WO2021174595A1 - 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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WO2021174595A1
WO2021174595A1 PCT/CN2020/079737 CN2020079737W WO2021174595A1 WO 2021174595 A1 WO2021174595 A1 WO 2021174595A1 CN 2020079737 W CN2020079737 W CN 2020079737W WO 2021174595 A1 WO2021174595 A1 WO 2021174595A1
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antibody
monoclonal antibody
seq
antigen
binding fragment
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French (fr)
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刘磊
刘映霞
沈晨光
吴燕
谭曙光
李德林
王非然
高福
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深圳市第三人民医院
中国科学院微生物研究所
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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
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—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
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of immunology and molecular virology, especially the field of diagnosis, prevention and treatment of new coronaviruses.
  • the present invention relates to monoclonal antibodies against the novel coronavirus, and compositions containing the antibodies (for example, diagnostic agents and therapeutic agents).
  • the present invention also relates to the use of the antibody.
  • the antibody of the present invention can be used to diagnose, prevent and/or treat a novel coronavirus infection and/or diseases caused by the infection (for example, novel coronavirus pneumonia).
  • the new type of coronavirus 2019-nCoV is the pathogen that causes the new type of coronavirus pneumonia (COVID-19). It is a single-stranded RNA virus. It is the same as the severe acute respiratory syndrome coronavirus (SARS-CoV) and The Middle East respiratory syndrome coronavirus (MERS-CoV) that caused the outbreak in 2012 belongs to the coronavirus family.
  • the spike protein (Spike, S protein) on the surface of the virus binds to the host cell receptor angiotensin-converting enzyme 2 (ACE2) molecule in the process of infecting the host, thereby initiating the fusion of the viral membrane with the host cell membrane, causing the host cell to be infected with the virus .
  • the S protein is divided into two parts, S1 and S2. Studies have confirmed that the receptor binding domain (RBD) of the C-terminal (CTD) of S1 binds to ACE2 and mediates the process of membrane fusion.
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair has a "light” (L) chain and a “heavy” (H) chain).
  • Antibody light chains can be classified into kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is composed of 3 domains (CH1, CH2, and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of a domain CL.
  • the constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (for example, effector cells) and the first component (C1q) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions with hyperdenaturation (called complementarity determining regions (CDR)), interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL is composed of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antibody binding site.
  • the allocation of amino acids to each region or domain follows 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 of Chothia et al. (1989) Nature 342:878-883.
  • antibody is not limited by any specific method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, 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 the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody It is also called “antigen binding part” for specific binding to antigen. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Recombinant DNA technology can be used. Or through the enzymatic or chemical cleavage of intact antibodies to produce antigen-binding fragments of antibodies.
  • antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining regions (CDR) Fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and such polypeptides, which comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • CDR complementarity determining regions
  • the antigen-binding fragment of the antibody is a single-chain antibody (e.g., scFv), in which the VL and VH domains pair to form a monovalent molecule by pairing a linker that enables it to be produced as a single polypeptide chain (see, e.g., Bird et al. , Science 242: 423 426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879 5883 (1988)).
  • scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having an amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448).
  • Other linkers that can be used 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, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the antigen-binding fragments of antibodies are diabodies, ie, diabodies, 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 two structures in the same chain Pairing between the domains, thereby forcing the domain to pair with the complementary domain of the other chain and creating two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444 6448 ( 1993), and Poljak RJ et al., Structure 2:1112 1123 (1994)).
  • antigen-binding fragments e.g., The above-mentioned antibody fragments
  • antigen-binding fragments of antibodies are specifically screened in the same manner as used for intact antibodies.
  • antibody includes not only intact antibodies but also antigen-binding fragments of antibodies.
  • the term "monoclonal antibody” refers to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibodies, except for natural mutations that may occur spontaneously. Antibody molecule.
  • the monoclonal antibody has high specificity for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which usually include at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the 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 (for example, see Journal of virological methods, 2009, 158(1-2): 171-179).
  • neutralizing antibody refers to an antibody or antibody fragment that can eliminate or significantly reduce the virulence (for example, the ability to infect cells) of the target virus.
  • E. coli expression system refers to an expression system composed of E. coli (strain) and a vector, wherein E. coli (strain) is derived from a strain available on the market, such as but not limited to: GI698 , ER2566, BL21 (DE3), B834 (DE3), BLR (DE3).
  • the term "vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; 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, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillary viruses.
  • Polyoma vacuole virus (such as SV40).
  • a vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, and fungal cells such as yeast cells or Aspergillus , Such as 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 Escherichia 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.
  • the term "specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed.
  • an antibody that specifically binds to a certain antigen means that the antibody has a concentration of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, The affinity (KD) of 10 -8 M, 10 -9 M, or 10 -10 M or less binds to the antigen.
  • KD refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • the antibody for example, the monoclonal antibody H4 of the present invention
  • the smaller dissociation equilibrium constant (KD) binds to the antigen (for example, the RBD of the new coronavirus S protein), for example, as measured in a BIACORE instrument using surface plasmon resonance (SPR).
  • amino acids are usually 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 the antigen protein on the virus, thereby preventing the virus from infecting cells and/or the maturation of the virus progeny and/or the release of the virus progeny Antibodies or antibody fragments with neutralizing activity can prevent the amplification of the virus, thereby inhibiting or eliminating the infection of the virus.
  • new coronavirus pneumonia and “COVID-19” refer to pneumonia caused by a new coronavirus infection. The two have the same meaning and can be used interchangeably.
  • the inventor 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 can block the S protein.
  • RBD receptor binding domain
  • ACE2 cell receptor angiotensin-converting enzyme 2
  • the antibody of the present invention is particularly suitable for diagnosing, preventing and treating novel coronavirus infection or diseases related to novel coronavirus infection (for example, novel coronavirus pneumonia).
