WO2023143176A1 - SARS-CoV-2病毒的广谱抗体及其应用 - Google Patents

SARS-CoV-2病毒的广谱抗体及其应用 Download PDF

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WO2023143176A1
WO2023143176A1 PCT/CN2023/072334 CN2023072334W WO2023143176A1 WO 2023143176 A1 WO2023143176 A1 WO 2023143176A1 CN 2023072334 W CN2023072334 W CN 2023072334W WO 2023143176 A1 WO2023143176 A1 WO 2023143176A1
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antibody
antigen
seq
binding fragment
strain
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PCT/CN2023/072334
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English (en)
French (fr)
Inventor
宋德勇
窦昌林
董创创
刘红
冯健霞
饶木顶
邢平平
于贝贝
张亚楠
胡凤娟
王毅云
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山东博安生物技术股份有限公司
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Priority to CN202380009441.5A priority Critical patent/CN116964103A/zh
Publication of WO2023143176A1 publication Critical patent/WO2023143176A1/zh

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the invention relates to the technical field of biomedicine or biopharmaceuticals, in particular to a broad-spectrum antibody against SARS-CoV-2 virus and its application.
  • COVID-19 Cornona Virus Disease-19
  • 2019-nCoV also known as SARS-CoV-2
  • SARS-CoV-2 novel coronavirus
  • S protein spike protein on the surface of the SARS-CoV-2 virus is combined into a trimer.
  • a single S protein contains about 1300 amino acids and belongs to a type of membrane fusion protein. It determines the host range and specificity of the virus and is the host neutralizer. The important site of action of the antibody.
  • S protein contains two subunits (subunit) S1 and S2, S1 mainly contains the receptor binding domain (RBD), which is responsible for recognizing the receptor of the cell, and S2 contains the basic elements required for the membrane fusion process.
  • RBD receptor binding domain
  • SARS-CoV-2 new coronavirus
  • mutations are fixed through antibody targeting sites, triggering antigenic epitope drift to evade recognition by related antibodies.
  • the main mutant strains of SARS-CoV-2 that have been reported so far include the British variant B.1.1.7, the South African variant B.1.351, and the Brazilian variant P.1.
  • the new coronavirus variant B.1.1.7 is a new coronavirus variant reported by the United Kingdom to the WHO in December 2020. Its transmission ability is about 70% higher than the original strain, and more than 60% of the new crown infection cases in London From a mutated virus. The biggest feature of this virus strain is that there are more than a dozen key site mutations. In the B.1.1.7 mutant lineage, the most notable mutation site is the key amino acid mutation N501Y in the RBD (receptor binding region). The structure of the virus protein changes and it is easier to bind to the ACE2 receptor in the human body. The practical consequence is that it has a stronger transmission ability.
  • the South African mutant strain B.1.351 appeared in August 2020. As of the end of December 2020, the proportion of infections caused by B.1.351 in South Africa has exceeded 80%.
  • the Brazilian mutant strain P.1 appeared in Manaus, Brazil, in early December 2020, and by mid-January 2021, it had caused a large-scale outbreak in the entire city.
  • the South African mutant strain B.1.351 and the Brazilian mutant strain P.1 were also found to have the E484K mutation, which has the effect of weakening anti-virus neutralizing antibodies and may also cause the virus to escape the recognition of the immune system.
  • Miao (B.1.621) was first discovered in Colombia. It has T95I, Y144S, Y145N, R346K, E484K, N501Y, D614G, P681H, D950N mutations, and has the potential of immune escape. This mutant strain also shows strong infectivity. It has similar characteristics with the two mutant strains previously found in the UK and South Africa.
  • the delta mutant strain B.1.617.2 was first discovered in India in October 2020, and it has 13 mutation sites.
  • the Delta mutant has some unique mutations, especially L4524, which makes the new coronavirus spike protein have a stronger affinity with the human cell ACE2 receptor; its P681R,
  • the ability of the mutant strain to infect cells can be enhanced by promoting the cleavage of the pre-spike protein into the active S1/S2 configuration.
  • Omicron variant B.1.1.529 was first discovered in South Africa and other places in November 2021, and it also has the first 4 VOCs
  • mutations in the Omicron variant may reduce the neutralizing activity of some monoclonal antibodies , the spreading power needs to be further monitored and studied.
  • the invention provides a broad-spectrum antibody or an antigen-binding fragment thereof, capable of binding to the S protein on the SARS-CoV-2 virus, blocking the cytopathy caused by the SARS-CoV-2 virus or neutralizing the SARS-CoV-2 virus.
  • the present invention also provides multispecific antibodies, bispecific antibodies, and antibody combinations derived from the broad-spectrum antibodies or antigen-binding fragments thereof, as well as encoding the broad-spectrum antibodies or antigen-binding fragments thereof, the multispecific antibodies , the nucleic acid of the bispecific antibody, or a nucleic acid combination; cells containing the nucleic acid or the nucleic acid combination; containing the broad-spectrum antibody or an antigen-binding fragment thereof, the multispecific antibody, the bispecific Antibody, said antibody combination, said nucleic acid, said nucleic acid combination, said cell pharmaceutical composition; containing said broad-spectrum antibody or its antigen-binding fragment, said multispecific antibody, said bispecific antibody, The antibody combination, the nucleic acid, the nucleic acid combination, the kit of the pharmaceutical composition; and the broad-spectrum antibody or antigen-binding fragment thereof, the multispecific antibody, the bispecific antibody, the The application of the antibody combination, the nucleic acid, the nucleic acid combination, and the pharmaceutical composition in the
  • One aspect of the present invention provides a broad-spectrum antibody or an antigen-binding fragment thereof, which binds to the S protein on a novel coronavirus (ie SARS-CoV-2, also known as 2019-nCoV) to block SARS-CoV-2 Virus-induced cytopathic or neutralizing SARS-CoV-2 virus.
  • SARS-CoV-2 also known as 2019-nCoV
  • the novel coronavirus that is, SARS-CoV-2 or 2019-nCoV
  • SARS-CoV-2 or 2019-nCoV is a collective term for the original strain of the novel coronavirus first discovered in 2019 and the subsequent mutant strain of the novel coronavirus.
  • one or more of the SARS-CoV-2 virus VOCs strains, VOIs strains, VUMs strains, and wild-type strains; more preferably, the VOCs strains include B.1.1.
  • the VOIs strains include C.37 strains, B One or more of 1.621 strains; the VUMs strains include one or more of B.1.617.1 strains, B.1.640 strains, C.1.2 strains, and B.1.630 strains ;
  • the wild-type strain is Wuhan-Hu-1 strain.
  • the SARS-CoV-2 virus includes Alpha (B.1.1.7) strain, Beta (B.1.351) strain, Gamma (p.1) strain, Delta ( B.1.617.2) strain, Lambda (C.37) strain, Mu (B.1.621) strain, Omicron strain, Kappa (B.1.617.1) strain, C.1.2 strain, B.
  • the Omicron strain includes BA.1 (B.1.1.529.1), BA.1.1, BA.2, BA.2.12.1, BA.2.13, BA.2.38 , BA.2.38.1, BA.2.74, BA.2.75, BA.2.76, BA.2.77, BA.2.79, BA.2.80, BA.3, BA.4, BA.5, BA.4.6, BA.4.7 , BA.5.5.1, BQ.1, BQ.1.1, one or more of XBB strains.
  • the S protein is the S protein (spike protein) on the surface of the SARS-CoV-2 virus, and the S protein contains two subunits (subunit) S1 and S2.
