WO2021180218A9 - 一种抗新型冠状病毒的单克隆抗体及其应用 - Google Patents

一种抗新型冠状病毒的单克隆抗体及其应用 Download PDF

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WO2021180218A9
WO2021180218A9 PCT/CN2021/080537 CN2021080537W WO2021180218A9 WO 2021180218 A9 WO2021180218 A9 WO 2021180218A9 CN 2021080537 W CN2021080537 W CN 2021080537W WO 2021180218 A9 WO2021180218 A9 WO 2021180218A9
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antibody
monoclonal antibody
seq
antigen
binding fragment
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WO2021180218A1 (zh
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谢晓亮
曹云龙
孙文洁
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北京大学
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    • 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
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—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]
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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
    • 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
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the present invention relates to the field of immunology and molecular virology, in particular to the fields of diagnosis, prevention and treatment of novel coronavirus.
  • the present invention relates to monoclonal antibodies against novel coronavirus, and compositions (eg, diagnostic and therapeutic agents) comprising the antibodies.
  • the present invention also relates to the use of said antibodies.
  • Antibodies of the present invention can be used to diagnose, prevent and/or treat infection with novel coronaviruses and/or diseases caused by such infections (eg, novel coronavirus pneumonia).
  • the novel coronavirus SARS-CoV-2 is the pathogen causing novel coronavirus pneumonia (COVID-19), a single-stranded RNA virus, which is closely related to the severe acute respiratory syndrome coronavirus (SARS-CoV) that caused the outbreak in 2002-2003. ) and the Middle East respiratory syndrome coronavirus (MERS-CoV) that caused the outbreak in 2012 belong to the family Coronaviridae.
  • Coronavirus particles are round or oval, also pleomorphic, 50-200nm in diameter, and belong to larger viruses.
  • Coronaviruses are enveloped viruses.
  • the viral capsid is covered with a lipid envelope, on which is arranged a wide spike protein (Spike, S protein) shaped like a sun halo.
  • S protein is located on the surface of the novel coronavirus SARS-CoV-2, which can bind the host cell receptor angiotensin through the receptor binding domain (RBD) it contains during the virus infection of the host.
  • Enzyme 2 (ACE2) molecule thereby initiating fusion of the viral membrane with the host cell membrane, resulting in the infection of the host cell with the virus.
  • Neutralizing antibodies have so far been shown to be an effective treatment for viral diseases.
  • B lymphocytes in patients will be activated, and then transformed and differentiated into a variety of different cells, and produce antibodies.
  • antibodies against the novel coronavirus in the peripheral blood of recovered patients with novel coronavirus pneumonia are produced and secreted by activated B cells.
  • B cells there are a variety of B cells in the plasma of recovered patients, and the binding activities and neutralizing titers of antibodies produced by different B cells are also different. So far, no studies have reported antibodies against 2019-nCoV with high binding activity and/or high neutralizing activity.
  • antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having one "light” (L) chain and one "heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Definition by Chothia et al. (1989) Nature 342:878-883.
  • antibody is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to, and/or compete with, the same antigen to which the full-length antibody binds Specific binding to an antigen, which is also referred to as an "antigen binding moiety".
  • an antigen binding moiety which is also referred to as an "antigen binding moiety”.
  • antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, dAb, and complementarity determining regions (CDRs) Fragments, single chain antibodies (eg, scFvs), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of the antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • CDRs complementarity determining regions
  • the antigen-binding fragment of an antibody is a single-chain antibody (eg, scFv) in which the VL and VH domains are paired to form a monovalent molecule by linkers that enable it to be produced as a single polypeptide chain (see, eg, Bird et al. , Science 242:423 426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879 5883 (1988)).
  • scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker with the amino acid sequence (GGGGS) 4 can be used, but also variants thereof (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.
  • the antigen-binding fragment of the antibody is a diabody, i.e., a diabody, in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short is used to allow for both structures on the same chain Pairing between domains forces the domains to pair with the complementary domains of the other chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444 6448 ( 1993), and Poljak R.J. et al., Structure 2:1121 1123 (1994)).
  • Antigen-binding fragments of an antibody e.g., Antibody fragments above
  • the antibody is screened for specificity for antigen-binding fragments in the same manner as is used for intact antibodies.
  • antibody includes not only whole antibodies but also antigen-binding fragments of antibodies.
  • the term "monoclonal antibody” refers to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, that is, a population that is identical except for natural mutations that may arise spontaneously Antibody molecule.
  • Monoclonal antibodies are highly specific for a single epitope on an antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which generally comprise at least two or more different antibodies that generally recognize different epitopes on an antigen.
  • Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (see, for example, Journal of virological methods, 2009, 158(1-2): 171-179).
  • neutralizing antibody refers to an antibody or antibody fragment that clears or significantly reduces the virulence (eg, ability to infect cells) of a target virus.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or artificial chromosomes of P1 origin (PAC); bacteriophages such as lambda Bacteriophage or M13 phage and animal virus, etc.
  • plasmids such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or artificial chromosomes of P1 origin (PAC)
  • bacteriophages such as lambda Bacteriophage or M13 phage and animal virus, etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg SV40
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, and fungal cells such as yeast cells or Aspergillus , insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • prokaryotic cells such as E. coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • an antibody that specifically binds to an antigen refers to an antibody that is less than about 10-5 M, such as less than about 10-6 M, 10-7 M, Binds the antigen with an affinity (KD) of 10-8 M, 10-9 M or 10-10 M or less.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • antibodies bind antigen with dissociation equilibrium constants (KD) less than about 10<" 5 >M.
  • KD dissociation equilibrium constant
  • an antigen eg, the S protein of the novel coronavirus
  • KD dissociation equilibrium constant
  • amino acids are generally represented by one-letter and three-letter abbreviations well known in the art.
  • alanine can be represented by A or Ala.
  • neutralizing activity refers to the ability of an antibody or antibody fragment to bind to an antigenic protein on a virus, thereby preventing viral infection of cells and/or maturation of viral progeny and/or release of viral progeny
  • the functional activity of the antibody or antibody fragment with neutralizing activity can prevent the amplification of the virus, thereby inhibiting or eliminating the infection of the virus.
  • COVID-19 and COVID-19 refer to pneumonia caused by novel coronavirus infection, both have the same meaning and can be used interchangeably.
  • the inventors of the present application discovered an antibody that can specifically recognize and target the S protein of the new coronavirus, especially the receptor binding domain (RBD) of the S protein, and shows high efficiency ability to neutralize viruses. Therefore, the antibodies of the present invention are particularly suitable for the diagnosis, prevention and treatment of novel coronavirus infection or diseases associated with novel coronavirus infection (eg, novel coronavirus pneumonia).
  • novel coronavirus infection or diseases associated with novel coronavirus infection eg, novel coronavirus pneumonia.
  • a monoclonal antibody or an antigen-binding fragment thereof comprising the heavy chain variable region (VH) complementarity determining region whose amino acid sequences are respectively shown in SEQ ID NOs: 1-3 1-3 (CDR1-3); and/or, light chain variable region (VL) complementarity determining regions 1-3 (CDR1-3) whose amino acid sequences are shown in SEQ ID NOs: 4-6, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • the monoclonal antibody comprises a heavy chain variable region (VH) as set forth in SEQ ID NO:7.
  • VH heavy chain variable region
  • the monoclonal antibody comprises a light chain variable region (VL) as set forth in SEQ ID NO:8.