  • a monoclonal antibody or an antigen-binding fragment thereof which comprises the variable heavy region (VH) complementarity determining region of the amino acid sequence shown in SEQ ID NO: 1-3, respectively 1-3 (CDR1-3); and/or, the amino acid sequence of the light chain variable region (VL) complementarity determining region 1-3 (CDR1-3) shown in SEQ ID NO: 4-6, respectively.
  • VH variable heavy region
  • VL light chain variable region
  • the monoclonal antibody includes a heavy chain variable region (VH) as shown in SEQ ID NO:7.
  • VH heavy chain variable region
  • the monoclonal antibody includes a light chain variable region (VL) as shown in SEQ ID NO: 8.
  • VL light chain variable region
  • the monoclonal antibody comprises: the amino acid sequence is shown in SEQ ID NO: 1-3, VH CDR1-3, and the amino acid sequence is shown in SEQ ID NO: 4-6, respectively VL CDR1-3.
  • the monoclonal antibody includes: VH as shown in SEQ ID NO: 7 and VL as shown in SEQ ID NO: 8.
  • the monoclonal antibody also has a leader sequence at the N-terminus of the variable region of the heavy chain.
  • the leader sequence has an amino acid sequence as shown in SEQ ID NO: 11.
  • the monoclonal antibody also has a leader sequence at the N-terminus of the variable region of the light chain.
  • the leader sequence has an amino acid sequence as shown in SEQ ID NO: 11.
  • 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 (e.g., scFv), human antibody, chimeric antibody or bispecific or multispecific antibody.
  • the monoclonal antibody further includes a heavy chain constant region.
  • the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 9.
  • the monoclonal antibody further includes a light chain constant region.
  • the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 10.
  • the light chain of the monoclonal antibody is of ⁇ type.
  • the monoclonal antibody or antigen-binding fragment thereof can specifically bind to the spike protein (S protein) of the novel coronavirus. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof can target the receptor binding domain (RBD) of the spike protein (S protein) of the novel coronavirus. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof can inhibit the receptor binding and/or membrane fusion process mediated by the receptor binding domain (RBD) of the S protein, and inhibit virus infection of cells .
  • RBD receptor binding domain
  • the monoclonal antibody or antigen-binding fragment thereof has a neutralizing ability (for example, it can neutralize a novel coronavirus). In some preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof can inhibit the infection or entry of the new coronavirus into host cells. Thus, the monoclonal antibody or its antigen-binding fragment can neutralize the novel coronavirus, and thereby prevent and treat the infection of the novel coronavirus.
  • the application also provides an isolated nucleic acid molecule, which encodes the monoclonal antibody or antigen-binding fragment thereof of the present invention.
  • nucleic acid molecules are not limited to the method of their production, and can be obtained using genetic engineering recombination 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 sequence is SEQ ID NO: VH CDR1-3 of 1-3.
  • the antibody heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:7.
  • the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 12.
  • the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence capable of encoding an antibody light chain variable region, wherein the antibody light chain variable region comprises: the amino acid sequence is SEQ ID NO: 4- 6 VL CDR1-3.
  • the antibody light chain variable region has an amino acid sequence as shown in SEQ ID NO: 8.
  • the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 13.
  • the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence capable of encoding a variable region of an antibody heavy chain as defined above, and a nucleotide sequence capable of encoding a variable region of an antibody light chain as defined above. Nucleotide sequence.
  • the antibody heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:7.
  • the nucleotide sequence capable of encoding the variable region of the antibody heavy chain has the nucleotide sequence shown in SEQ ID NO: 12.
  • the nucleic acid molecule further comprises a nucleotide sequence encoding a leader sequence, which is located at the 5'end of the nucleotide sequence capable of encoding the variable region of the antibody heavy chain.
  • the leader sequence has an amino acid sequence as shown in SEQ ID NO: 11.
  • the nucleotide sequence encoding the leader sequence has the nucleotide sequence shown in SEQ ID NO: 16.
  • the antibody light chain variable region includes the amino acid sequence shown in SEQ ID NO: 8.
  • the nucleotide sequence capable of encoding the variable region of the antibody light chain has the nucleotide sequence shown in SEQ ID NO: 13.
  • the nucleic acid molecule further comprises a nucleotide sequence encoding a leader sequence, which is located at the 5'end of the nucleotide sequence capable of encoding the variable region of the antibody light chain.
  • the leader sequence has an amino acid sequence as shown in SEQ ID NO: 11.
  • the nucleotide sequence encoding the leader sequence has the nucleotide sequence shown in SEQ ID NO: 16.
  • the isolated nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 12 and the nucleotide sequence shown in SEQ ID NO: 13.
  • the isolated nucleic acid molecule comprises a first polynucleotide comprising a nucleotide sequence encoding a leader sequence and a nucleotide sequence capable of encoding the variable region of an antibody heavy chain; and,
  • the second polynucleotide includes a nucleotide sequence encoding a leader sequence and a nucleotide sequence capable of encoding the variable region of the antibody light chain.
  • the isolated nucleic acid molecule comprises a first polynucleotide, which comprises the nucleotide sequence shown in SEQ ID NO: 16 and the nucleoside shown in SEQ ID NO: 12 Acid sequence; and, the second polynucleotide, which comprises the nucleotide sequence shown in SEQ ID NO: 16 and the nucleotide sequence shown in SEQ ID NO: 13.
  • the isolated nucleic acid molecule further comprises a nucleotide sequence capable of encoding the constant region of the antibody heavy chain.
  • the heavy chain constant region has an amino acid sequence as shown in SEQ ID NO:9.
  • the nucleotide sequence capable of encoding the constant region of the antibody heavy chain has the nucleotide sequence shown in SEQ ID NO: 14.
  • the isolated nucleic acid molecule further comprises a nucleotide sequence capable of encoding the constant region of the antibody light chain.
  • the light chain constant region has an amino acid sequence as shown in SEQ ID NO: 10.