  • the antibody can bind to the SARS-CoV-2 virus
  • the S protein refers to one or more of the S1 and S2 subunits that bind the S protein, or the RBD protein that binds the S1 subunit.
  • the antibodies or antigen-binding fragments described in this application can block the pathological changes of ACE2-expressing cells caused by SARS-CoV-2 virus, or block the infection and invasion of ACE2-expressing cells by SARS-CoV-2 virus.
  • the cells include cells that naturally express ACE2 or cells that artificially express ACE2.
  • the cells are mammalian cells. Further, the mammals include humans, and non-human animals such as mice or monkeys.
  • the antibody or antigen-binding fragment thereof still blocks the cellular activity caused by SARS-CoV-2 virus at concentrations below 50nM, 40nM, 30nM, 20nM, 10nM, 5nM, 1nM or 0.1nM. lesions, or neutralize the SARS-CoV-2 virus.
  • the present invention provides a broad-spectrum antibody comprising the following 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions that can bind to SARS-CoV-2 virus S protein or its Antigen-binding fragments, wherein,
  • the three light chain complementary determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO:38, LCDR2 shown in SEQ ID NO:39, and LCDR3 shown in SEQ ID NO:40, and /or the three heavy chain complementary determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:41, HCDR2 shown in SEQ ID NO:42 and HCDR3 shown in SEQ ID NO:43 ;
  • the three light chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO:21, LCDR2 shown in SEQ ID NO:22 and LCDR3 shown in SEQ ID NO:23, and /or the three heavy chain complementary determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:27, HCDR2 shown in SEQ ID NO:28 and HCDR3 shown in SEQ ID NO:29 ;
  • the three light chain complementarity determining regions of the broad-spectrum antibody or its antigen-binding fragment comprise LCDR1 shown in SEQ ID NO:9, LCDR2 shown in SEQ ID NO:10, and LCDR3 shown in SEQ ID NO:11, and /or the three heavy chain complementary determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:12, HCDR2 shown in SEQ ID NO:13 and HCDR3 shown in SEQ ID NO:14 ;
  • the three light chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO:15, LCDR2 shown in SEQ ID NO:16, and LCDR3 shown in SEQ ID NO:17, and /or the three heavy chain complementary determining regions of the broad-spectrum antibody or its antigen-binding fragment comprise HCDR1 shown in SEQ ID NO:18, HCDR2 shown in SEQ ID NO:19, and HCDR3 shown in SEQ ID NO:20 ;or
  • the three light chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO:21, LCDR2 shown in SEQ ID NO:22 and LCDR3 shown in SEQ ID NO:23, and /or the three heavy chain complementary determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:24, HCDR2 shown in SEQ ID NO:25 and HCDR3 shown in SEQ ID NO:26 .
  • VL light chain variable region
  • VH heavy chain variable region
  • LCDR light chain complementarity determining region
  • HCDR heavy chain complementarity determining region
  • LCDR1, LCDR2, LCDR3, HCDR1 Each embodiment of HCDR2 and HCDR3 can be implemented independently or in any combination.
  • the antibody or antigen-binding fragment thereof comprises:
  • the light chain variable region shown in SEQ ID NO:36, and/or the heavy chain variable region shown in SEQ ID NO:37; the antibody or its antigen-binding fragment binds SARS-CoV-2 virus S protein; preferably, the SARS-CoV-2 virus includes Alpha (B.1.1.7) strain, Beta (B.1.351) strain, Gamma (p.1) strain, Delta (B.1.617.2 ) strain, Lambda (C.37) strain, Mu (B.1.621) strain, Omicron strain, Kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B .1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and one or more of wild-type strains; preferably, the wild-type strain is Wuhan-Hu- 1 strain;
  • the Omicron strain comprises BA.1(B.1.1.529.1), BA.1.1, BA.2, BA.2.12.1, BA.2.13, BA.2.38, BA.2.38.1, BA.2.7
  • SARS-CoV-2 virus S protein preferably, the SARS-CoV-2 virus includes Alpha (B.1.1.7) strain, Beta (B.1.351) strain, Gamma (p.1) strain, Delta (B.1.617.2 ) strain, Lambda (C.37) strain, Mu (B.1.621) strain, Omicron strain, Kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B .1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and one or more of wild-type strains; preferably, the wild-type strain is Wuhan-Hu- 1 strain; preferably, the Omicron strain comprises BA.1 (B.1.1.529.1), BA.1.1, BA.2, BA.2.12.1, BA.2.13, BA.2.38, BA.2.38.
  • the light chain variable region shown in SEQ ID NO:1, and/or the heavy chain variable region shown in SEQ ID NO:2; the antibody or its antigen-binding fragment is combined with SARS-CoV-2 virus S protein; preferably, the SARS-CoV-2 virus includes Alpha (B.1.1.7) strain, Beta (B.1.351) strain, Gamma (p.1) strain, Delta (B.1.617.2 ) strain, Lambda (C.37) strain, Mu (B.1.621) strain, Omicron strain, Kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B .1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and one or more of wild-type strains; preferably, the wild-type strain is Wuhan-Hu- 1 strain; preferably, the Omicron strain comprises BA.1 (B.1.1.529.1), BA.1.1, BA.2, BA.2.12.1, BA.2.13, BA.2.38, BA.2.38.
  • the light chain variable region shown in SEQ ID NO:3, and/or the heavy chain variable region shown in SEQ ID NO:4; the antibody or its antigen-binding fragment is combined with SARS-CoV-2 virus S protein; preferably, the SARS-CoV-2 virus includes Alpha (B.1.1.7) strain, Beta (B.1.351) strain, Gamma (p.1) strain, Delta (B.1.617.2 ) strain, Lambda (C.37) strain, Mu (B.1.621) strain, Omicron strain, Kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B .1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and one or more of wild-type strains; preferably, the wild-type strain is Wuhan-Hu- 1 strain; preferably, the Omicron strain comprises BA.1 (B.1.1.529.1), BA.1.1, BA.2, BA.2.12.1, BA.2.13, BA.2.38, BA.2.38.
  • the light chain variable region shown in SEQ ID NO:5, and/or the heavy chain variable region shown in SEQ ID NO:6; the antibody or its antigen-binding fragment binds SARS-CoV-2 virus S protein; preferably, the SARS-CoV-2 virus includes Alpha (B.1.1.7) strain, Beta (B.1.351) strain, Gamma (p.1) strain, Delta (B.1.617.2 ) strain, Lambda (C.37) strain, Mu (B.1.621) strain, Omicron strain, Kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B .1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and one or more of wild-type strains; preferably, the wild-type strain is Wuhan-Hu- 1 strain; preferably, the Omicron strain comprises BA.1 (B.1.1.529.1), BA.1.1, BA.2, BA.2.12.1, BA.2.13, BA.2.38, BA.2.38.
  • the light chain variable region shown in SEQ ID NO:3, and/or the heavy chain variable region shown in SEQ ID NO:35; the antibody Or its antigen-binding fragment is combined with SARS-CoV-2 virus S protein;
  • the SARS-CoV-2 virus includes Alpha (B.1.1.7) strain, Beta (B.1.351) strain, Gamma (p .1) strain, Delta (B.1.617.2) strain, Lambda (C.37) strain, Mu (B.1.621) strain, Omicron strain, Kappa (B.1.617.1) strain, One or more of C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and wild-type strain; preferably Yes, the wild-type strain is Wuhan-Hu-1 strain; preferably, the Omicron strain includes BA.1 (B.1.1.529.1), BA.1.1, BA.2, BA.2.12.1 , BA.2.13, BA.2.38, BA.2.38.1, BA.2.74,
  • sequence of the heavy chain constant region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 30.