  • VL light chain variable region
  • the monoclonal antibody comprises: VH CDR1-3 whose amino acid sequences are shown in SEQ ID NOs: 1-3, respectively, and VH CDRs 1-3 whose amino acid sequences are shown in SEQ ID NOs: 4-6, respectively VL CDR1-3.
  • the monoclonal antibody comprises: VH as set forth in SEQ ID NO:7 and VL as set forth in SEQ ID NO:8.
  • the monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv, dAb, complementarity determining region fragments, single chain antibodies (eg, scFv), human antibodies, chimeric antibodies or bispecific or multispecific antibodies.
  • the monoclonal antibody further comprises a heavy chain constant region.
  • the amino acid sequence of the heavy chain constant region is set forth in SEQ ID NO:9.
  • the monoclonal antibody further comprises a light chain constant region.
  • the amino acid sequence of the light chain constant region is set forth in SEQ ID NO:10.
  • the monoclonal antibody or antigen-binding fragment thereof can specifically bind to the spike protein (S protein) of the novel coronavirus.
  • the monoclonal antibody or antigen-binding fragment thereof is capable of targeting the receptor binding domain (RBD) of the spike protein (S protein) of the novel coronavirus.
  • the monoclonal antibody or antigen-binding fragment thereof is capable of inhibiting receptor binding and/or membrane fusion mediated by the receptor binding domain (RBD) of the S protein, thereby inhibiting viral infection of cells .
  • the monoclonal antibody or antigen-binding fragment thereof has neutralizing ability (eg, capable of neutralizing novel coronavirus). In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof is capable of inhibiting novel coronavirus infection or entry into host cells. Thus, the monoclonal antibody or antigen-binding fragment thereof can neutralize the novel coronavirus, thereby preventing and treating the infection of the novel coronavirus.
  • the application also provides isolated nucleic acid molecules encoding the monoclonal antibodies or antigen-binding fragments thereof of the invention.
  • nucleic acid molecules are not limited by the method of their production, and can be obtained using genetic engineering recombinant techniques or chemical synthesis methods.
  • the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence capable of encoding an antibody heavy chain variable region, wherein the antibody heavy chain variable region comprises: the amino acid sequences of SEQ ID NOs, respectively : VH CDR1-3 of 1-3.
  • the VH CDR1-3 are encoded by the nucleotide sequences shown in SEQ ID NOs: 11-13, respectively. Accordingly, in certain preferred embodiments, the isolated nucleic acid molecule comprises the nucleotide sequences set forth in SEQ ID NOs: 11-13.
  • the antibody heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:7.
  • the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO:17.
  • the antibody light chain variable region has the amino acid sequence set forth in SEQ ID NO:8.
  • the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO:18.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence capable of encoding an antibody heavy chain variable region, as defined above, and a nucleotide sequence capable of encoding an antibody light chain variable region, as defined above Nucleotide sequence.
  • the antibody heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:7.
  • the nucleotide sequence capable of encoding an antibody heavy chain variable region has the nucleotide sequence shown in SEQ ID NO:17.
  • the antibody light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8.
  • the nucleotide sequence capable of encoding the variable region of an antibody light chain has the nucleotide sequence shown in SEQ ID NO:18.
  • the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:17 and the nucleotide sequence set forth in SEQ ID NO:18.
  • the isolated nucleic acid molecule further comprises a nucleotide sequence capable of encoding an antibody heavy chain constant region.
  • the heavy chain constant region has the amino acid sequence set forth in SEQ ID NO:9.
  • the nucleotide sequence capable of encoding an antibody heavy chain constant region has the nucleotide sequence shown in SEQ ID NO:19.
  • the isolated nucleic acid molecule further comprises a nucleotide sequence capable of encoding an antibody light chain constant region.
  • the light chain constant region has the amino acid sequence set forth in SEQ ID NO:10.
  • the nucleotide sequence capable of encoding the constant region of an antibody light chain has the nucleotide sequence shown in SEQ ID NO:20.
  • the present invention provides an isolated nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof of the present invention as defined above.
  • the present invention provides a vector comprising an isolated nucleic acid molecule as defined above.
  • the vector of the present invention may be a cloning vector or an expression vector.
  • the vectors of the present invention are, for example, plasmids, cosmids, phages, and the like.
  • host cells comprising the isolated nucleic acid molecules or vectors of the present invention are also provided.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (eg, mammalian cells, eg, mouse cells, human cells, etc.).
  • the cells of the invention may also be cell lines, such as HEK293 cells.
  • a method of preparing a monoclonal antibody of the present invention or an antigen-binding fragment thereof comprising, culturing a host cell of the present invention under suitable conditions, and recovering the monoclonal antibody of the present invention from the cell culture Antibodies or antigen-binding fragments thereof.
  • the present invention provides a composition comprising a monoclonal antibody or antigen-binding fragment thereof, an isolated nucleic acid molecule, a vector or a host cell as described above.
  • the present invention provides a kit comprising the monoclonal antibody or antigen-binding fragment thereof of the present invention.
  • the monoclonal antibodies or antigen-binding fragments thereof of the invention further comprise a detectable label.
  • the kit further comprises a second antibody that specifically recognizes the monoclonal antibody or antigen-binding fragment thereof of the invention.
  • the second antibody further comprises a detectable label.
  • detectable labels are well known to those skilled in the art and include, but are not limited to, radioisotopes, fluorescent substances, luminescent substances, colored substances, and enzymes (eg, horseradish peroxidase), and the like.
  • the sample includes, but is not limited to, excreta, oral or nasal secretions, bronchoalveolar lavage fluid, and the like from a subject (eg, a mammal, preferably a human).
  • a subject eg, a mammal, preferably a human.
  • the monoclonal antibody is an antibody comprising: VH CDRs 1-3 having amino acid sequences set forth in SEQ ID NOs: 1-3, respectively, and/or amino acid sequences set forth in SEQ ID NOs, respectively VL CDR1-3 shown in NO:4-6; preferably, it comprises: VH shown in SEQ ID NO:7 and/or VL shown in SEQ ID NO:8.
  • the detection method can use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescence immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, competition assay, and similar assays .
  • ELISA enzyme-linked immunosorbent assay
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of the present invention, and a pharmaceutically acceptable carrier and/or excipient.
  • the monoclonal antibody comprises: VH CDRs 1-3 whose amino acid sequences are set forth in SEQ ID NOs: 1-3, respectively, and/or whose amino acid sequences are set forth in SEQ ID NOs: 4-6, respectively The VL CDR1-3 shown; preferably, the monoclonal antibody comprises: VH shown in SEQ ID NO:7 and/or VL shown in SEQ ID NO:8.
  • the present invention provides a method for neutralizing the virulence of a novel coronavirus in a sample, comprising contacting a sample comprising the novel coronavirus with a monoclonal antibody or antigen-binding fragment thereof of the present invention.
  • a sample comprising the novel coronavirus with a monoclonal antibody or antigen-binding fragment thereof of the present invention.
  • Such methods may be used for therapeutic purposes, or non-therapeutic purposes (eg, the sample is a cell sample rather than a patient or a sample from a patient).
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention for preparing a medicament for neutralizing the virulence of a novel coronavirus in a sample.
  • the present invention provides a monoclonal antibody or antigen-binding fragment thereof as described above for use in neutralizing the virulence of a novel coronavirus in a sample.
  • the present invention provides a monoclonal antibody or an antigen-binding fragment thereof as described above for use in preventing or treating a novel coronavirus infection in a subject or a disease associated with a novel coronavirus infection (e.g., novel coronavirus) viral pneumonia).