  • the nucleotide sequence capable of encoding the constant region of the antibody light chain has the nucleotide sequence shown in SEQ ID NO: 15.
  • the isolated nucleic acid molecule comprises a first polynucleotide, which comprises a nucleotide sequence encoding a leader sequence, a nucleotide sequence capable of encoding the variable region of an antibody heavy chain, and a nucleotide sequence capable of encoding The nucleotide sequence of the constant region of the antibody heavy chain; and, the second polynucleotide comprising a nucleotide sequence encoding a leader sequence, a nucleotide sequence capable of encoding the variable region of the antibody light chain, and a nucleotide sequence capable of encoding the antibody light chain The nucleotide sequence of the constant region.
  • the isolated nucleic acid molecule comprises a first polynucleotide, which comprises the nucleotide sequence shown in SEQ ID NO: 16, SEQ ID NO: 12 and SEQ ID NO: 14 ; And, the second polynucleotide, which comprises the nucleotide sequence shown in SEQ ID NO: 16, SEQ ID NO: 13 and SEQ ID NO: 15.
  • the present invention provides an isolated nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention as defined above.
  • the invention provides a vector comprising an isolated nucleic acid molecule as defined above.
  • the vector of the present invention can be a cloning vector or an expression vector.
  • the vectors of the present invention are, for example, plasmids, cosmids, bacteriophages and the like.
  • a host cell comprising the isolated nucleic acid molecule or vector of the present invention.
  • 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 (such as mammalian cells, such as mouse cells, human cells, etc.).
  • the cells of the present invention can also be cell lines, such as 293T cells.
  • a method for preparing the monoclonal antibody of the present invention or an antigen-binding fragment thereof which comprises culturing the 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 the monoclonal antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector or 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 antibody or antigen-binding fragment thereof of the present invention further includes a detectable label.
  • the kit further includes a second antibody, which specifically recognizes the monoclonal antibody or antigen-binding fragment thereof of the present invention.
  • the second antibody further includes 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, enzymes (such as horseradish peroxidase) and the like.
  • the present invention provides a method for detecting the presence or level of a novel coronavirus or its S protein or S protein RBD in a sample, which includes using the monoclonal antibody or antigen-binding fragment thereof of the present invention.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention further includes a detectable label.
  • the method further includes using a second antibody carrying a detectable label to detect the monoclonal antibody or antigen-binding fragment thereof of the present invention.
  • the method can be used for diagnostic purposes (for example, the sample is a sample from a patient), or for non-diagnostic purposes (for example, the sample is a cell sample, not a sample from a patient).
  • the present invention provides a method for diagnosing whether a subject is infected with a novel coronavirus, which comprises: using the monoclonal antibody or antigen-binding fragment thereof of the present invention to detect the RBD of the novel coronavirus or its S protein or S protein Presence in a sample from the subject.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention further includes a detectable label.
  • the method further includes using a second antibody carrying a detectable label to detect the monoclonal antibody or antigen-binding fragment or anti-idiotypic antibody of the present invention.
  • the sample includes, but is not limited to, excrement, oral or nasal secretions, alveolar lavage fluid, etc. from a subject (such as a mammal, preferably a human).
  • the monoclonal antibody is an antibody comprising: the amino acid sequence of the VH CDR1-3 shown in SEQ ID NO: 1-3, and/or the amino acid sequence of the VH CDR1-3 shown in SEQ ID, respectively VL CDR1-3 shown in NO: 4-6; preferably, it includes: VH shown in SEQ ID NO: 7 and/or VL shown in SEQ ID NO: 8.
  • the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescence immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, competition method and similar detection methods .
  • 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 includes: the amino acid sequence of the VH CDR1-3 shown in SEQ ID NO: 1-3, and/or the amino acid sequence of the VH CDR1-3 shown in SEQ ID NO: 4-6, respectively
  • the monoclonal antibody includes: VH as shown in SEQ ID NO: 7 and/or VL as shown in SEQ ID NO: 8.
  • the present invention provides a method for neutralizing the virulence of a novel coronavirus in a sample, which comprises contacting a sample containing the novel coronavirus with the monoclonal antibody or antigen-binding fragment thereof of the present invention.
  • a sample is a cell sample, not a patient or a sample from a patient.
  • the present invention provides the monoclonal antibody or antigen-binding fragment thereof as described above, which is used to neutralize the virulence of the novel coronavirus in a sample.
  • the present invention provides the monoclonal antibody or antigen-binding fragment thereof as described above, which is used to prevent or treat a novel coronavirus infection in a subject or a disease related to a novel coronavirus infection (such as a novel coronavirus infection). Viral pneumonia).
  • the present invention provides a method for preventing or treating a novel coronavirus infection or a novel coronavirus infection-related disease (such as novel coronavirus pneumonia) in a subject, which includes, giving a subject in need The person administers a prophylactic 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.
  • the subject is a mammal, such as a human.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention can be administered to a subject by any appropriate route of administration.
  • administration routes include, but are not limited to, oral, buccal, sublingual, topical, parenteral, rectal, intrathecal, or nasal routes.
  • the monoclonal antibody is an antibody comprising: the amino acid sequence of the VH CDR1-3 shown in SEQ ID NO: 1-3, and/or the amino acid sequence of the VH CDR1-3 shown in SEQ ID, respectively VL CDR1-3 shown in NO: 4-6; preferably, it includes: VH shown in SEQ ID NO: 7 and/or VL shown in SEQ ID NO: 8.
  • the drugs and pharmaceutical compositions provided by the present invention can be used alone or in combination, and can also be used in combination with other pharmacologically active agents (for example, antiviral drugs, such as fapilavir, remdesivir, interferon, etc.).
  • the pharmaceutical composition further contains a pharmaceutically acceptable carrier and/or excipient.