  • sequence of the light chain constant region of the antibody or its antigen-binding fragment is SEQ ID NO: 31.
  • the broad-spectrum antibody or antigen-binding fragment thereof of the present invention includes monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, Fab, Fab', F(ab')2, Fv, scFv or dsFv fragments, etc.
  • the SARS-CoV-2 virus includes Alpha (B.1.1.7) strain, Beta (B.1.351) strain, Gamma (p.1) strain, Delta ( B.1.617.2) strain, Lambda (C.37) strain, Mu (B.1.621) strain, Omicron strain, Kappa (B.1.617.1) strain, C.1.2 strain, B.
  • the Omicron strain includes BA.1 (B.1.1.529.1), BA.1.1, BA.2, BA.2.12.1, BA.2.13, BA.2.38 , BA.2.38.1, BA.2.74, BA.2.75, BA.2.76, BA.2.77, BA.2.79, BA.2.80, BA.3, BA.4, BA.5, BA.4.6, BA.4.7 One or more of , BA.5.5.1, BQ.1, BQ.1.1, XBB strains;
  • the antibody or its antigen-binding fragment binds T345, R346, K444, R403, K417, Y453, K458, G476, Y489, F490, Y505, K440, S443, T415, One or more of D420, Y421, A475, N487 and R493 residues;
  • the antibody or its antigen-binding fragment binds to T345, R346 and K444 residues on the Delta RBD; the antibody or its antigen-binding fragment binds to T345, R346, K440, S443 and K444 on the Omicorn BA.1 RBD residues; the antibody or its antigen-binding fragment binds to the R403, K417, Y453, K458, G476, Y489, F490 and Y505 residues on the Delta RBD; or the antibody or its antigen-binding fragment binds to the Omicron BA.2 RBD T415, D420, Y421, A475, N487, Y489 and R493 residues;
  • the antigen-binding fragment is a Fab, Fab', F(ab')2, Fv, scFv or dsFv fragment;
  • BA7208 or its antigen-binding fragment binds to T345, R346 and K444 residues on Delta RBD; BA7208 or its antigen-binding fragment binds to T345, R346, K440, S443 and K444 residues on Omicorn BA.1 RBD; BA7125V1 or its antigen-binding fragment binds to residues R403, K417, Y453, K458, G476, Y489, F490 and Y505 on Delta RBD; or BA7535 or its antigen-binding fragment binds to T415, D420, Y421, A475, N487, Y489 and R493 residues.
  • the second aspect of the present invention provides a multispecific antibody derived from the broad-spectrum antibody or antigen-binding fragment of the first aspect; preferably, the multispecific antibody includes a bispecific Antibody; more preferably, the multispecific antibody is derived from one or more of BA7054, BA7125, BA7134, BA7208, BA7125V1, BA7535.
  • a third aspect of the present invention provides a bispecific antibody, including a first antibody or an antigen-binding fragment that binds to the S protein on the SARS-CoV-2 virus, and an antibody that binds to the S protein on the SARS-CoV-2 virus.
  • a second antibody or antigen-binding fragment wherein the first antibody or antigen-binding fragment is the broad-spectrum antibody or antigen-binding fragment of the first aspect, and/or the second antibody or antigen-binding fragment is the first antibody or antigen-binding fragment A broad spectrum antibody or antigen-binding fragment of one aspect.
  • the first antibody or antigen-binding fragment is the same as or different from the second antibody or antigen-binding fragment; preferably, the first antibody or antigen-binding fragment binds the same as the second antibody or antigen-binding fragment Or the S protein of different SARS-CoV-2 viruses; more preferably, the first antibody or antigen-binding fragment binds to the same or different epitopes on the S protein as the second antibody or antigen-binding fragment.
  • the bispecific antibody binds T345, R346, K444, R403, K417, Y453, K458, G476, Y489, F490, Y505, K440, S443, T415, D420, Y421 of the RBD of SARS-CoV-2 virus One or more of , A475, N487 and R493 residues.
  • the SARS-CoV-2 virus includes Alpha (B.1.1.7) strain, Beta (B.1.351) strain, Gamma (p.1) strain, Delta (B.1.617.2) virus Strain, Lambda (C.37) strain, Mu (B.1.621) strain, Omicron strain, Kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and one or more of wild-type strains; preferably, the wild-type strain is Wuhan-Hu-1 virus strain; preferably, the Omicron strains include BA.1 (B.1.1.529.1), BA.1.1, BA.2, BA.2.12.1, BA.2.13, BA.2.38, BA.2.38.1, BA.2.74, BA.2.75, BA.2.76, BA.2.77, BA.2.79, BA.2.80, BA.3, BA.4, BA.5, BA.4.6, BA.4.7, BA.5.5.1, One or more of BQ.1,
  • the first antigen-binding fragment is Fab
  • the second antigen-binding fragment is scfv
  • the bispecific antibody has a knob-hole Fc region
  • the heavy chain constant region connected to the first antigen-binding fragment is a heavy chain constant region with a hole
  • the heavy chain constant region connected to the second antigen-binding fragment is a heavy chain constant region with a knob
  • the second antigen-binding fragment is connected to the heavy chain constant region with knob through VL or VH; more preferably, the VL or VH is connected to the heavy chain constant region with knob through a linker;
  • the bispecific antibody also has a light chain constant region
  • the first antibody or antigen-binding fragment comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, and the 3 light chain complementarity determining regions comprise SEQ ID NO:21 LCDR1, LCDR2 shown in SEQ ID NO:22 and LCDR3 shown in SEQ ID NO:23, and/or three heavy chain complementarity determining regions comprising HCDR1 shown in SEQ ID NO:27, shown in SEQ ID NO:28 HCDR2 and HCDR3 shown in SEQ ID NO: 29; the second antibody or antigen-binding fragment comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, and the 3 light chain complementarity determining regions Comprising LCDR1 shown in SEQ ID NO:15, LCDR2 shown in SEQ ID NO:16 and LCDR3 shown in SEQ ID NO:17, and/or three heavy chain complementarity determining regions include SEQ ID NO:18 HCDR1, HCDR2 shown in SEQ ID NO: 19 and
  • the first antibody or antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO:7, and/or the heavy chain variable region shown in SEQ ID NO:8;
  • the second antibody Or the antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO:3, and/or the heavy chain variable region shown in SEQ ID NO:35;
  • the first antibody or antigen-binding fragment comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, and the 3 light chain complementarity determining regions comprise SEQ ID NO: 21 LCDR1, LCDR2 shown in SEQ ID NO:22 and LCDR3 shown in SEQ ID NO:23, and/or the three heavy chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise SEQ ID NO:27 HCDR1 shown in SEQ ID NO:28 and HCDR3 shown in SEQ ID NO:29;
  • the second antibody or antigen-binding fragment comprises 3 light chain complementarity determining regions comprising SEQ ID NO:38 LCDR1, LCDR2 shown in SEQ ID NO:39, and LCDR3 shown in SEQ ID NO:40, and/or the three heavy chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise SEQ ID NO:41 HCDR1 shown, HCDR2 shown in SEQ ID NO:42, and
  • the first antibody or antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO:7, and/or the heavy chain variable region shown in SEQ ID NO:8;
  • the second antibody Or the antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO:36, and/or the heavy chain variable region shown in SEQ ID NO:37;
  • the first antibody or antigen-binding fragment is BA7208 Fab
  • the second antibody or antigen-binding fragment is BA7125V1scfv
  • the first antibody or antigen-binding fragment is BA7208 Fab
  • the second antibody or antigen-binding The binding fragment is BA7535scfv.