  • the present invention provides a method for preventing or treating a novel coronavirus infection or a novel coronavirus infection-related disease (eg, novel coronavirus pneumonia) in a subject, comprising, administering to a subject in need thereof or administering a prophylactically or therapeutically effective amount of the monoclonal antibody or antigen-binding fragment thereof of the present invention, or the pharmaceutical composition of the present invention.
  • a novel coronavirus infection or a novel coronavirus infection-related disease eg, novel coronavirus pneumonia
  • the subject is a mammal, such as a human.
  • the monoclonal antibody is an antibody comprising: VH CDRs 1-3 having amino acid sequences set forth in SEQ ID NOs: 1-3, respectively, and/or amino acid sequences set forth in SEQ ID NOs, respectively VL CDR1-3 shown in NO:4-6; preferably, it comprises: VH shown in SEQ ID NO:7 and/or VL shown in SEQ ID NO:8.
  • the medicaments and pharmaceutical compositions provided by the present invention can be used alone or in combination, or can be used in combination with other pharmaceutically active agents (eg, antiviral drugs, such as favipiravir, remdesivir, and interferon, etc.).
  • the pharmaceutical composition further contains a pharmaceutically acceptable carrier and/or excipient.
  • the monoclonal antibody of the present application (eg, BD23 antibody) can bind to the novel coronavirus S protein with high affinity, and has strong neutralizing activity against the novel coronavirus. Therefore, the monoclonal antibody (eg, BD23 antibody) of the present application has clinical application value for diagnosis, prevention and treatment of novel coronavirus infection.
  • Figure 1 shows the results of SDS-PAGE detection of recombinantly expressed BD23 antibody, wherein "NR” means non-reducing SDS-PAGE; “R” means reducing SDS-PAGE.
  • the results in Figure 1 show that under non-reducing SDS-PAGE conditions, a single band of about 190.88 KDa was formed; under reducing SDS-PAGE conditions, two bands of about 47.75 KDa and 25.70 KDa were formed (respectively Corresponding to the heavy and light chains of the antibody); and, the purity of the purified BD23 antibody was 97.7%.
  • FIG. 2 shows the results of the determination of the affinity of the BD23 antibody to the S protein using a microcalorimeter.
  • Figure 3 shows the measurement results of the neutralizing inhibitory activity of BD23 antibody against SARS-CoV-2 pseudovirus.
  • the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Refined Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995, was performed as described; restriction enzymes were used according to the conditions recommended by the product manufacturer. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer.
  • the reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7 (its encoding gene is shown in SEQ ID NO:17), wherein, the amino acid sequence of CDR1-3 of the heavy chain variable region is shown in SEQ ID NO:1 -3 shown (its encoding genes are shown in SEQ ID NOs: 11-13 respectively);
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO:8 (its encoding gene is shown in SEQ ID NO:18), wherein, the amino acid sequence of CDR1-3 of the light chain variable region is shown in SEQ ID NO:4 Shown in -6 (its encoding genes are shown in SEQ ID NOs: 14-16 respectively);
  • amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:9 (its encoding gene is shown in SEQ ID NO:19);
  • nucleic acid molecules encoding antibody heavy and light chains are synthesized in vitro and then cloned into expression vectors, respectively, resulting in recombinant expression vectors encoding antibody heavy and light chains, respectively.
  • HEK293 cells were co-transfected with the above-obtained recombinant expression vectors encoding antibody heavy and light chains, respectively.
  • the cell culture medium was changed to serum-free medium, and cultivation was continued at 37°C for 6 days.
  • the antibody protein expressed by the cells is purified from the culture by an affinity purification column.
  • the purified target protein was detected by reducing and non-reducing SDS-PAGE.
  • the results are shown in Figure 1.
  • the results in Figure 1 show that purified BD23 antibody was obtained with a purity of 97.7%.
  • the monoclonal antibody to be tested (irrelevant control antibody or BD23 antibody; the concentration is 0.1 ⁇ g/ml) was added and incubated. After washing with ELISA wash solution, horseradish peroxidase (HRP)-labeled Goat anti-human IgG Fc was added as a secondary antibody (diluted at 1:500), and the incubation was continued. Then, the ELISA plate was washed with PBST, and a chromogenic reagent was added to develop the color. Then read the absorbance at OD450nm on a microplate reader. The results are shown in Table 2. The results in Table 2 show that the BD23 antibody can specifically recognize and bind to the S protein RBD.
  • HRP horseradish peroxidase
  • a highly sensitive microcalorimetry molecular interaction analysis system is used to detect the binding ability of antibody BD23 and S protein.
  • the analysis system can directly quantitatively analyze the affinity of biomolecular interactions in solution simply, quickly and accurately. .
  • the recombinantly expressed S protein with His-tag (its amino acid sequence is shown in SEQ ID NO: 22) was labeled with Cy5 Fluorescent dyes. Briefly, the Cy5 fluorochrome was diluted to 100 nM using 1x PBS-T buffer. Then, 90 ⁇ L of His-tagged S protein (at a concentration of 200 nM) was mixed with 90 ⁇ L of diluted dye (100 nM) and incubated at room temperature for 30 minutes. Subsequently, the incubated samples were centrifuged at 15000 g for 10 minutes at 4°C. Collect the supernatant into a new tube for use.
  • the affinity of the BD23 antibody to the S protein was detected using a microcalorimeter (MONT.115PICO) according to the manufacturer's instructions. Specific steps are as follows:
  • the dilution method is as follows: prepare 16 PCR tubes, add 10 ⁇ l PBST buffer (PBS+0.005% Tween 20) to No. 2-16 PCR tubes; take 20 ⁇ l BD23 antibody (concentration of 1 ⁇ M) to No. 1 tube; then, from No. 1 tube Pipette 10 ⁇ l to the No. 2 tube and mix well; then, pipette 10 ⁇ l from the No. 2 tube to the No. 3 tube and mix well; operate sequentially, and finally take 10 ⁇ l of the mixed liquid from the No. 16 tube and discard.
  • Binding affinity mode measure the Kd value of the interaction between antibody BD23 and S protein in a microcalorimeter.
  • Example 5 Assessment of the ability of BD23 antibodies to neutralize the SARS-CoV-2 pseudovirus
  • the microporous cell neutralization assay was used to detect the effect of monoclonal antibody BD23 on SARS-CoV-2 false Virus neutralizing activity.
  • the SARS-CoV-2 pseudovirus used in this example was provided by the China National Institute for Food and Drug Control. It has similar cell infection characteristics to the true virus, can simulate the early process of the true virus infecting cells, and carries the reporter gene luciferase. Quick and easy detection analysis. The safety of the pseudovirus is high, and the neutralization experiment can be completed in the P2-level laboratory to detect the neutralization activity of the antibody (Neutralization titer).
  • the specific steps of the experimental method are as follows.
  • the reagents (0.25% trypsin-EDTA, DMEM complete medium) stored at 2-8°C were taken out and equilibrated at room temperature for more than 30 minutes.
  • the wells of A2-H2 are set as cell control wells (CC), which only contain experimental cells; the wells of A3-H3 are set as Virus control wells (VV) containing experimental cells and pseudovirus; wells for A4-A11, B4-B11, C4-C11, D4-D11, E4-E11, F4-F11, G4-G11, H4-H11 were set to Experimental wells, which contain experimental cells, pseudoviruses, and different concentrations of the antibody to be tested; the remaining wells are set as blank.
  • the experimental cells and pseudoviruses used in this example are Huh-7 cells and SARS-CoV-2 virus (both provided by the China Institute for Food and Drug Control).
  • the 96-well plate was placed in a cell culture incubator (37°C, 5% CO 2 ) and incubated for 1 hour.
