，用SuperScript III RT逆转录酶(Invitrogen,18080-044)反转获得cDNA。
之后，以cDNA为模板按商业说明书使用TransTaq High Fidelity(HiFi)PCR SuperMix II(Transgen,AS131-22)，进行1st PCR，测序PCR和克隆PCR，通过跑琼脂糖胶鉴定轻重链。挑选可匹配的轻重链对(图4)。
实施例5：构建表达载体，抗体表达及纯化
以AbVec hIgG抗体表达质粒为骨架，插入实施例4中获得的成对轻重链序列分别构轻链表达质粒和重链表达质粒，按ExpiCHO表达系统商业说明书所述方法(ExpiCHO TM Expression System Kit，A29133)，转染并表达CHO细胞。500g，10min离心分离收集细胞悬液，并按Protein G Sepharose商业说明书(Protein G
Figure PCTCN2022074998-appb-000003
4 Fast Flow，17-0618-01)所述方法，纯化IgG抗体
实施例6：抗体中和效果检测
[根据细则26改正12.04.2022]　
制备SARS-Cov-2假病毒，构建表达载体，包含：小鼠白血病病毒(MLV)gag/pol，逆转录病毒EGFP，SARS-CoV-2 S蛋白全长序列(GenBank:QHD43419.1)或SARS-CoV S蛋白全长序列(AY569693)。按商业说明书方法，使用PEI转染试剂，转染HEK293T，并于48小时后，收集上清液中的假病毒。之后使用预混匀的假病毒/抗体悬液侵染高表达hACE2的VeroE6细胞，为实验组(SD,n＝3)(附图2A)，单独假病毒悬液侵染为对照组(SD,n＝3)(附图2B)通过流式分析可得抗体中和效果约为～10nM(附图2C)。
实施例7：抗体亲和力检测
按Octet RED 96蛋白质相互作用工作站商业使用说明书所述，准备Biotin溶液(10mM)，ZB脱盐柱130ul。使用Biotin溶液1:1孵育标记S蛋白，用脱盐柱去除多余未结合Biotin。在96孔板中依次加入S蛋白样品，XG-Ab008梯度稀释溶液50mM、25mM、12.5mM、6.25mM、3.125mM、1.563mM。根据仪器度值拟合得XG-Ab008对S蛋白的结合系数(附图2D)。
讨论
现有常用的SARS-CoV-2抗体筛选技术包括杂交瘤、噬菌体、转基因小鼠及单个B细胞克隆技术等。通过对传统鼠源抗体进行人源化改造生产出的全人抗体，可以大幅降低抗体的免疫原性(再生元，REGN-COV2)。随着流式细胞术的发展，单个B细胞分离技术已被广泛使用。通过标记B细胞表面抗原，筛选的到抗原特异性单个B细胞，之后分别对分选单个B细胞进行抗体基因扩增。之后通过载体构建，抗体表达等方法获得抗原特异性中和抗体(清华大学，BRII-198、BRII-196)。也有通过高通量单细胞测序的方法筛选有效的中和抗体(百济神州，DXP-593)
虽然靶向SARS-CoV-2病毒S蛋白的单克隆抗体技术以日趋完善，但采用不同的分选策略，扩增条件仍然会出现差异巨大的筛选结果。对此，本发明提供了一种基于单个B细胞扩增并表达单克隆抗体的策略，以及筛选出的靶向S蛋白的高效中和单克隆抗体。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种抗体的重链可变区，其特征在于，所述的重链可变区具有选自下组的互补决定区CDR：
    SEQ ID NO:3所示的VH-CDR1、SEQ ID NO:4所示的VH-CDR2，和SEQ ID NO:5所示的VH-CDR3；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留与SARS-CoV-2 S蛋白RBD结构域的结合亲和力的衍生序列。
  2. 如权利要求1所述的抗体的重链可变区，其特征在于，所述重链可变区具有如SEQ ID NO:2所示的氨基酸序列。
  3. 一种抗体的重链，其特征在于，所述的重链具有如权利要求1所述的重链可变区。
  4. 一种抗体的轻链可变区，其特征在于，所述的轻链可变区具有选自下组的互补决定区CDR：
    SEQ ID NO:8所示的VL-CDR1、SEQ ID NO:9所示的VL-CDR2，和SEQ ID NO:10所示的VL-CDR3；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留与SARS-CoV-2 S蛋白RBD结构域的结合亲和力的衍生序列。
  5. 如权利要求4所述的抗体的轻链可变区，其特征在于，所述轻链可变区具有如SEQ ID NO:7所示的氨基酸序列。
  6. 一种抗体的轻链，其特征在于，所述的轻链具有如权利要求4所述的轻链可变区。
  7. 一种抗体，其特征在于，所述抗体具有如权利要求1所述的重链可变区，和/或如权利要求4所述的轻链可变区；
    或者，所述抗体具有如权利要求3所述的重链，和/或如权利要求6所述的轻链；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留与SARS-CoV-2 S蛋白RBD结构域的结合亲和力的衍生序列。
  8. 如权利要求7所述的抗体，其特征在于，所述的所述抗体具有所述的重链可变区和所述的轻链可变区；其中，所述的重链可变区和所述的轻链可变区具有以下CDR：如SEQ ID NO:3所示的VH-CDR1、如SEQ ID NO:4所示的VH-CDR2、如SEQ ID NO:5所示的VH-CDR3、如SEQ ID NO:8所示的VL-CDR1、如SEQ ID NO:9 所示的VL-CDR2，和如SEQ ID NO:10所示的VL-CDR3。
  9. 如权利要求7所述的抗体，其特征在于，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:7所示。
  10. 一种重组蛋白，其特征在于，所述的重组蛋白包括：
    (i)如权利要求1所述的重链可变区、如权利要求3所述的重链、如权利要求4所述的轻链可变区、如权利要求6所述的轻链、或如权利要求7所述的抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  11. 一种多核苷酸，其特征在于，所述多核苷酸编码选自下组的多肽：如权利要求1所述的重链可变区、如权利要求3所述的重链、如权利要求4所述的轻链可变区、如权利要求6所述的轻链、如权利要求7所述的抗体，或如权利要求11所述的重组蛋白。
  12. 一种载体，其特征在于，所述载体含有如权利要求11所述的多核苷酸。
  13. 一种遗传工程化的宿主细胞，其特征在于，所述宿主细胞含有如权利要求12所述的载体或基因组中整合有如权利要求11所述的多核苷酸。
  14. 一种抗体偶联物，其特征在于，抗体偶联物含有：
    (a)抗体部分，所述抗体部分选自下组：如权利要求1所述的重链可变区、如权利要求3所述的重链、如权利要求4所述的轻链可变区、如权利要求6所述的轻链、如权利要求7所述的抗体、如权利要求11所述的重组蛋白，或其组合；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP，或其组合。
  15. 一种药物组合物，所述药物组合物含有：
    (i)活性成分，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求3所述的重链、如权利要求4所述的轻链可变区、如权利要求6所述的轻链、如权利要求7所述的抗体、如权利要求11所述的重组蛋白、权利要求14所述的抗体偶联物、或其组合；以及
    (ii)药学上可接受的载体。
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