  • the monoclonal antibody (such as the H4 antibody) of the present application can bind to the new coronavirus S protein RBD with high affinity, and has strong neutralizing activity against the new coronavirus.
  • the affinity of the H4 antibody of the present invention to RBD is 4.48 nM
  • the neutralization titer (half inhibitory concentration, IC 50 ) of the novel coronavirus is 0.896 ⁇ g/mL. Therefore, the monoclonal antibody (such as H4 antibody) of the present application has clinical application value for preventing and treating novel coronavirus infection.
  • Figure 1 shows the results of molecular sieve chromatography and SDS-PAGE detection of the new coronavirus S protein RBD.
  • Figure 2 shows the molecular sieve chromatography results and SDS-PAGE detection results of the recombinantly expressed H4 antibody.
  • the "-" on the gel chart indicates that DTT (non-reducing SDS-PAGE) is not added; “+” indicates that DTT (reduced SDS-PAGE).
  • Figure 3 shows the results of the kinetic curve of H4 antibody binding to RBD protein.
  • Figure 4 shows the cell surface fluorescence detected by BD FACSCanto in Example 6.
  • Figure 5 shows the neutralizing activity of different concentrations of H4 antibody against 2019-nCoV live virus.
  • the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Compiled Molecular Biology Experiment Guide, 3rd Edition, John Wiley & Sons, Inc., 1995; the restriction enzymes are used in accordance with the conditions recommended by the product manufacturer. If no specific conditions are indicated in the examples, it shall be carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer.
  • the reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased commercially.
  • the inventors of the present application first used the S protein RBD of 2019-nCoV expressed in E. coli as the antigen, and through flow sorting, from the periphery of people who were infected with 2019-nCoV and recovered and discharged. From blood mononuclear cells (PBMCs), memory B cells that can specifically bind to the S protein RBD are screened, and then RT-PCR is performed on the single B cells obtained from the screening to obtain the sequence encoding the variable region of the antibody. Furthermore, the sequence encoding the variable region of the antibody and the constant region gene are linked to an expression vector, and expressed and purified in mammalian cells, thereby obtaining antibody H4.
  • PBMCs blood mononuclear cells
  • antibody H4 can specifically bind to S protein RBD, block the binding of S protein RBD to ACE2, inhibit the infection of human cells by 2019-nCoV, and has anti-2019-nCoV infection. Neutralizing activity.
  • the DNA fragment encoding the 2019-nCoV/2019 strain spike protein S protein RBD (the amino acid sequence of which is shown in SEQ ID NO: 17) was ligated to the pET21a vector, and the DNA fragment was in the coding region A nucleotide sequence encoding a 6*histidine tag (6*His tag) and a stop codon are also attached to the 3'end of the.
  • IPTG was added to the culture to a final concentration of 1 mM, and the culture was continued at 37 degrees Celsius for 4-6 hours. After the cultivation, the inclusion bodies were harvested and renatured. The refolded protein solution was concentrated and dialyzed into 20mM Tris, 150mM NaCl, pH9.0 buffer.
  • the protein in the solution was purified by molecular sieve chromatography, in which AKTA-purifier (GE) and superdex200Hiload 16/60 column (GE) and buffer A (20mM Tris, 150mM NaCl, pH9.0) were used, and in the purification During the process, the UV absorption value at 280nm was monitored at the same time, and the fraction containing the target protein was collected. After purification, the purity of the target protein (RBD of S protein) was identified by SDS-PAGE. The result is shown in Figure 1. The results in Figure 1 show that a high-purity RBD protein with a size of 32 kDa was obtained.
  • Example 2 Isolation of memory B cells that specifically recognize RBD protein
  • PBMCs were washed twice with PBS. Subsequently, PBMCs were sorted with FACSAria III, PE - Cy5 - APC - APC - Cy7 + Pacific Blue + FITC + PE + cells (ie, B cells) were collected, and directly collected into a 96-well plate, 1 cell/well.
  • Example 3 Isolation and identification of H4 antibody and construction of recombinant expression vector
  • Superscript III reverse transcriptase (Invitrogen) was used to perform reverse transcription on the B cells obtained in Example 2 (at 55° C. for 60 minutes), wherein the reverse transcription primers used are shown in Table 2.
  • PCRa PCRa
  • HotStar Tap Plus enzyme QIAgen
  • the reaction conditions used As follows: 95°C, 5min; 35 cycles (95°C 30s, 55°C (heavy chain/ ⁇ chain) 30s, 72°C 90s); 72°C, 7min.
  • PCRb the second round of PCR
  • the primers used are shown in Table 4; the reaction conditions used are as follows: 95°C, 5min; 35 cycles of (95°C) 30s, 58°C (heavy chain)/60°C ( ⁇ chain)/64°C ( ⁇ chain) 30s, 72°C (90s); 72°C, 7min.
  • the PCR products were separated by 1% agarose gel electrophoresis.
  • the PCR product with a band size of 400-500bp was recovered and sent to a sequencing company for sequencing.
  • the sequencing results were analyzed with NCBI online software.
  • H4 antibody strain The amino acid sequence of the heavy chain variable region of the H4 antibody is shown in SEQ ID NO: 7 (the coding gene is shown in SEQ ID NO: 12), and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8 (coding The gene is shown in SEQ ID NO: 13).
  • SEQ ID NO: 7 The amino acid sequence of the heavy chain variable region of the H4 antibody is shown in SEQ ID NO: 12
  • SEQ ID NO: 8 coding The gene is shown in SEQ ID NO: 13
  • sequence identity between H4 antibody and germline genes is shown in Table 5-6 below.
  • the nucleotide sequence encoding the heavy chain/light chain variable region obtained by the analysis was connected to the corresponding nucleotide sequence encoding the heavy chain/ ⁇ chain constant region by bridging PCR, and then cloned into the expression vector pCAGGS( Purchased from Addgene) to obtain recombinant expression vectors encoding antibody heavy and light chains, respectively.