  • Inhibition rate [1-(mean luminescence intensity of experimental wells-mean luminescence intensity of CC wells)/(mean luminescence intensity of VV wells-mean luminescence intensity of CC wells)] ⁇ 100%.
  • Example 6 Assessment of the ability of BD23 antibodies to neutralize SARS-CoV-2 true virus
  • the SARS-CoV-2 virus used was provided by the Military Medical Research Institute, and its titer (TCID50) was 10 5 /ml, and all experimental operations were completed in the BSL-3 laboratory.
  • TCID50 titer
  • Vero E6 cells were added to each well of a 96-well culture plate at a concentration of 5 ⁇ 10 4 /ml, and cultured at 37° C., 5% CO 2 for 24 hours.
  • step (3) After the culture in step (1), the cell culture medium in the 96-well culture plate was discarded, and the mixture (200 ⁇ l) containing the antibody to be tested and the true virus prepared in step (2) was added to serve as the experimental group. After 1 h of incubation, the supernatant was aspirated from the wells, and 200 ⁇ l of DMEM medium (containing 2% antibiotics and 16 ⁇ g/ml trypsin) was added to each well.
  • DMEM medium containing 2% antibiotics and 16 ⁇ g/ml trypsin
  • the cell control group and the virus control group were set in parallel.
  • 200 ⁇ l of DMEM medium (containing 2% antibiotics and 16 ⁇ g/ml trypsin) was added to each well.
  • 100 TCID50 of true virus 100 ⁇ l was added to each well, and incubate at 37°C for 1 h; after the incubation, aspirate the supernatant from the wells , 200 ⁇ l of DMEM medium (containing 2% antibiotics and 16 ⁇ g/ml trypsin) was added to each well.
  • CPE cytopathic effect
  • the experimental results are shown in Figure 4.
  • the results show that the monoclonal antibody BD23 has good neutralizing activity against the SARS-CoV-2 true virus, and can effectively inhibit virus infection and cell invasion (IC50 is 20.4 ⁇ g/ml). At a concentration of 50 ⁇ g/ml, the inhibition rate of mAb BD23 against SARS-CoV-2 true virus was approximately 70%.

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Abstract

提供了抗新型冠状病毒的单克隆抗体以及包含该抗体的组合物，该抗体可用于诊断、预防和/或治疗新型冠状病毒感染和/或由感染引起的疾病。

Description

一种抗新型冠状病毒的单克隆抗体及其应用 技术领域
本发明涉及免疫学领域和分子病毒学领域，特别是新型冠状病毒的诊断、预防和治疗领域。具体而言，本发明涉及抗新型冠状病毒的单克隆抗体，以及包含所述抗体的组合物(例如，诊断剂和治疗剂)。此外，本发明还涉及所述抗体的用途。本发明的抗体可用于诊断、预防和/或治疗新型冠状病毒的感染和/或由所述感染引起的疾病(例如，新型冠状病毒肺炎)。
背景技术
[根据细则26改正12.04.2021]　
新型冠状病毒SARS-CoV-2是导致新型冠状病毒肺炎(COVID-19)的病原体，是一种单链RNA病毒，它与2002-2003年引发疫情的重症急性呼吸综合征冠状病毒(SARS-CoV)以及2012年引发疫情的中东呼吸综合征冠状病毒(MERS-CoV)同属冠状病毒科。冠状病毒颗粒呈圆形或椭圆形，亦为多形性，直径50-200nm，属于尺寸较大的病毒。冠状病毒是包膜病毒，病毒衣壳外面包裹着脂质包膜，其上排列较宽的刺突蛋白(Spike,S蛋白)，形状如太阳光环。已有研究证实，S蛋白位 于新型冠状病毒SARS-CoV-2的病毒表面，其能够在病毒感染宿主的过程中通过其包含的受体结合结构域(RBD)结合宿主细胞受体血管紧张素转换酶2(ACE2)分子，从而启动病毒膜与宿主细胞膜发生融合，导致宿主细胞感染病毒。
迄今为止，中和抗体已被证明是治疗病毒性疾病的有效方法。一般来说，患者体内的B淋巴细胞受到抗原的刺激后，会被活化，进而转变和分化成多种不同的细胞，并产生抗体。据现有的研究报道，新型冠状病毒肺炎康复者的外周血内抗新型冠状病毒的抗体，它们由经活化的B细胞产生和分泌。然而，康复者血浆内存在着多种B细胞，并且不同的B细胞所产生的抗体的结合活性和中和效价也有所不同。到目前为止，尚无研究报道，具有高结合活性和/或高中和活性的抗新型冠状病毒的抗体。
因此，需要开发能够抗新型冠状病毒SARS-CoV-2的具有高结合活性和/或高中和活性的抗体，以提供有效的诊断、预防和/或治疗新型冠状病毒感染的手段。
发明内容
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒 定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab'、F(ab') 2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
在一些情况下，抗体的抗原结合片段是单链抗体(例如，scFv)，其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单 价分子(参见，例如,Bird等人,Science 242:423 426(1988)和Huston等人,Proc.Natl.Acad.Sci.USA 85:5879 5883(1988))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS) 4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