  • the linker between the VL or VH and the heavy chain constant region with knob is a connecting polypeptide, preferably, the sequence may be AA. Furthermore, the connecting sequence of VL and VH in the scfv may be SEQ ID NO:32.
  • sequence of the constant region of the heavy chain (knob chain) of the antibody may be SEQ ID NO: 33; the sequence of the constant region of the heavy chain (hole chain) of the antibody may be SEQ ID NO: 34.
  • the third aspect of the present invention also provides an antibody combination comprising a combination of two or more antibodies or antigen-binding fragments that bind to the S protein on the SARS-CoV-2 virus;
  • the antibody combination is a combination of two antibodies or antigen-binding fragments; wherein, the first antibody or antigen-binding fragment is the broad-spectrum antibody or antigen-binding fragment of the first aspect, and/or the second The second antibody or antigen-binding fragment is the broad-spectrum antibody or antigen-binding fragment of the first aspect; more preferably, the first antibody or antigen-binding fragment is the same or different from the second antibody or antigen-binding fragment; more preferably Yes, the first antibody or antigen-binding fragment binds to the S protein of the same or different SARS-CoV-2 virus as the second antibody or antigen-binding fragment; more preferably, the first antibody or antigen-binding fragment binds to The second antibody or antigen-binding fragment binds to the same or different epitope on the S protein;
  • the first antibody or antigen-binding fragment comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, and the 3 light chain complementarity determining regions comprise SEQ ID NO: 21 LCDR1, LCDR2 shown in SEQ ID NO:22 and LCDR3 shown in SEQ ID NO:23, and/or three heavy chain complementarity determining regions comprising HCDR1 shown in SEQ ID NO:27, shown in SEQ ID NO:28 HCDR2 and HCDR3 shown in SEQ ID NO: 29; the second antibody or antigen-binding fragment comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, and the 3 light chain complementarity determining regions Comprising LCDR1 shown in SEQ ID NO:15, LCDR2 shown in SEQ ID NO:16 and LCDR3 shown in SEQ ID NO:17, and/or three heavy chain complementarity determining regions include SEQ ID NO:18 HCDR1, HCDR2 set forth in SEQ ID NO: 19, and
  • the first antibody or antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO:7, and/or the first antibody or antigen of the heavy chain variable region shown in SEQ ID NO:8 Binding fragment; said second antibody or antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO:3, and/or the heavy chain variable region shown in SEQ ID NO:35;
  • the first antibody or antigen-binding fragment comprises 3 light chain complementarity determining regions and/or 3 heavy chain complementarity determining regions, and the 3 light chain complementarity determining regions comprise SEQ ID NO: 21 LCDR1, LCDR2 shown in SEQ ID NO:22 and LCDR3 shown in SEQ ID NO:23, and/or the three heavy chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise SEQ ID NO:27 HCDR1 shown in SEQ ID NO:28 and HCDR3 shown in SEQ ID NO:29;
  • the second antibody or antigen-binding fragment comprises 3 light chain complementarity determining regions comprising SEQ ID NO:38 LCDR1, LCDR2 shown in SEQ ID NO:39, and LCDR3 shown in SEQ ID NO:40, and/or the three heavy chain complementarity determining regions of the broad-spectrum antibody or antigen-binding fragment thereof comprise SEQ ID NO:41 HCDR1 shown, HCDR2 shown in SEQ ID NO:42, and
  • the first antibody or antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO:7, and/or the heavy chain variable region shown in SEQ ID NO:8;
  • the second antibody Or the antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO:36, and/or the heavy chain variable region shown in SEQ ID NO:37;
  • the first antibody or antigen-binding fragment is BA7208 antibody or antigen-binding fragment
  • the second antibody or antigen-binding fragment is BA7125V1 antibody or antigen-binding fragment
  • the first antibody or antigen-binding fragment is BA7208 an antibody or antigen-binding fragment
  • the second antibody or antigen-binding fragment is the BA7535 antibody or antigen-binding fragment
  • the antibody combination binds T345, R346, K444, R403, K417, Y453, K458, G476, Y489, F490, Y505, K440, S443, T415, D420, Y421, One or more of A475, N487 and R493 residues;
  • the SARS-CoV-2 virus includes Alpha (B.1.1.7) strain, Beta (B.1.351) strain, Gamma (p.1) strain, Delta (B.1.617.2) virus Strain, Lambda (C.37) strain, Mu (B.1.621) strain, Omicron strain, Kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, and one or more of wild-type strains; preferably, the wild-type strain is Wuhan-Hu-1 strain; preferably, The Omicron strains include BA.1 (B.1.1.529.1), BA.1.1, BA.2, BA.2.12.1, BA.2.13, BA.2.38, BA.2.38.1, BA.2.74, BA .2.75, BA.2.76, BA.2.77, BA.2.79, BA.2.80, BA.3, BA.4, BA.5, BA.4.6, BA.4.7, BA.5.5.1, BQ.1, BQ .1.1, one or more of
  • BA7208 or its antigen-binding fragment binds to T345, R346 and K444 residues on Delta RBD; BA7208 or its antigen-binding fragment binds to T345, R346, K440, S443 and K444 residues on Omicorn BA.1
  • RBD BA7125V1 or its antigen-binding fragments bind to R403, K417, Y453, K458, G476, Y489, F490 and Y505 residues on Delta RBD; or BA7535 or its antigen-binding fragments bind to T415, D420, Y421, A475, N487, Y489 and R493 residues.
  • the epitopes of the antibodies or antigen-binding fragments of the present application are analyzed by cryo-electron microscopy.
  • the fourth aspect of the present invention provides a nucleic acid encoding the broad-spectrum antibody or an antigen-binding fragment thereof, or the multispecific antibody, the bispecific antibody, or the antibody combination.
  • the fourth aspect of the present invention also provides a nucleic acid combination, which includes a combination of nucleic acids encoding each antibody in the antibody combination described in the third aspect.
  • a fifth aspect of the present invention provides a vector comprising nucleic acid encoding the broad-spectrum antibody or its antigen-binding fragment, or the multispecific antibody, or the bispecific antibody; or comprising the nucleic acid encoding the antibody Combination of nucleic acids from individual antibodies in the combination.
  • Said vector can be used to express said broad-spectrum antibody or antigen-binding fragment thereof, or said multispecific antibody, or said bispecific antibody, or said antibody combination.
  • the vector can be a viral vector; preferably, the viral vector includes but is not limited to a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a retroviral vector, etc.; preferably, the vector can be a non-viral Vector; preferably, the vector can be a mammalian cell expression vector; preferably, the expression vector can be a bacterial expression vector; preferably, the expression vector can be a fungal expression vector.
  • the sixth aspect of the present invention provides a cell comprising the nucleic acid, the nucleic acid combination or the vector, the cell can express the broad-spectrum antibody or an antigen-binding fragment thereof, or the multispecific antibody , or the bispecific antibody, or the combination of antibodies.
  • the cells are bacterial cells; preferably, the bacterial cells are Escherichia coli cells, etc.; preferably, the cells are fungal cells; preferably, the fungal cells are yeast cells; preferably, the yeast Cells are Pichia cells, etc.; preferably, the cells are mammalian cells; preferably, the mammalian cells are Chinese hamster ovary cells (CHO), human embryonic kidney cells (293), B cells, T cells, DC cells or NK cells, etc.
  • the seventh aspect of the present invention provides a pharmaceutical composition, which includes the broad-spectrum antibody or its antigen-binding fragment, the multispecific antibody, the bispecific antibody, the antibody combination, nucleic acid, nucleic acid combination, carrier or cell, preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, preferably, the pharmaceutically acceptable carrier includes one or more of the following: a pharmaceutically acceptable solvent , dispersants, additives, plasticizers, pharmaceutical excipients.