  • the construction scheme of the construct expressing the heavy chain and the light chain is as follows:
  • Heavy chain coding sequence (5’-3’): CMV promoter-EcoR I restriction site-Leader sequence gene-VH gene-CH gene-Xho I restriction site;
  • Light chain ( ⁇ ) coding sequence (5’-3’): CMV promoter-Sac I restriction site-Leader sequence gene-VL gene-CL ( ⁇ ) gene-Xho I restriction site;
  • the amino acid sequence of the leader sequence is shown in SED ID NO: 11 (the coding gene is shown in SEQ ID NO: 16), and the amino acid sequence of CH is shown in SED ID NO: 9 (the coding gene is shown in SEQ ID NO: 14). (Shown), the amino acid sequence of CL is shown in SED ID NO: 10 (the coding gene is shown in SEQ ID NO: 15).
  • 293T cells were cultured in DMEM containing 10% FBS. 293T cells were co-transfected with the recombinant expression vectors obtained in Example 3 encoding the antibody heavy chain and light chain respectively. After 4-6 hours of transfection, the cell culture medium was replaced with serum-free DMEM, and the culture was continued for 3 days. Collect the supernatant, add DMEM, continue the culture for 4 days, and then collect the supernatant again.
  • the collected supernatant was centrifuged at 5000 rpm for 30 min, and then mixed with an equal volume of buffer containing 20 mM sodium phosphate (pH 7.0), and then filtered with a 0.22 ⁇ m filter membrane, and then loaded on the protein A pre-packed column (5 mL, GE Healthcare ).
  • the protein bound to the pre-packed column was eluted with 10 mM glycine (pH 3.0).
  • the eluted fraction is concentrated and then purified by molecular sieve chromatography. Subsequently, the purified target protein was detected by SDS-PAGE (reducing and non-reducing). The result is shown in Figure 2.
  • the results in Figure 2 show that purified H4 antibody was obtained.
  • Biacore 8K (Biacore Inc.) is used to perform surface plasmon resonance analysis. Specific steps are as follows:
  • the anti-human IgG antibody is immobilized on the flow cell (Fc) of the CM5 chip by means of amino coupling. The fixed amount is controlled at around 8,000 response units (RU). Then, the purified H4 antibody is bound by antibody capture.
  • the RBD protein was diluted with a solution of 20 mM HEPES, 150 mM NaCl, pH 7.4 in successive times. Then, the serially diluted RBD protein was sequentially passed through each channel (starting with a low concentration and loaded one by one). Record the kinetic curve of H4 antibody binding to RBD protein ( Figure 3), and use BIA evaluation software 8K (Biacore, Inc.) software to calculate the kinetic constants (as shown in Table 4). The results in Figure 3 and Table 4 show that the H4 antibody can bind to the RBD of the S protein of 2019-nCoV with higher affinity.
  • the gene encoding hACE2 protein (Genbank accession number: NP_068576.1) was cloned into the pEGFP-N1 vector (purchased from Addgene), and the gene can be fused and expressed with the gene encoding GFP to construct a plasmid pEGFP-hACE2.
  • the plasmid pEGFP-hACE2 was transfected into HEK293T cells. After 24h, the expression of GFP can be observed under a fluorescence microscope. Collect HEK293T-hACE2 cells. The irrelevant antibody was incubated with 200ng/mL RBD protein at a molar ratio of 10:1 for 1h at room temperature.
  • HEK293T-hACE2 cells (2x10 5 /reaction) were incubated with the RBD protein (200ng/ml, carrying 6*His tag) incubated with irrelevant antibodies for 30min at room temperature. After centrifugation at 500xg for 5 min, the supernatant was removed, and the cells were washed twice with PBS. Subsequently, the cells were incubated with anti-His/APC (Miltenyi Biotec, 130-119-820) at room temperature for 30 minutes, and then washed twice with PBS, and then the fluorescence on the cell surface was detected with BD FACSCanto.
  • RBD protein 200ng/ml, carrying 6*His tag
  • the H4 antibody purified in Example 4 was incubated with 200 ng/mL RBD protein at a molar ratio of 10:1 for 1 h at room temperature, and then incubated with HEK293T-hACE2 cells. Then, as described above, use anti-His/APC to detect the binding of RBD protein to cells. The result is shown in Figure 4.
  • Figure 4 shows the cell surface fluorescence detected by BD FACSCanto.
  • Example 7 Evaluation of the ability of H4 antibody to neutralize live 2019-nCoV virus
  • the H4 antibody purified in Example 4 was diluted from 200 ⁇ g/mL to the 12th gradient (0.098 ⁇ g/mL), and then respectively compared with the BetaCoV/Shenzhen/SZTH-003/2020 of half the tissue culture infection dose (TCID 50).
  • the virus obtained from Shenzhen Third People's Hospital, GISAID number: EPI_ISL_406594
  • the virus was mixed and incubated at 37 degrees Celsius for 2 hours. After incubation, the virus was added to a 96-well plate pre-seeded with Vero cells, and cultured in a 37°C, 5% CO 2 incubator for 4 days to observe the cytopathic effect (CPE), and calculate the neutralization of the H4 antibody Titer.
  • CPE cytopathic effect
  • Figure 5 shows the neutralizing activity of different concentrations of H4 antibody against 2019-nCoV live virus.
  • the results showed that the neutralizing titer (half inhibitory concentration, IC 50 ) of the H4 antibody to the live 2019-nCoV virus was 0.896 ⁇ g/mL, and it had excellent neutralizing activity.