在一些情况下，抗体的抗原结合片段是双抗体，即，双价抗体，其中VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444 6448(1993)，和Poljak R.J.等人,Structure 2:1121 1123(1994))。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的单克隆抗体BD23)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即，除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单 克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Nature,256:495,1975)，但也可采用重组DNA技术获得(如参见Journal of virological methods,2009,158(1-2):171-179)。
如本文中所使用的，“中和抗体”是指，能清除或显著降低目标病毒的毒力(例如，感染细胞的能力)的抗体或抗体片段。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草芽孢杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK293细胞或人细胞等的动物细胞。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(KD)结合该抗原。
如本文中所使用的，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M的解离平衡常数(KD)结合抗原。例如，本发明的单克隆抗体BD23能够以大约10 -9M(nM水平)的解离平衡常数(KD)结合抗原(例如，新型冠状病毒的S蛋白)。
在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“中和活性”是指抗体或抗体片段具有与病毒上的抗原蛋白相结合，从而阻止病毒感染细胞和/或病毒子代的成熟和/或病毒子代的释放的功能活性，具有中和活性的抗体或抗体片段可以阻止病毒的扩增，从而抑制或消除病毒的感染。
[根据细则26改正12.04.2021]　
如本文中所使用的，术语“新型冠状病毒肺炎”和“COVID-19”是指，因新型冠状病毒感染而导致的肺炎，二者具有相同的含义，可互换使用。
本申请发明人经过大量的实验研究后发现了一种抗体，其能够特异性识别和靶向新型冠状病毒的S蛋白，特别是S蛋白的受体结合结构域(RBD)，并且显示出了高效的中和病毒的能力。因此，本发明的抗体特别适合用于诊断、预防和治疗新型冠状病毒感染或与新型冠状病毒感染相关的疾病(例如新型冠状病毒肺炎)。
在本申请的第一个方面，提供了一种单克隆抗体或其抗原结合片段，其包含，氨基酸序列分别如SEQ ID NO:1-3所示的重链可变区(VH)互补决定区1-3(CDR1-3)；和/或，氨基酸序列分别如SEQ ID NO:4-6所示的轻链可变区(VL)互补决定区1-3(CDR1-3)。
在某些优选的实施方案中，所述的单克隆抗体包括如SEQ ID NO:7所示的重链可变区(VH)。
在某些优选的实施方案中，所述的单克隆抗体包括如SEQ ID NO:8所示的轻链可变区(VL)。
在某些优选的实施方案中，所述的单克隆抗体包含：氨基酸序列分别如SEQ ID NO:1-3所示的VH CDR1-3，和氨基酸序列分别如SEQ ID NO:4-6所示的VL CDR1-3。
在某些优选的实施方案中，所述的单克隆抗体包括：如SEQ ID NO:7所示的VH和如SEQ ID NO:8所示的VL。
在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段选自Fab、Fab'、F(ab') 2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、人抗体、嵌合抗体或双特异或多特异抗体。
在某些优选的实施方案中，所述的单克隆抗体还包括重链恒定区。在某些优选的实施方案中，所述重链恒定区的氨基酸序列如SEQ ID NO:9所示。
在某些优选的实施方案中，所述的单克隆抗体还包括轻链恒定区。在某些优选的实施方案中，轻链恒定区的氨基酸序列如SEQ ID NO:10所示。
在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段能够特异性结合新型冠状病毒的刺突蛋白(S蛋白)。在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段能够靶向新型冠状病毒的刺突蛋白(S蛋白)的受体结合域(RBD)。在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段能够抑制S蛋白的受体结合域(RBD)介导的受体结合和/或膜融合过程，抑制病毒对细胞的感染。
在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段具有中和能力(例如，能够中和新型冠状病毒)。在某些优选的实施方案中，所述单克隆抗体或其抗原结合片段能够抑制新型冠状病毒感染或进入宿 主细胞。由此，所述单克隆抗体或其抗原结合片段能够中和新型冠状病毒，并由此预防和治疗新型冠状病毒的感染。
本申请还提供了分离的核酸分子，其编码本发明的单克隆抗体或其抗原结合片段。此类核酸分子不受限于其产生的方法，并且可以利用基因工程重组技术或化学合成方法获得。
因此，在另一个方面，本发明提供了分离的核酸分子，其包含能够编码抗体重链可变区的核苷酸序列，其中所述抗体重链可变区包含：氨基酸序列分别为SEQ ID NO:1-3的VH CDR1-3。
在某些优选的实施方案中，所述VH CDR1-3分别由SEQ ID NO:11-13所示的核苷酸序列编码。因此，在某些优选的实施方案中，所述分离的核酸分子包含SEQ ID NO:11-13所示的核苷酸序列。
在某些优选的实施方案中，所述抗体重链可变区具有如SEQ ID NO:7所示的氨基酸序列。
在某些优选的实施方案中，所述核酸分子具有如SEQ ID NO:17所示的核苷酸序列。
在另一个方面，本发明提供了分离的核酸分子，其包含能够编码抗体轻链可变区的核苷酸序列，其中所述抗体轻链可变区包含：氨基酸序列分别为SEQ ID NO:4-6的VL CDR1-3。
在某些优选的实施方案中，所述述VL CDR1-3分别由SEQ ID NO:14-16所示的核苷酸序列编码。因此，在某些优选的实施方案中，所述分离的核酸分子包含SEQ ID NO:14-16所示的核苷酸序列。
在某些优选的实施方案中，所述抗体轻链可变区具有如SEQ ID NO:8所示的氨基酸序列。
在某些优选的实施方案中，所述核酸分子具有如SEQ ID NO:18所示的核苷酸序列。
在另一个方面，本发明提供了分离的核酸分子，其包含如上文所定义的能够编码抗体重链可变区的核苷酸序列，以及如上文所定义的能够编码抗体轻链可变区的核苷酸序列。
在某些优选的实施方案中，所述抗体重链可变区具有如SEQ ID NO:7所示的氨基酸序列。在某些优选的实施方案中，所述能够编码抗体重链可变区的核苷酸序列具有如SEQ ID NO:17所示的核苷酸序列。
在某些优选的实施方案中，所述抗体轻链可变区包括如SEQ ID NO:8所示的氨基酸序列。在某些优选的实施方案中，所述能够编码抗体轻链可变区的核苷酸序列具有如SEQ ID NO:18所示的核苷酸序列。
在某些优选的实施方案中，所述分离的核酸分子包含如SEQ ID NO:17所示的核苷酸序列和如SEQ ID NO:18所示的核苷酸序列。
在某些优选的实施方案中，所述分离的核酸分子还包含，能够编码抗体重链恒定区的核苷酸序列。在某些优选的实施方案中，所述重链恒定区具有如SEQ ID NO:9所示的氨基酸序列。在某些优选的实施方案中，所述能够编码抗体重链恒定区的核苷酸序列具有如SEQ ID NO:19所示的核苷酸序列。