  • the pharmaceutical composition is a nasal spray, nasal drops, atomization or injection preparation; preferably, the injection preparation is an intravenous injection preparation; more preferably, the pharmaceutical composition contains a therapeutically effective amount of, preferably , about 30 mg to about 2400 mg, preferably about 1200 mg or about 2400 mg of said antibody or antigen-binding fragment, said multispecific antibody, said bispecific antibody, or said antibody combination.
  • the antibody combination includes a combination of BA7208 and BA7535 monoclonal antibodies, or a combination of BA7208 and BA7125V1 monoclonal antibodies; preferably, in the antibody combination, the molar ratio of BA7208 and BA7535 is 1:1; preferably, in In the antibody combination, the molar ratio of BA7208 and BA7125V1 is 1:1; more preferably, the two antibodies in the antibody combination are mixed and administered together.
  • the pharmaceutical composition is a unit preparation, the unit preparation is a nasal spray, nasal drop, atomization or injection preparation, and the unit preparation contains a therapeutically effective amount, preferably, 30 mg to 2400 mg, preferably , about 1200mg or about 2400mg of said antibody or antigen-binding fragment thereof, said multispecific antibody, said bispecific antibody or said antibody combination; preferably, said pharmaceutical composition contains One of the antibody or its antigen-binding fragment, the multispecific antibody, the bispecific antibody and the antibody combination, and a buffer; more preferably, the buffer includes trehalose and polysorbate One or more of 80; more preferably, the pH of the pharmaceutical composition is 5.5-6.5; more preferably, the buffer also includes one or more of histidine hydrochloride and histidine; More preferably, the molar ratio of histidine hydrochloride to histidine is 10.5:9.5; more preferably, based on the total volume of the pharmaceutical composition, the pharmaceutical composition comprises 0.04-0.1 g/mL trehalose ,
  • the pharmaceutical composition comprises 10.5mM histidine hydrochloride, 9.5mM histidine, 0.08g/mL trehalose, 0.0002g/mL polysorbate 80, and 40 ⁇ 4 mg/mL of said antibody or antigen-binding fragment thereof.
  • the administration object of the pharmaceutical composition is a treatment subject or a prevention subject;
  • the treatment subject includes asymptomatic, mild, common, severe or critical SARS-CoV-2 virus infection type patients; preferably, the treatment subjects are those with onset time ⁇ 7 days and confirmed infection with SARS-CoV-2 virus within 72 hours according to the "New Coronavirus Pneumonia Diagnosis and Treatment Program (Trial Ninth Edition)" Symptomatic, mild, common, severe or critical patients;
  • the treatment subjects include asymptomatic, mild, moderate, severe and critical patients infected with SARS-CoV-2 virus; preferably, the treatment subjects are laboratory tests (such as RT-PCR) within 72 hours Check) confirmed infection with SARS-CoV-2, and asymptomatic, mild, moderate, severe and critical COVID-19 patients confirmed according to NIH guidelines; or
  • the prophylaxis subjects include pre-exposure prophylaxis subjects, or post-exposure prophylaxis subjects; preferably, the pre-exposure prophylaxis Anti-exposure subjects include high-risk groups of new crown virus exposure, healthy subjects, or other subjects who are not suitable for vaccination; the post-exposure prophylaxis subjects include close contacts of confirmed patients with new coronary pneumonia and/or asymptomatic infections. contacts.
  • the eighth aspect of the present invention provides a kit comprising the broad-spectrum antibody or antigen-binding fragment thereof of the present invention, or the multispecific antibody, or the bispecific antibody, or the antibody combination, or comprising a nucleic acid encoding a broad-spectrum antibody or an antigen-binding fragment thereof or said multispecific antibody, or said bispecific antibody, or a combination of nucleic acids encoding each antibody in said antibody combination, or said cell, or said pharmaceutical composition.
  • the kit also includes a container or instructions filled with appropriate buffer reagents.
  • the ninth aspect of the present invention provides that the broad-spectrum antibody or its antigen-binding fragment, the multispecific antibody, the bispecific antibody, the antibody combination, nucleic acid, nucleic acid combination, vector or cell is used in the preparation of therapeutic or Use in medicines for the prevention of disease.
  • the tenth aspect of the present invention provides the broad-spectrum antibody or its antigen-binding fragment, the multispecific antibody, the bispecific antibody, the antibody combination, nucleic acid, or nucleic acid combination in the preparation of diagnostic and detection kits in the application.
  • the eleventh aspect of the present invention provides a method for treating or preventing a disease, comprising administering the broad-spectrum antibody or antigen-binding fragment, the multispecific antibody, the bispecific antibody of the present invention to a subject in need Specific antibodies, said antibody combinations, nucleic acids, nucleic acid combinations, vectors, cells or pharmaceutical compositions.
  • the method includes administering to a subject in need a pharmaceutical composition containing an effective amount of the antibody or antigen-binding fragment; preferably, administering to a subject in need a pharmaceutical composition containing about 30 mg to about 2400 mg, preferably The pharmaceutical composition of said antibody or antigen-binding fragment, said multispecific antibody, said bispecific antibody or said antibody combination of about 1200 mg or about 2400 mg; more preferably, said pharmaceutical composition is a nasal spray, nasal drop, atomization or injection preparation; preferably, the injection preparation is an intravenous injection preparation; more preferably, the pharmaceutical composition is a unit preparation, and the unit preparation is nasal spray, nasal drop, atomization or An injectable formulation, and the unit formulation contains about 30 mg to about 2400 mg, preferably about 1200 mg or about 2400 mg of said antibody or antigen-binding fragment, said multispecific antibody, said bispecific antibody or said antibody combination; More preferably, the pharmaceutical composition is a nasal spray, nasal drops, atomization or injection preparation, preferably, the injection preparation is an intravenous injection preparation;
  • the twelfth aspect of the present invention provides a method for diagnosis and detection, comprising combining the broad-spectrum antibody or antigen-binding fragment, the multispecific antibody, the bispecific antibody, and the antibody of the present invention , nucleic acid, nucleic acid combination, kit or pharmaceutical composition administered to a subject or sample in need.
  • the thirteenth aspect of the present invention provides the broad-spectrum antibody or its antigen-binding fragment, the multispecific antibody, the bispecific antibody, the antibody combination, nucleic acid, nucleic acid combination, vector, cell or drug
  • the composition is used for treating and preventing diseases.
  • the fourteenth aspect of the present invention provides the broad-spectrum antibody or its antigen-binding fragment, the multispecific antibody, the bispecific antibody, the antibody combination, nucleic acid, nucleic acid combination, kit, or drug
  • the composition is used for detection and diagnosis.
  • a fifteenth aspect of the present invention provides the broad-spectrum antibody or antigen-binding fragment thereof, the multispecific antibody, the bispecific antibody, the antibody combination, the nucleic acid, the nucleic acid combination, or
  • the pharmaceutical composition is used for preventing, treating, detecting or diagnosing diseases related to SARS-CoV-2 virus.
  • the disease is COVID-19 pneumonia and other related complications.
  • the broad-spectrum antibody or antigen-binding fragment, the multispecific antibody, the bispecific antibody, and the antibody combination can block the infection of cells by SARS-CoV-2 virus or its pseudovirus, Invasion, etc., or neutralize SARS-CoV-2 virus or its pseudovirus.