Abstract

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，还涉及所述抗体的用途。所述抗体可用于诊断、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)。

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21.05.2020]　
由新型冠状病毒2019-nCoV导致的肺炎已经在全国及世界范围内广泛传播。截止到2020年3月3日，全国确诊病例为80303例，累计死亡病例2947例，在中国以外国家(泰国、美国、德国、澳大利亚、伊朗等54个国家)累计确诊病例9742例，累计死亡171例，对公众的生命和健康造成重大威胁。目前对于新型冠状病毒的治疗仍未有特效药物。
新型冠状病毒2019-nCoV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。该病毒表面的刺突蛋白(Spike,S蛋白)在感染宿主的过程中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。S蛋白分为S1和S2两部分，已有研究证实S1的C端(CTD)的受体结合结构域(RBD)与ACE2结合，介导膜融合过程。
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。目前已经上市的治疗和预防病毒感染的药物有预防小儿呼吸道合胞病毒(RSV)感染的帕利珠单抗(Synagis)，治疗HIV感染的艾巴利珠单抗(Trogarzo)，以及用于狂犬病毒暴露后预防的Rabishield。此外，还有多种针对不同病毒的单抗处于临床研究的不同阶段(https://clinicaltrials.gov/)。抗体主要通过两方面起作用。一方面，具有中和活性的抗体可通过结合病毒囊膜蛋白，阻断病毒与细胞受体的结合，从而阻断病毒感染。另一方面，抗体依赖的细胞介导的细胞毒性作用(ADCC)和补体依赖的细胞毒性作用(CDC)可募集巨噬细胞或是补体等免疫细胞和免疫分子，从而清除游离的病毒以及被感染的细胞。
因此，需要开发能够抗新型冠状病毒2019-nCoV的中和抗体，以提供有效预防和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H4)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“大肠杆菌表达系统”是指由大肠杆菌(菌株)与载体组成的表达系统，其中大肠杆菌(菌株)来源于市场上可得到的菌株，例如但不限于：GI698,ER2566,BL21(DE3),B834(DE3),BLR(DE3)。
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(例如，本发明的单克隆抗体H4)以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的解离平衡常数(KD)结合抗原(例如，新型冠状病毒S蛋白的RBD)，例如，如使用表面等离子体共振术(SPR)在BIACORE仪中测定的。
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21.05.2020]　
如本文中所使用的，术语“新型冠状病毒”和“2019-nCoV”是指，2019年底发现的一类冠状病毒，二者具有相同的含义，可互换使用。
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)，并且能够阻断S蛋白的RBD与细胞受体血管紧张素转换酶2(ACE2)的结合，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，所述单克隆抗体在重链可变区的N端还具有前导序列。在某些优选的实施方案中，所述前导序列具有如SEQ ID NO:11所示的氨基酸序列。
在某些优选的实施方案中，所述单克隆抗体在轻链可变区的N端还具有前导序列。在某些优选的实施方案中，所述前导序列具有如SEQ ID NO:11所示的氨基酸序列。
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，所述的单克隆抗体的轻链为κ型。
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：氨基酸序列为SEQ ID NO:1-3的VH CDR1-3。
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:12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：氨基酸序列为SEQ ID NO:4-6的VL CDR1-3。
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:13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:12所示的核苷酸序列。
在某些优选的实施方案中，所述核酸分子还包含编码前导序列的核苷酸序列，其位于所述能够编码抗体重链可变区的核苷酸序列的5’端。在某些优选的实施方案中，所述前导序列具有如SEQ ID NO:11所示的氨基酸序列。在某些优选的实施方案中，所述编码前导序列的核苷酸序列具有如SEQ ID NO: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:13所示的核苷酸序列。
在某些优选的实施方案中，所述核酸分子还包含编码前导序列的核苷酸序列，其位于所述能够编码抗体轻链可变区的核苷酸序列的5’端。在某些优选的实施方案中，所述前导序列具有如SEQ ID NO:11所示的氨基酸序列。在某些优选的实施方案中，所述编码前导序列的核苷酸序列具有如SEQ ID NO:16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:12所示的核苷酸序列和如SEQ ID NO:13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第一多核苷酸，其包含编码前导序列的核苷酸序列和能够编码抗体重链可变区的核苷酸序列；以及，第二多核苷酸，其包含编码前导序列的核苷酸序列和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，所述分离的核酸分子包含第一多核苷酸，其包含如SEQ ID NO:16所示的核苷酸序列和如SEQ ID NO:12所示的核苷酸序列；以及，第二多核苷酸，其包含如SEQ ID NO:16所示的核苷酸序列和如SEQ ID NO:13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:14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:15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第一多核苷酸，其包含编码前导序列的核苷酸序列、能够编码抗体重链可变区的核苷酸序列和能够编码抗体重链恒定区的核苷酸序列；以及，第二多核苷酸，其包含编码前导序列的核苷酸序列、能够编码抗体轻链可变区的核苷酸序列和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，所述分离的核酸分子包含第一多核苷酸，其包含如SEQ ID NO:16、SEQ ID NO:12和SEQ ID NO:14所示的核苷酸序列；以及，第二多核苷酸，其包含如SEQ ID NO:16、SEQ ID NO:13和SEQ ID NO:15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293T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或者抗独特型抗体。