在某些优选的实施方案中，所述分离的核酸分子还包含，能够编码抗体轻链恒定区的核苷酸序列。在某些优选的实施方案中，所述轻链恒定区具有如SEQ ID NO:10所示的氨基酸序列。在某些优选的实施方案中，所述能够编码抗体轻链恒定区的核苷酸序列具有如SEQ ID NO:20所示的核苷酸序列。
在另一个方面，本发明提供了分离的核酸分子，其编码如上文所定义的本发明的单克隆抗体或其抗原结合片段。
在另一个方面，本发明提供了一种载体，其包含如上文所定义的分离的核酸分子。本发明的载体可以是克隆载体，也可以是表达载体。在某些优选的实施方案中，本发明的载体是例如质粒，粘粒，噬菌体等等。
在另一个方面，还提供了包含本发明的分离的核酸分子或载体的宿主细胞。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的细胞还可以是细胞系，例如HEK293细胞。
在另一个方面，还提供了制备本发明的单克隆抗体或其抗原结合片段的方法，其包括，在合适的条件下培养本发明的宿主细胞，和从细胞培养物中回收本发明的单克隆抗体或其抗原结合片段。
在另一个方面，本发明提供了一种组合物，其包含如上文所描述的单克隆抗体或其抗原结合片段、分离的核酸分子、载体或宿主细胞。
在另一个方面，本发明提供了一种试剂盒，其包括本发明的单克隆抗体或其抗原结合片段。在某些优选的实施方案中，本发明的单克隆抗体或其抗原结合片段还包括可检测的标记。在某些优选的实施方案中，所述试剂盒还包括第二抗体，其特异性识别本发明的单克隆抗体或其抗原结合片段。优选地，所述第二抗体还包括可检测的标记。此类可检测的标记是本领域技术人员熟知的，包括但不限于，放射性同位素，荧光物质，发光物质，有色物质和酶(例如辣根过氧化物酶)等。
在另一个方面，本发明提供了检测新型冠状病毒或其S蛋白或S蛋白的RBD在样品中的存在或其水平的方法，其包括，使用本发明的单克隆抗体或其抗原结合片段。在某些优选的实施方案中，本发明的单克隆抗体或其抗原结合片段还包括可检测的标记。在另一个优选的实施方案中，所述方法还包括，使用携带可检测的标记的第二抗体来检测本发明的单克隆抗体或其抗原结合片段。所述方法可以用于诊断目的(例如，所述样品是来自患者的样品)，或者非诊断目的(例如，所述样品是细胞样品，而非来自患者的样品)。
在另一个方面，本发明提供了诊断受试者是否感染了新型冠状病毒的方法，其包括：使用本发明的单克隆抗体或其抗原结合片段检测新型冠状病毒或其S蛋白或S蛋白的RBD在来自所述受试者的样品中的存在。在某些优选的实施方案中，本发明的单克隆抗体或其抗原结合片段还包括可检测的标记。在另一个优选的实施方案中，所述方法还包括，使用携带可检测的标记的第二抗体来检测本发明的单克隆抗体或其抗原结合片段。
在另一个方面，提供了本发明的单克隆抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测新型冠状病毒或其S蛋白或S蛋白的RBD在样品中的存在或其水平，或用于诊断受试者是否感染了新型冠状病毒。
在某些优选的实施方案中，所述样品包括但不限于来自受试者(例如哺乳动物，优选人)的排泄物、口腔或鼻腔分泌物、肺泡灌洗液等。
在某些优选的实施方案中，所述单克隆抗体是这样的抗体，其包括：氨基酸序列分别如SEQ ID NO:1-3所示的VH CDR1-3，和/或氨基酸序列分别如SEQ ID NO:4-6所示的VL CDR1-3；优选地，其包括：如SEQ ID NO:7所示的VH和/或如SEQ ID NO:8所示的VL。
使用抗体或其抗原结合片段来检测目标病毒或抗原(例如，新型冠状病毒或其S蛋白或S蛋白的RBD)在样品中的存在或其水平的一般方法是本领域技术人员所熟知的。在某些优选的实施方案中，所述检测方法可以使用酶联免疫吸附(ELISA)、酶免疫检测、化学发光免疫检测、放射免疫检测、荧光免疫检测、免疫色谱法、竞争法及类似检测方法。
在另一个方面，本发明提供了一种药物组合物，其包含本发明的单克隆抗体或其抗原结合片段，以及药学上可接受的载体和/或赋形剂。在某些优选的实施方案中，所述单克隆抗体包括：氨基酸序列分别如SEQ ID NO:1-3所示的VH CDR1-3，和/或氨基酸序列分别如SEQ ID NO:4-6所示的VL CDR1-3；优选地，所述单克隆抗体包括：如SEQ ID NO:7所示的VH和/或如SEQ ID NO:8所示的VL。
在另一个方面，本发明提供了用于中和样品中新型冠状病毒的毒力的方法，其包括，将包含新型冠状病毒的样品与本发明的单克隆抗体或其抗原结合片段接触。此类方法可以用于治疗目的，或非治疗目的(例如所述样品是细胞样品，而不是患者或来自患者的样品)。
在另一个方面，提供了本发明的单克隆抗体或其抗原结合片段用于制备药物的用途，所述药物用于中和样品中新型冠状病毒的毒力。在另一个方面，本发明提供了如上文所描述的单克隆抗体或其抗原结合片段，其用于中和样品中新型冠状病毒的毒力。
在另一个方面，提供了本发明的单克隆抗体或其抗原结合片段在制备药物组合物中的用途，所述药物组合物用于预防或治疗受试者的新型冠状病毒感染或与新型冠状病毒感染相关的疾病(例如新型冠状病毒肺炎)。在另一个方面，本发明提供了如上文所描述的单克隆抗体或其抗原结合片段，其用于预防或治疗受试者的新型冠状病毒感染或与新型冠状病毒感染相关的疾病(例如新型冠状病毒肺炎)。
在另一个方面，本发明提供了用于预防或治疗受试者的新型冠状病毒感染或新型冠状病毒感染相关的疾病(例如新型冠状病毒肺炎)的方法，其包括，给有此需要的受试者施用预防或治疗有效量的本发明的单克隆抗体或其抗原结合片段，或者本发明的药物组合物。
在某些优选的实施方案中，所述受试者是哺乳动物，例如人。
可通过任何适当的施用途径来将本发明的单克隆抗体或其抗原结合片段或者本发明的药物组合物施用给受试者。此类施用途径包括但不限于，口服、口腔、舌下、局部、肠胃外、直肠、叶鞘内、或鼻腔途径。
在某些优选的实施方案中，所述单克隆抗体是这样的抗体，其包括：氨基酸序列分别如SEQ ID NO:1-3所示的VH CDR1-3，和/或氨基酸序列分别如SEQ ID NO:4-6所示的VL CDR1-3；优选地，其包括：如SEQ ID NO:7所示的VH和/或如SEQ ID NO:8所示的VL。
本发明所提供的药物和药物组合物可以单独使用或联合使用，也可以与其他药学活性剂(例如抗病毒药物，如法匹拉韦、瑞德西韦和干扰素等)联合使用。在某些优选的实施方案中，所述药物组合物还含药学上可接受的载体和/或赋形剂。
序列信息
本申请所涉及的部分序列的信息如下面的表1所示。
表1.部分序列的信息
Figure PCTCN2021080537-appb-000001
Figure PCTCN2021080537-appb-000002
Figure PCTCN2021080537-appb-000003
有益效果
本申请的单克隆抗体(例如BD23抗体)能够以高亲和力与新型冠状病毒S蛋白结合，并且对新型冠状病毒具有很强的中和活性。因此，本申请的单克隆抗体(例如BD23抗体)具有诊断、预防和治疗新型冠状病毒感染的临床应用价值。
附图说明
图1显示了重组表达的BD23抗体的SDS-PAGE检测结果，其中，“NR”表示非还原性SDS-PAGE；“R”表示还原性SDS-PAGE。图1的结果显示，在非还原性SDS-PAGE条件下，形成了约190.88KDa的单一条带；在还原性SDS-PAGE条件下，形成了约47.75KDa和25.70KDa的两个条带(分别对应于抗体的重链和轻链)；并且，所纯化的BD23抗体的纯度为97.7％。
图2显示了使用微量热泳动仪检测BD23抗体与S蛋白的亲和力的测定结果。
图3显示了BD23抗体对SARS-CoV-2假病毒的中和抑制活性的测定结果。
图4显示了BD23抗体对SARS-CoV-2真病毒的中和抑制活性的测定结果。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪 器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：记忆B细胞的分离