  • the SARS-CoV-2 virus includes Alpha (B.1.1.7) strain, Beta (B.1.351) strain, Gamma(p.1) strain, Delta(B.1.617.2) strain, Lambda(C.37) strain, Mu(B.1.621) strain, Omicron strain, Kappa(B.1.617.1)
  • the wild-type strain is Wuhan-Hu-1 strain
  • the Omicron strain includes BA.1 (B.1.1.529.1), BA.1.1, BA.2, BA.
  • the present invention also provides BA7054, BA7125, BA7134, BA7208, BA7125V1, BA7535, BA7208-7125V1-linker 4, BA7208-7125V1-linker 6 antibody, BA7208-7535-linker 4, BA7208 and BA7125V1 antibody combination, BA7535 and The application of BA7208 antibody combination in the prevention, treatment, detection or diagnosis of diseases related to SARS-CoV-2 virus; further, the SARS-CoV-2 virus includes Alpha (B.1.1.7) strain, Beta (B.1.351) strain, Gamma (p.1) strain, Delta (B.1.617.2) strain, Lambda (C.37) strain, Mu (B.1.621) strain, Omicron strain , Kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and wild One or more of the type strains; preferably, the wild-type strain is Wuhan-Hu-1 strain
  • the present invention also provides BA7054, BA7125, BA7134, BA7208, BA7125V1, BA7535, BA7208-7125V1-linker 4, BA7208-7125V1-linker 6 antibody, BA7208-7535-linker 4, BA7208 and BA7125V1 antibody combination, BA7535 and
  • the BA7208 antibody combination is used for the prevention, treatment, detection or diagnosis of SARS-CoV-2 virus-related diseases; further, the SARS-CoV-2 virus includes Alpha (B.1.1.7) strains, Beta (B.1.351) strain, Gamma (p.1) strain, Delta (B.1.617.2) strain, Lambda (C.37) strain, Mu (B.1.621) strain, Omicron strain, Kappa (B.1.617.1) strain, C.1.2 strain, B.1.630 strain, B.1.640 strain, B.1.526 strain, B.1.525 strain, AZ.5 strain, and wild type One or more of the strains; preferably, the wild-type strain is Wuhan-Hu
  • Table 1 shows the names of the main strains of the above-mentioned SARS-CoV-2 virus, their places of occurrence and their main mutations.
  • Figure 1 shows the serum titer of S protein immunized mice
  • Fig. 2 shows that ELISA detection candidate antibody BA7054, BA7125, BA7134, BA7208 block wild type, B.1.351 and the combination of the RBD protein of B.1.617.2 strain and hACE2;
  • Figure 3A-3C shows that antibody BA7054, BA7134, BA7208, BA7125V1 block the RBD of B.1.351, B.1.617.2 and B.1.1.529.1 strain, and the combination of 3 control antibodies and hACE2;
  • Figure 3D shows It is shown that antibody BA7535 blocks the binding of 11 kinds of strain proteins to hACE2;
  • Figures 3E-3M show that antibody BA7535 blocks the binding of 9 kinds of strain proteins to hACE2.
  • Figures 4A-4I show graphs of the binding kinetics of the bis-antibody BA7208/7125V1 and its parental mAb to the RBDs of B.1.617.2, B.1.529.1 and B.1.621 variants.
  • Figures 4J-4K show graphs of the binding kinetics of BA7535, BA7208 to the BA.1 (B.1.1.529.1) RBD.
  • Figures 5A-5H show the pseudovirus neutralization curves of BA7208, BA7125V1, BA7054, BA7134, BA7208/BA7125V1 antibodies and three control antibodies against various SARS-CoV-2 variants in Table 13, the data were collected from two biological Repeats are expressed as mean ⁇ SD.
  • Figure 5I shows pseudovirus neutralization curves of BA7535 against 2 SARS-CoV-2 variants.
  • Figure 6 shows the neutralizing activity of BA7208 antibody against multiple pseudoviruses.
  • FIG. 7 shows that BA7208 has broad-spectrum and excellent neutralizing activity against various Omicron mutants.
  • Figure 8 shows that BA7535 has a broad spectrum and excellent neutralizing activity.
  • Figures 9A-9D show the neutralizing activity of antibodies BA7535, BA7208, the BA7535+BA7208 combination, and LY-COV1404 (an antibody marketed by Eli Lilly) against more than 30 previous and emerging variants in a pseudovirus system.
  • Figures 10A-10G show live virus neutralization of SARS-CoV-2 variants by anti-SARS-CoV-2 antibodies in FRNT assays.
  • Figures 11A-11B show the neutralizing activity of BA7208 and BA7208/7125V1 against SARS-CoV-2 variants Omicron BA.1 and BA.2.
  • Figures 11C-11E show the euvirus neutralization curves of BA7535 against Omicron BA.1, BA.2, and BA.5 mutant strains, with three biological replicates.
  • Figure 12A shows the pharmacokinetic curves of a single intravenous injection of antibody drugs BA7208, BA7125V1, BA7054, BA7134, BA7208/7125V1 in Balb/c mice; Pharmacokinetic profile;
  • Figure 12C shows the pharmacokinetic profile of a single intravenous injection of BA7535.
  • 12D-12F show the results of single-dose toxicity test of BA7208 mice.
  • Figure 12G shows the mouse PK data-neutralization curve for bis-antibody BA7208/7535-Linker 4 (BA7208 Fab, 7535scfv).
  • Figure 12H- Figure 12L shows the experimental results of BA7208-mediated ADCC, ADCP, wherein Figure 12H shows the ADCC activity of BA7208 with CHO-K1 cells expressing SARS-CoV-2 Spike (wild type) as target cells .
  • Figure 12I shows the ADCP activity of BA7208 with CHO-K1 cells expressing SARS-CoV-2 Spike (wild type) as target cells.
  • Figure 12J shows the ADCP activity of BA7208 with HEK293T cells expressing SARS-CoV-2 BA.1 Spike as target cells.
  • Figure 12K shows the ADCC activity of BA7208 using HEK293T cells expressing SARS-CoV-2 BA.1 Spike as target cells.
  • Experiments were performed in duplicate using an unrelated mAb with the same constant region as an isotype control (not shown). Data are presented as mean ⁇ SD.
  • Figure 13 shows a schematic diagram of the structure of an anti-2019-nCoV (or SARS-CoV-2) bispecific antibody.
  • Figure 14A shows that ELISA detection of double antibody BA7208-7125V1-linker 4 and BA7208-7125V1-linker 6 blocks the binding of B.1.1.529 (also known as Omicron (Omicron) strain) RBD protein to hACE2 .
  • B.1.1.529 also known as Omicron (Omicron) strain
  • FIG. 14B shows that ELISA detection of double antibodies BA7208-7125V1-linker 4 and BA7208-7125V1-linker 6 blocks the binding of B.1.621 (also known as Mu strain) RBD protein to hACE2.
  • Figure 15 shows the neutralization curve of the pseudovirus of the double antibody BA7208/7535-linker 4 (BA7208 Fab, 7535scfv).
  • Figure 16 shows the cryo-electron microscope observation of Delta Spike Trimer with BA7208-Fab/BA7125V1-Fab (BA7125-Fab on the figure is the Fab of BA7125V1 described in the Examples).
  • Figure 17 shows the cryo-electron microscope observation of Omicron Spike Trimer with BA7208-Fab.
  • Figure 18 shows the interaction of Omicorn RBD mutation site and BA7208 Fab heavy chain.
  • Figure 19A-19H shows BA7208, BA7125V1, BA7254 structural overlay analysis diagram (BA7125 on Figure 19A-19H is BA7125V1 described in the embodiment).
  • Figure 20 shows the results of comparing the competitive binding of BA7535 and BA7208 antibodies in a biolayer interferometer (BLI)-based competitive binding assay.