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或者抗独特型抗体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或者抗独特型抗体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 PCTCN2020079737-appb-000001
Figure PCTCN2020079737-appb-000002
Figure PCTCN2020079737-appb-000003
有益效果
本申请的单克隆抗体(例如H4抗体)能够以高亲和力与新型冠状病毒S蛋白RBD结合，并且对新型冠状病毒具有很强的中和活性。例如，本发明H4抗体对RBD的亲和力为4.48nM，对新型冠状病毒的中和滴度(半抑制浓度，IC 50)为0.896μg/mL。因此，本申请的单克隆抗体(例如H4抗体)具有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显示了新型冠状病毒S蛋白RBD的分子筛层析结果与SDS-PAGE检测结果。
图2显示了重组表达的H4抗体的分子筛层析结果与SDS-PAGE检测结果，其中，凝胶图上的“-”表示没有添加DTT(非还原性SDS-PAGE)；“+”表示添加了DTT(还原性SDS-PAGE)。
图3显示了H4抗体结合RBD蛋白的动力学曲线结果。
图4显示了实施例6中用BD FACSCanto检测的细胞表面荧光情况。
图5显示了不同浓度的H4抗体抗2019-nCoV活病毒的中和活性。
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。
为了获得具有保护效果的中和抗体，本申请发明人首先以大肠杆菌表达的2019-nCoV的S蛋白RBD作为抗原，通过流式分选术，从感染了2019-nCoV且痊愈出院的人员的外周血单核细胞(PBMCs)中筛选到能够特异性结合S蛋白RBD的记忆B细胞，然后，对筛选获得的单一B细胞进行RT-PCR，获得编码抗体可变区的序列。进一步，将编码抗体可变区的序列与恒定区基因连接至表达载体中，并在哺乳动物细胞中进行表达和纯化，从而获得抗体H4。对抗体H4进行一系列的功能检测，结果显示，抗体H4能够特异性结合S蛋白RBD，阻断S蛋白RBD与ACE2的结合，抑制2019-nCoV对人细胞的感染，具有抗2019-nCoV感染的中和活性。
实施例1：2019-nCoV病毒S蛋白RBD的表达与纯化
使用NdeI和XhoI酶，将编码2019-nCoV/2019毒株刺突蛋白S蛋白RBD(其氨基酸序列如SEQ ID NO:17所示)的DNA片段连接到pET21a载体上，所述DNA片段在编码区的3'端还连接有编码6*组氨酸标签(6*His标签)的核苷酸序列及终止密码子。将连接产物转化到BL21大肠杆菌感受态细胞中。然后，挑取单克隆，接种到40mL LB培养基中，培养6-8小时；然后再转接种到4L的LB培养基中，并在37摄氏度培养至OD600＝0.4-0.6。随后，向培养物中加入IPTG至终浓度1mM，并在37摄氏度继续培养4-6小时。培养结束后，收获包涵体，并进行复性。将复性后的蛋白溶液浓缩，并透析至20mM Tris，150mM NaCl，pH9.0缓冲液中。随后，通过分子筛层析法纯化溶液中的蛋白，其中，使用AKTA-purifier(GE)和superdex200Hiload 16/60柱子(GE)以及缓冲液A(20mM Tris，150mM NaCl，pH9.0)，并且在纯化过程中，同时监测280nm处的紫外吸收值，收取含有目的蛋白的级分。纯化结束后，通过SDS-PAGE鉴定目的蛋白(S蛋白的RBD)的纯度。结果如图1所示。图1的结果显示，获得了高纯度的RBD蛋白，其大小为32kDa。
实施例2：特异性识别RBD蛋白的记忆B细胞的分离
在感染2019-nCoV病毒且痊愈出院的人员的知情同意下，采集10mL的血液，分离PBMCs。将分离的PBMCs以10 7/mL的密度与终浓度为400nM的RBD蛋白(如实施例1制备的)在冰上孵育结合半小时；然后用PBS洗2次，再与下列抗体(均购自BD)孵育：anti-human CD3/PE-Cy5，anti-human CD16/PE-Cy5，anti-human CD235a/PE-Cy5，anti-human CD19/APC-Cy7，anti-human CD27/Pacific Blue，anti-human CD38/APC，anti- human IgG/FITC，以及anti-His/PE。在冰上孵育半小时后，用PBS洗PBMCs 2次。随后，用FACSAria III分选PBMCs，收集PE -Cy5 -APC -APC -Cy7 +Pacific Blue +FITC +PE +的细胞(即B细胞)，直接将其收集到96孔板内，1细胞/孔。
实施例3：H4抗体的分离和鉴定以及重组表达载体的构建
使用Superscript III reverse transcriptase(Invitrogen)对实施例2获得的B细胞进行逆转录(在55℃，进行60分钟)，其中，所使用的逆转录引物如表2所示。
表2.所使用的逆转录引物的序列信息
Figure PCTCN2020079737-appb-000004
以逆转录产物作为模板，用HotStar Tap Plus酶(QIAgen)进行第一轮PCR(PCRa)，扩增抗体可变区的序列；其中，所使用的引物如表3所示；所使用的反应条件如下：95℃ ，5min；35个循环的(95℃ 30s，55℃(重链/κ链)30s，72℃ 90s)；72℃，7min。随后，以该扩增产物作为模板再进行第二轮PCR(PCRb)；其中，所使用的引物如表4所示；所使用的反应条件如下：95℃，5min；35个循环的(95℃ 30s，58℃(重链)/60℃(κ链)/64℃(λ链)30s，72℃ 90s)；72℃，7min。
通过1％的琼脂糖凝胶电泳，分离PCR产物。回收条带大小在400-500bp的PCR产物，并送测序公司测序。测序结果用NCBI在线软件进行分析。
通过序列测定，获得一株抗体的序列，命名为H4。H4抗体的重链可变区的氨基酸序列如SEQ ID NO:7所示(编码基因如SEQ ID NO:12所示)，轻链可变区的氨基酸序列如SEQ ID NO:8所示(编码基因如SEQ ID NO:13所示)。H4抗体与胚系基因的序列一致性如下面的表5-6所示。
表3.第一轮PCR(PCRa)所使用的引物
Figure PCTCN2020079737-appb-000005
Figure PCTCN2020079737-appb-000006
表4.第二轮PCR(PCRb)所使用的引物
Figure PCTCN2020079737-appb-000007
表5.H4抗体重链与胚系基因的比较