采集感染SARS-CoV-2病毒且痊愈出院的人员的血液(由北京佑安医院提供)，并在P2+生物安全实验室中，利用STEMCELL SepMate TM-15(Stemcell Technologies，产品目录号：86415)进行PBMCs的提取。随后，根据制造商的说明书，使用STEMCELL EasySep Human Memory B Cell Isolation Kit(Stemcell Technologies，产品目录号：17864)对提取的PBMCs进行记忆B细胞的富集。
实施例2：抗体序列的获得与鉴定
根据制造商的说明书，使用Chromium Single Cell V(D)J Reagent Kits(购自10X genomics，产品目录号：100006)对上述富集后的记忆B细胞进行单细胞转录组的VDJ测序。对测序结果进行分析，获得一株抗体，命名为BD23。BD23抗体的序列信息如下：
重链可变区的氨基酸序列如SEQ ID NO:7所示(其编码基因如SEQ ID NO:17所示)，其中，重链可变区的CDR1-3的氨基酸序列如SEQ ID NO:1-3所示(其编码基因分别如SEQ ID NO:11-13所示)；
轻链可变区的氨基酸序列如SEQ ID NO:8所示(其编码基因如SEQ ID NO:18所示)，其中，轻链可变区的CDR1-3的氨基酸序列如SEQ ID NO:4-6所示(其编码基因分别如SEQ ID NO:14-16所示)；
重链恒定区的氨基酸序列如SEQ ID NO:9所示(其编码基因如SEQ ID NO:19所示)；
轻链恒定区的氨基酸序列如SEQ ID NO:10所示(其编码基因如SEQ ID NO:20所示)。
实施例3：抗体BD23的制备和纯化
根据实施例2中鉴定的BD23抗体的序列信息，委托北京义翘神州有限公司表达和纯化BD23抗体，并检测BD23抗体的抗原反应性。
简言之，在体外合成编码抗体重链和轻链的核酸分子，然后分别克隆至表达载体中，从而得到分别编码抗体重链和轻链的重组表达载体。将上述得到的分别编码抗体重链和轻链的重组表达载体共转染HEK293细胞。转染4-6小时后，将细胞培养液更换成无血清的培养基，并且继续在37℃下培养6天。培养结束后，通过亲和纯化柱从培养物中纯化细胞所表达的抗体蛋白。随后，通过还原性和非还原性SDS-PAGE检测所纯化的目的蛋白。结果如图1所示。图1的结果显示，获得了经纯化的BD23抗体，其纯度为97.7％。
随后，使用重组表达的S蛋白RBD作为包被抗原，使用辣根过氧化物酶(HRP)标记的Goat anti-human IgG Fc作为二抗，通过ELISA实验，检测经纯化的BD23抗体的抗原反应性。简言之，用重组表达的S蛋白RBD(其氨基酸序列如SEQ ID NO:21所示，浓度为0.01μg/ml或1μg/ml)包被96孔板，随后用封闭液对96孔板进行封闭。然后，分别加入待测单抗(无关对照抗体或BD23抗体；浓度为0.1μg/ml)，并孵育。用ELISA洗涤液进行洗涤后，加入辣根过氧化物酶(HRP)标记的Goat anti-human IgG Fc作为二抗(以1:500稀释)，并继续孵育。然后，用PBST洗涤酶标板，并加入显色剂显色。随后在酶标仪上读取OD450nm的吸收值。结果如表2所示。表2的结果显示，BD23抗体能够特异性识别并结合S蛋白RBD。
表2：通过ELISA检测的BD23抗体与S蛋白RBD的反应性(OD450读数)
Figure PCTCN2021080537-appb-000004
Figure PCTCN2021080537-appb-000005
实施例4：抗体BD23与S蛋白的结合能力的评估
本实施例采用高灵敏型微量热泳动分子互作分析系统，检测抗体BD23与S蛋白的结合能力，所述分析系统能够直接在溶液中简单、快速、精确地定量分析生物分子相互作用的亲和力。
(1)用Cy5荧光染料标记带有组氨酸标签(his-tag)的S蛋白
根据制造商的说明书，使用Monolith His-tag标记试剂盒(Cat#MO-L018)，将重组表达的带有His-tag的S蛋白(其氨基酸序列如SEQ ID NO:22所示)标记上Cy5荧光染料。简言之，使用1x PBS-T buffer，将Cy5荧光染料稀释至100nM。然后，取90μL带有His-tag的S蛋白(浓度为200nM)，与90μL经稀释的染料(100nM)混匀，并在室温孵育30分钟。随后，将经孵育的样品于4℃，以15000g离心10分钟。收集上清至新的管中，备用。
(2)检测抗体BD23与S蛋白结合的亲和力
根据制造商的说明书，使用微量热泳动仪(MO NT.115PICO)，检测BD23抗体与S蛋白的亲和力。具体步骤如下：
a.将抗体BD23连续倍比稀释(共计16个浓度)，稀释的起始浓度为1μM。稀释方式如下：准备16个PCR管，取10μl PBST buffer(PBS+0.005％Tween 20)加入2-16号PCR管；取20μl BD23抗体(浓度为1μM)至1号管；然后，从1号管移液10μl到2号管，混匀；然后，从2号管移液10μl到3号管，混匀；顺次操作，最后从16号管取10μl混匀液体，丢弃。
b.向每个PCR管(1-16号管)中加入10μl荧光分子(步骤(1)中制备的标记有Cy5荧光染料的S蛋白)，混匀。
c.室温放置5分钟，然后使用毛细管(cat#MO-K025)将样品加载至微量热泳动仪。
d.使用Binding affinity模式，在微量热泳动仪中测量抗体BD23 与S蛋白相互作用的Kd值。
测量结果如图2所示。结果显示，BD23抗体与S蛋白相互作用的Kd为4.344nM。这表明，BD23抗体与新型冠状病毒S蛋白具有极强的亲和力。
实施例5：BD23抗体中和SARS-CoV-2假病毒的能力的评估
在本实施例中，参照Temperton N J等人,Emerg Infect Dis,2005,11(3),411-416的描述，利用微孔细胞中和实验法，检测单抗BD23对SARS-CoV-2假病毒的中和活性。本实施例所应用的SARS-CoV-2假病毒由中国食品药品检定研究院提供，其具有与真病毒相似的细胞感染特点，能够模拟真病毒感染细胞的早期过程，并且携带报告基因luciferase,可以快速方便地进行检测分析。操作假病毒的安全性高，在P2级实验室内就可完成中和实验，检测抗体的中和活性(Neutralization titer)。实验方法的具体步骤如下。
1.平衡试剂
将保存于2-8℃的试剂(0.25％胰酶-EDTA，DMEM完全培养基)取出，室温平衡30分钟以上。
2.试验操作
(1)取96孔板，按照表3所示，设置样品的排布方式；其中，A2-H2的孔设置为细胞对照孔(CC)，其仅含有实验细胞；A3-H3的孔设置为病毒对照孔(VV)，其含有实验细胞和假病毒；A4-A11、B4-B11、C4-C11、D4-D11、E4-E11、F4-F11、G4-G11、H4-H11的孔设置为实验孔，其含有实验细胞、假病毒以及不同浓度的待测抗体；其余的孔设置为空白。本实施例中所使用的实验细胞和假病毒分别为Huh-7细胞和SARS-CoV-2病毒(均由中国食品药品检定研究院提供)。
表3. 96孔板中样品的排布方式
Figure PCTCN2021080537-appb-000006
Figure PCTCN2021080537-appb-000007
(2)在细胞对照孔中加入100μl/孔的DMEM完全培养基(含有1％的抗生素，25mM HEPES，10％FBS)；在病毒对照孔中加入100μl/孔的DMEM完全培养基；并且，在实验孔中加入50μl/孔的指定浓度的、稀释于DMEM完全培养基中的待测抗体。表3中所使用的稀释度1-8的抗体浓度分别为1/30μg/μl,1/90μg/μl,1/270μg/μl,1/810μg/μl,1/2430μg/μl,1/7290μg/μl,1/21870μg/μl,1/65610μg/μl。
(3)用DMEM完全培养基将SARS-CoV-2假病毒稀释至约1.3×10 4/ml(TCID50)；然后向病毒对照孔和实验孔中添加50μl/孔的SARS-CoV-2假病毒。
(4)将96孔板置于细胞培养箱中(37℃，5％CO 2)孵育1小时。
(5)用DMEM完全培养基将预先培养好的Huh-7细胞稀释至2×10 5个/ml。在前一步骤的孵育结束后，向细胞对照孔、病毒对照孔和实验孔中添加100μl/孔的细胞。