  • Figures 21A-21B show the live virus titers in the lungs of the prevention group and the treatment group after BA7208 was injected into the mice.
  • Figures 21C-21D show the live virus titers in the lungs of the prevention group and the treatment group after BA7208 nasal drop and aerosol administration to mice.
  • Figure 22A shows the prophylactic and therapeutic trial routes of BA7535 and BA7535/BA7208 combination in hACE2 transgenic mice.
  • Figure 22C shows the viral load (Viral burden) in the lungs and brain analyzed by the focus formation assay (FFA) at 2 and 4 dpi, the dotted line represents the limit of detection (LOD).
  • Figure 23 shows the mutation sites on the RBD of various SARS-CoV-2 variant strains.
  • Figure 24A shows the crystal structure of BA.2 Spike trimer in complex with BA7535-Fab.
  • Figure 24B shows the crystal structure of BA.2 RBD and BA7535-Fab complex.
  • Figure 25 shows that BA7535 partially overlaps with the binding epitope of RBD and ACE2.
  • Figure 26 shows a comparison of the crystal structures of RBD/BA7535-Fab and REGN10987 (PDB ID: 6XDG).
  • Spike RBD antigen proteins of mutant strains There are 5 kinds of Spike RBD antigen proteins of mutant strains, which are B.1.617.1 RBD, B.1.617.2 RBD, B.1.351 RBD and p.1 RBD protein, B.1.1.529.1 RBD, and 2 kinds of mutant strains
  • the Spike proteins are B.1.617.2 Spike and B.1.351 Spike proteins, and each mouse was immunized according to the method in Table 3.
  • mice were killed, the spleen was dissected, and the spleen was crushed with a syringe rubber stopper and filtered through a filter.
  • the filtered spleen cells were frozen for preparation, and the cDNA was obtained after RNA extraction.
  • the establishment of the phage library was carried out according to the usual method.
  • the storage capacity data of the constructed library are shown in Table 4.
  • Table 4 The capacity of the phage library constructed by immunizing mice with each virus strain
  • 177 positive IgG1 clones were constructed and sequenced.
  • the amino acid sequences of the variable regions of the 4 lead antibodies are shown in Table 6: (CDR regions are underlined, and the analysis system is the IMGT system).
  • BA7125V1 is an antibody obtained by a point mutation in the variable region of the heavy chain of BA7125 .
  • the antibody variable region gene was amplified by conventional molecular biology technique PCR (2 ⁇ Phanta Max Master Mix manufacturer: Vazyme product number: P515-P1-AA batch number: 7E351H9), and the antibody heavy chain variable region gene was separately synthesized by homologous recombination.
  • the antibody light chain variable region gene was connected into the vector pCDNA3.4 with the nucleic acid sequence of the antibody light chain constant region sequence.
  • the positive clones after sequencing were extracted with plasmids, co-transfected into HEK293 cells and cultured in a shaker at 37°C ⁇ 8%CO 2 ⁇ 125rpm. After transient expression for 7 days, the supernatant was purified by Protein A affinity chromatography to obtain antibodies, and passed UV280 combined with theoretical extinction coefficient to determine antibody concentration.
  • Table 9 detects the protein binding sensitivity of antibody drug blocking B.1.351 (also known as Beta (Beta) strain) Spike RBD and hACE2
  • Table 10 detects the protein binding sensitivity of antibody drug blocking B.1.617.2 (also known as Delta (Delta) strain) Spike RBD and hACE2
  • each antibody can block B.1.351 (also known as Beta (Beta) strain) RBD protein and B.1.617.2 (also known as Delta (Delta) strain) RBD protein and ACE2
  • B.1.1.529.1 also known as Omicron (Omicron) BA.1 strain
  • BA7208 and BA7134 can block its binding to ACE2, and other antibodies have no blocking activity .
  • the detection method is the same as 3.2.1, except that the Spike RBD protein includes Gamma (p.1) strain, Delta (B.1.617.2) strain, Omicron B.1.1.529.1 (ie BA.1 strain), Lambda (C.37) strain, Mu (B.1.621), Kappa (B.1.617.1) strain, C.12 strain, B.1.630 strain, B.1.640 strain, wild type strain ( That is, Wuhan-Hu-1 strain or Original WT), Beta (B.1.351) RBD protein, each RBD protein was purchased from Beijing Sino Biological, and the test results are shown in Figure 3D. It can be seen from Figure 3D that the antibody BA7535 can block Break the binding of each strain protein to hACE2.
  • the IC50 ( ⁇ g/mL) of BA7535 blocking the binding between RBD and ACE2 of each mutant strain is shown in Table 10-1.
  • the detection method is the same as 3.2.1, the difference is that Spike RBD proteins include P.1, B.1.617.2, C.37, B.1.621, B.1.617.1, C.1.2, B.1.630, B.1.640 , RBD proteins of the BA.1 strain, and each RBD protein was purchased from Beijing Yiqiao Shenzhou.
  • the test results are shown in Figures 3E-3M. It can be seen from Figure 3E-3M that the antibody BA7535 is effective against 9 SARS-CoV-2 variants (P .1, B.1.617.2, C.37, B.1.621, B.1.617.1, C.1.2, B.1.630, B.1.640, BA.1) RBDs all exhibited broad blocking activity.
  • the Spike RBD protein of each strain was serially diluted 2-fold with HBS-EP + 1 ⁇ (cytiva, BR-1006-69) buffer, starting at 50nM, 2-fold dilution 4 concentration gradients, and set 0 concentration. Startup 3 times.
  • Antibody 2 ⁇ g/ml, injection time 100s, flow rate 10 ⁇ L/min, capture with ProA chip (cytiva, 29127556); antigen protein: binding 120s, flow rate 30 ⁇ L/min, dissociation 600s; regeneration: regeneration with MgCl 2 buffer for 30s , flow rate 30 ⁇ L/min.
  • association constant (ka) and dissociation constant (kd) were calculated using the 1:1binding binding model (BIAcore Evaluation Software version 3.2), and the equilibrium dissociation constant (KD) was calculated as the ratio kd/ka. Affinity data are shown in Table 11 and Table 12, respectively.
  • Table 12 shows the comparison of the affinity of the monoclonal antibody and the double antibody.
  • BA7208-7125V1 For the sequence and structure information of the double antibody BA7208-7125V1, please refer to the following examples 4-5. Binding kinetics of the RBD of the B.1.529.1 and B.1.621 variants, see Figures 4A-4I for results. From Table 12 and Figures 4A-4I, it can be seen that the affinity of the double antibody is better than that of the two maternal monoclonal antibodies, and the advantages of the two maternal monoclonal antibodies are combined to have the best broad-spectrum.
  • the detection method is the same as 3.3.1, and the results are shown in Figure 4J-4K. It can be seen that the affinity of BA7535 to BA.1 RBD is higher than that of BA7208, and the measured equilibrium constants (KD) are 0.10 ⁇ 0.02nM and 1.81 ⁇ 0.26nM, respectively.
  • VSV vesicular stomatitis virus
  • the reagent consumables are shown in Table 12. After 50 ⁇ L of pseudoviruses and 100 ⁇ L of the antibody to be tested were incubated at 37°C for 1 hour, 100 ⁇ L of 293T-ACE2 cells were infected. Quantity: 4E5cells/well, after incubation at 37°C for 20-28h, the luminescence value RLU of Luciferase was detected by chemiluminescence method, and the pseudovirus inhibition rate of the antibody to be tested was calculated according to the RLU reading value.