Figure PCTCN2020079737-appb-000008
表6.H4抗体轻链与胚系基因的比较
Figure PCTCN2020079737-appb-000009
将分析得到的编码重链/轻链可变区的核苷酸序列分别与相应的编码重链/κ链的恒定区的核苷酸序列通过搭桥PCR进行连接，然后分别克隆至表达载体pCAGGS(购自Addgene)中，从而得到分别编码抗体重链和轻链的重组表达载体。表达重链和轻链的构建体的构建方案如下：
重链编码序列(5’-3’)：CMV启动子-EcoR I酶切位点-前导序列基因-VH基因-CH基因-Xho I酶切位点；
轻链(κ)编码序列(5’-3’)：CMV启动子-Sac I酶切位点-前导序列基因-VL基因-CL(κ)基因-Xho I酶切位点；
其中，前导序列的氨基酸序列如SED ID NO:11所示(编码基因如SEQ ID NO:16所示)，CH的氨基酸序列如SED ID NO:9所示(编码基因如SEQ ID NO:14所示)，CL的氨基酸序列如SED ID NO:10所示(编码基因如SEQ ID NO:15所示)。
实施例4：H4抗体的表达
在含10％FBS的DMEM中培养293T细胞。用实施例3得到的分别编码抗体重链和轻链的重组表达载体共转染293T细胞。转染4-6小时后，将细胞培养液更换成无血清的DMEM，并且继续培养3天。收集上清，然后补加DMEM，继续培养4天，然后再次收集上清。
将收集的上清以5000rpm离心30min，然后与含有20mM磷酸钠(pH 7.0)的缓冲液等体积混合，随后用0.22μm滤膜进行过滤，然后装载至与protein A预装柱(5mL，GE  Healthcare)。以10mM甘氨酸(pH 3.0)洗脱结合至预装柱的蛋白。将洗脱级分浓缩，然后通过分子筛层析法进行纯化。随后，通过SDS-PAGE(还原性和非还原性)检测所纯化的目的蛋白。结果如图2所示。图2的结果显示，获得了经纯化的H4抗体。
实施例5：H4抗体与S蛋白RBD的结合能力的评估
在本实施例中，利用Biacore 8K(Biacore Inc.)进行表面等离子共振分析。具体步骤如下：
首先，将anti-human IgG的抗体以氨基偶联的方式固定在CM5芯片的通道(flow cell,Fc)。固定量控制在8,000响应值(response units,RU)左右。然后，以抗体捕获的方式，结合纯化的H4抗体。另外，以20mM HEPES，150mM NaCl，pH 7.4溶液连续倍比稀释RBD蛋白。然后，将连续稀释的RBD蛋白依次通过各通道(从低浓度开始逐一上样)。记录H4抗体结合RBD蛋白的动力学曲线(图3)，并利用BIAevaluation software 8K(Biacore，Inc.)软件计算动力学常数(如表4所示)。图3和表4的结果显示，H4抗体能够以较高的亲和力结合2019-nCoV的S蛋白的RBD。
表4.抗体与RBD蛋白的结合动力学常数
  ka(1/Ms) kd(1/s) KD(M)
H4 8.47E+04 3.79E-04 4.48E-09
实施例6：H4阻断RBD与ACE2结合的能力的评估
利用XhoI和BamHI，将编码hACE2蛋白的基因(Genbank登录号：NP_068576.1)克隆入pEGFP-N1载体(购自Addgene)中，且所述基因能够与编码GFP的基因融合表达，从而构建获得质粒pEGFP-hACE2。将质粒pEGFP-hACE2转染入HEK293T细胞。24h后，可在荧光显微镜下观察到GFP的表达。收集HEK293T-hACE2细胞。将无关抗体按10:1的摩尔比与200ng/mL的RBD蛋白在室温下孵育1h。然后，将HEK293T-hACE2细胞(2x10 5/反应)与同无关抗体孵育后的RBD蛋白(200ng/ml,携带6*His标签)在室温条件下孵育30min。500xg离心5min后，去掉上清，细胞用PBS洗2次。随后，将细胞与anti-His/APC(美天旎，130-119-820)在室温下孵育30min，然后用PBS洗2次，然后用BD FACSCanto检测细胞表面的荧光情况。
为了检测H4抗体的阻断效果，将实施例4纯化的H4抗体按10:1的摩尔比与200ng/mL的RBD蛋白在室温下孵育1h，再与HEK293T-hACE2细胞孵育。然后，如上所述，用 anti-His/APC检测RBD蛋白与细胞的结合情况。结果如图4所示。图4显示了用BD FACSCanto检测的细胞表面荧光情况。结果显示，右侧图框中hACE2阳性且RBD阳性的细胞(即，携带双荧光的细胞)的数目显著少于左侧图框；这表明，H4抗体能有效阻断2019-nCoV的S蛋白RBD与HEK293T-hACE2细胞的结合。
实施例7：H4抗体中和2019-nCoV活病毒的能力的评估
将实施例4纯化的H4抗体从200μg/mL开始倍比稀释至第12个梯度(0.098μg/mL)，然后分别与半数组织培养感染剂量(TCID 50)的BetaCoV/Shenzhen/SZTH-003/2020病毒(获自深圳市第三人民医院，GISAID号:EPI_ISL_406594)在37摄氏度混合孵育2小时。孵育后，将病毒加入到预先接种了Vero细胞的96孔板中，并于37摄氏度，5％CO 2培养箱中培养4天，观察致细胞病变效应(CPE)，并计算H4抗体的中和滴度。结果如图5所示。图5显示了不同浓度的H4抗体抗2019-nCoV活病毒的中和活性。结果显示，H4抗体对2019-nCoV活病毒的中和滴度(半抑制浓度，IC 50)为0.896μg/mL，具有优良的中和活性。

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，所述单克隆抗体在重链可变区的N端还具有前导序列，和/或，所述单克隆抗体在轻链可变区的N端还具有前导序列；
    优选地，所述前导序列具有如SEQ ID NO:11所示的氨基酸序列；
    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：氨基酸序列为SEQ ID NO:1-3的VH CDR1-3；
    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:12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：氨基酸序列为SEQ ID NO:4-6的VL CDR1-3；
    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:13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或抗独特型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；
    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或抗独特型抗体；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或抗独特型抗体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或抗独特型抗体。
  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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