(6)将96孔板置于细胞培养箱中(37℃，5％CO 2)培养20-28小时。
(7)从细胞培养箱中取出96孔板，从每个孔中吸弃150μl上清，然后加入100μl荧光素酶检测试剂，室温避光反应2min。
(8)反应结束后，用移液器将各个孔中的液体反复吹吸6～8次，使细胞充分裂解。然后，从每孔中吸出150μl液体，转移至对应的96孔化学发光检测板中，用化学发光检测仪(Perkinelmer EnSight多功能酶标仪)读取发光值。
(9)计算中和抑制率：
抑制率＝[1－(实验孔的发光强度均值－CC孔的发光强度均值)/(VV孔的发光强度均值－CC孔的发光强度均值)]×100％。
(10)根据中和抑制率的结果，利用Reed-Muench法计算待测抗体的IC50。
实验结果如图3所示。结果显示，单抗BD23对SARS-CoV-2假病毒具有良好的中和活性，其IC50为8.78nM(即1.317μg/ml)。
实施例6：BD23抗体中和SARS-CoV-2真病毒的能力的评估
在本实施例中，所使用的SARS-CoV-2病毒由军事医学研究院提供，其滴度(TCID50)为10 5/ml，并且，所有实验操作均在BSL-3实验室内完成。中和实验方法的具体步骤如下。
(1)以5×10 4/ml的浓度，向96孔培养板的每孔中加入100μl Vero E6细胞，并在37℃，5％CO 2的条件下培养24小时。
(2)将待测抗体稀释成3个浓度：为50μg/ml；10μg/ml；2μg/ml。取100μl指定浓度的待测抗体，加入等体积的SARS-CoV-2真病毒(100TCID50)，并在37℃，5％CO 2的条件下孵育1h。
(3)在步骤(1)的培养结束后，弃去96孔培养板中的细胞培养液，加入步骤(2)制备的含有待测抗体和真病毒的混合液(200μl)，作为实验组。孵育1h后，从孔中吸出上清，每孔加入200μl DMEM培养基(含有2％抗生素和16μg/ml胰蛋白酶)。
在实验过程中，平行设置细胞对照组和病毒对照组。在细胞对照组(4个复孔)中，弃去孔中的细胞培养液后，每孔添加200μl DMEM培养基(含有2％抗生素和16μg/ml胰蛋白酶)。在病毒对照组(4个复孔)中，弃去孔中的细胞培养液后，每孔添加100TCID50的真病毒(100μl)，并在37℃孵育1h；孵育结束后，从孔中吸出上清，每孔加入200μl DMEM培养基(含有2％抗生素和16μg/ml胰蛋白酶)。
(4)在37℃，5％CO 2的条件下培养细胞4-5天。
(5)在光学显微镜下观察细胞病变(CPE)，并根据细胞病变情况， 评估不同浓度的单抗BD23对CPE的抑制活性。
实验结果如图4所示。结果显示，单抗BD23对SARS-CoV-2真病毒具有良好的中和活性，能够有效抑制病毒感染和侵入细胞(IC50为20.4μg/ml)。在50μg/ml的浓度下，单抗BD23对SARS-CoV-2真病毒的抑制率为大约70％。

Claims (14)

  1. 一种单克隆抗体或其抗原结合片段，其包含，氨基酸序列分别如SEQ ID NO：1-3所示的重链可变区(VH)互补决定区1-3(CDR1-3)；和/或，氨基酸序列分别如SEQ ID NO：4-6所示的轻链可变区(VL)互补决定区1-3(CDR1-3)；
    优选地，所述的单克隆抗体包括，如SEQ ID NO：7所示的重链可变区(VH)，和/或，如SEQ ID NO：8所示的轻链可变区(VL)；
    优选地，所述的单克隆抗体包含：氨基酸序列分别如SEQ ID NO：1-3所示的VH CDR1-3，和氨基酸序列分别如SEQ ID NO：4-6所示的VL CDR1-3；
    优选地，所述的单克隆抗体包括：如SEQ ID NO：7所示的VH和如SEQ ID NO：8所示的VL；
    优选地，所述单克隆抗体或其抗原结合片段选自Fab、Fab′、F(ab′) 2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、人抗体、嵌合抗体或双特异或多特异抗体；
    优选地，所述的单克隆抗体还包括重链恒定区；优选地，所述重链恒定区的氨基酸序列如SEQ ID NO：9所示；
    优选地，所述的单克隆抗体还包括轻链恒定区；优选地，所述轻链恒定区的氨基酸序列如SEQ ID NO：10所示。
  2. 分离的核酸分子，其包含能够编码抗体重链可变区的核酸序列，其中，所述抗体重链可变区包含：氨基酸序列分别为SEQ ID NO：1-3的VH CDR1-3；
    例如，所述分离的核酸分子包含SEQ ID NO：11-13所示的核苷酸序列；
    例如，所述抗体重链可变区具有如SEQ ID NO：7所示的氨基酸序 列；
    例如，所述核酸分子具有如SEQ ID NO：17所示的核苷酸序列。
  3. 分离的核酸分子，其包含能够编码抗体轻链可变区的核酸序列，其中所述抗体轻链可变区包含：氨基酸序列分别为SEQ ID NO：4-6的VL CDR1-3；
    例如，所述分离的核酸分子包含SEQ ID NO：14-16所示的核苷酸序列；
    例如，所述抗体轻链可变区具有如SEQ ID NO：8所示的氨基酸序列；
    例如，所述核酸分子具有如SEQ ID NO：18所示的核苷酸序列。
  4. 分离的核酸分子，其编码权利要求1的单克隆抗体或其抗原结合片段。
  5. 一种载体，其包含权利要求2-4任一项的分离的核酸分子。
  6. 一种宿主细胞，其包含权利要求2-4任一项的分离的核酸分子或权利要求5的载体。
  7. 制备权利要求1的单克隆抗体或其抗原结合片段的方法，其包括，在合适的条件下培养权利要求6的宿主细胞，和从细胞培养物中回收所述单克隆抗体或其抗原结合片段。
  8. 一种组合物，其包含权利要求1的单克隆抗体或其抗原结合片段，权利要求2-4任一项的分离的核酸分子，权利要求5的载体，权利要求6的宿主细胞。
  9. 试剂盒，其包括权利要求1的单克隆抗体或其抗原结合片段；
    例如，所述单克隆抗体或其抗原结合片段还包括可检测的标记，例如放射性同位素，荧光物质，发光物质，有色物质和酶；
    例如，所述试剂盒还包括第二抗体，其特异性识别所述单克隆抗体或其抗原结合片段；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素，荧光物质，发光物质，有色物质和酶。
  10. 用于检测新型冠状病毒或其S蛋白或S蛋白的RBD在样品中的存在或其水平的方法，其包括使用权利要求1的单克隆抗体或其抗原结合片段；
    例如，所述单克隆抗体或其抗原结合片段还包括可检测的标记，例如放射性同位素，荧光物质，化学发光物质，有色物质和酶；
    例如，所述方法还包括，使用携带可检测的标记(例如放射性同位素，荧光物质，发光物质，有色物质和酶)的第二抗体来检测所述单克隆抗体或其抗原结合片段。
  11. 权利要求1的单克隆抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测新型冠状病毒或其S蛋白或S蛋白的RBD在样品中的存在或其水平，或用于诊断受试者是否感染了新型冠状病毒；
    优选地，所述样品为来自受试者(例如哺乳动物，优选人)的排泄物、口腔或鼻腔分泌物、或肺泡灌洗液。
  12. 一种药物组合物，其包含权利要求1的单克隆抗体或其抗原结合片段，以及药学上可接受的载体和/或赋形剂；
    优选地，所述药物组合物还包含其他药学活性剂，例如法匹拉韦，瑞德西韦，干扰素等。
  13. 用于中和样品中的新型冠状病毒的毒力的方法，其包括，将包含新型冠状病毒的样品与权利要求1的单克隆抗体或其抗原结合片段接触。
  14. 权利要求1的单克隆抗体或其抗原结合片段用于制备药物的用途，所述药物用于中和样品中新型冠状病毒的毒力，或用于预防或治疗受试者的新型冠状病毒感染或与新型冠状病毒感染相关的疾病(例如新型冠状病毒肺炎)；
    优选地，所述受试者是哺乳动物，例如人；
    优选地，所述药物单独使用，或与其他药学活性剂(例如法匹拉韦，瑞德西韦，干扰素等)联合使用。
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