  • the buffer system of the antibody is pH7.4, 0.01M PBS buffer solution
  • the reagent consumables are shown in Table 13
  • the test results of the pseudovirus neutralization activity of each antibody are shown in Table 14.
  • BA7208, BA7125V1, BA7054, and BA7134 have a wide range of pseudovirus neutralizing activities.
  • BA7208 and BA7125V1 have broad-spectrum and complementary neutralizing activities. Except for B.1.640 and Mu, BA7208 could neutralize 11 of the 13 mutants with IC50 ranging from 2.81ng/mL to 6.22ng/mL, and And it has the best neutralizing activity on BA.1 and BA.2, with IC50 of 3.52ng/mL and 3.43ng/mL, respectively.
  • BA7125V1 can neutralize 11 mutants except 2 Omicorn mutants, with IC50 ranging from 11.36ng/mL to 42.31ng/mL.
  • BA7054 can neutralize nine mutant strains except Mu, Omicorn and B.1.640, with IC50 ranging from 4.47ng/mL to 10.11ng/mL.
  • BA7134 can neutralize 11 mutant strains except B.1.640 and Mu, with IC50 ranging from 3.81ng/mL to 147.60ng/mL.
  • BA7208, BA7125V1 and BA7054 have significantly higher neutralizing activities than VIR-7381, REGN10933 and REGN10987.
  • the double-antibody BA7208-7125V1 has the broadest spectrum and can neutralize all 13 mutant strains, with IC50 ranging from 2.33ng/mL to 116.10ng/mL; the double-antibody BA7208-7125V1 neutralizes most active viruses
  • the activity of the best monoclonal antibody in the strain was slightly lower than that of BA7208 and BA7125V1 (the activity of 10.8ng/mL was also very good); on the key strain Omcicron, the activity of the double antibody BA7208-7125V1 was 3 times lower than that of the monoclonal antibody BA7208.
  • the combination of BA7125V1+BA7208 is similar to the double antibody BA7208-7125V1, and also has the broadest spectrum of neutralizing activity, which can make up for the respective disadvantages of the two monoclonal antibodies.
  • Figure 5I shows that antibody BA7535 has neutralizing activity against BA.1.1 and BA.2 SARS-Cov-2 pseudoviruses. It can be seen that BA7535 has BA.1.1 and BA.2 SARS-Cov-2 pseudoviruses. Good neutralizing activity.
  • Table 15 shows the SARS-Cov-2 pseudovirus IC50 ( ⁇ g/mL) of antibody BA7535 to BA.1.1 and BA.2
  • BA7208 has broad-spectrum and excellent neutralizing activity, with IC50 ⁇ 10 ng/mL for 15 tested strains.
  • BA7208 has broad-spectrum and excellent neutralizing activity against Omicron mutants, and the neutralizing activity IC50 of BA.2.75 among the detected strains is ⁇ 10ng/mL.
  • Pseudovirus neutralization activity test steps 50 ⁇ L of each new coronavirus pseudovirus (purchased from Beijing Sanyao) was mixed with 100 ⁇ L of serially diluted BA7208 antibody and incubated at 37°C for 1 hour. 50 ⁇ L+100 ⁇ L DMEM was used as a negative control, and 150 ⁇ L DMEM was used as a blank.
  • the pseudovirus neutralizing activity data of the BA7535 antibody is shown in Table 18, where BA.4/5 represents BA.4 and BA.4, and the RBD mutations of BA.4 and BA.5 are the same, so the pseudoviruses of BA.4 and BA.5 Like a virus.
  • BA7535 has broad-spectrum and excellent neutralizing activity, with IC50 ⁇ 10 ng/mL for 19 tested strains.
  • BA7535 and BA7208 in the antibody combination, the molar ratio of BA7208 and BA7535 is 1:1; the two antibodies in the antibody combination are mixed and administered together
  • BA7208 has excellent neutralizing activity against true viruses of Omicron BA.1 and BA.2 mutant strains
  • BA7208 and the double antibody BA7208/7125V1 have a strong neutralizing effect on SARS-CoV-2 variants Omicron BA.1 and BA.2, with IC50 values between 0.0329 and 0.316 ⁇ g/ml. It can be seen from Table 21-1 that the IC50 values of the BA7208 antibody for the neutralization of true viruses of Omciron BA.1 and BA.2 mutant strains are: 53.2 and 18.17 ng/mL, respectively.
  • BA7535 has excellent euvirus neutralizing activity against Omicron BA.1, BA.2, and BA.5 mutant strains
  • Balb/c mice (body weight 25 ⁇ 3g) were used to inject five kinds of antibodies (BA7208, BA7125V1, BA7054, BA7134 and BA7208/7125V1 double antibodies) intravenously at 10 mg/kg respectively, and 5 minutes after the end of administration, 30 Minutes, 1 hour, 4 hours, 8 hours, 1 (24h), 3 (72h), 5 (120h), 7 (168h), 10 (240h), 14 (336h) days, blood samples were collected, and serum levels were determined by ELISA method.
  • Pharmacokinetic parameters were calculated with Phoenix WinNonlin 6.4 for drug concentration. The pharmacokinetic parameters of each antibody are shown in Table 22, and the pharmacokinetic curve is shown in Figure 12A. The results showed that, except for BA7054, all antibodies had good metabolic stability in mice.
  • Figure 12B shows the pharmacokinetic test results of multiple administrations of BA7208 in rats.
  • the mouse T1/2 of LY-CovMab is 9.5 days
  • the human T1/2 is 28 days
  • the rat T1/2 of BA7208 is 8.4-11.2 days
  • the human T1/2 can reach 28 days.
  • the IC50 of BA7208 against BA.2 true virus is 18.17ng/mL (effective concentration)
  • the blood concentration will still be higher than the IC50 after half a year of prophylactic administration in the human body, predicting the protection of a single administration to the human body The effect can last for more than half a year.
  • mice (body weight 25 ⁇ 3g)
  • inject antibody BA7535 once at 10 mg/kg intravenously, and after the end of administration, 5 minutes, 1 hour, 6 hours, 1 (24h) day, 3 (72h) day , 5 (120h) days, 7 (168h) days, 10 (240h) days, 14 (336h) days
  • blood samples were collected, the drug concentration in serum was determined by ELISA method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin6.4.
  • the pharmacokinetic curve is shown in Figure 12C, and it can be seen that the BA7535 antibody has good metabolic stability in mice.
  • BA7535 showed satisfactory half-life and AUC(0-t), the terminal half-life (t 1/2 , ⁇ z) was about 95 hours, and AUC(0-t) was about 12785 hours* ⁇ g/mL. See Table 24-1 below for the results.
  • the mouse PK experiment process of the double antibody BA7208/7535-linker 4 is the same as the mouse PK experiment of 3.6.3 BA7535 monoclonal antibody, and the tail vein injection was administered.
  • the results are shown in Figure 12G.
  • the terminal half-life (t1/2, ⁇ z) is about 92.3 hours, and AUC(0-t) is about 12108 hours* ⁇ g/mL, similar to monoclonal antibodies.
  • PK parameters of the double antibody BA7208/7535-linker 4 are as follows in Table 24-2:
  • ADCC reporter bioassay was performed using CHO-K1-Spike cells (Genscript) as target cells and Jurkat cells (G7011, Promega) as effector cells.
  • CHO-K1-Spike cells (Genscript) were used as target cells and Jurkat-FcyRIIA-H131 cells (Vazyme) as effector cells.
  • FIG. 13 The schematic diagram of the structure of the anti-2019-nCoV (or SARS-CoV-2) double antibody of the present application is shown in Figure 13.
  • Figure 13 is only used as an example and should not be construed as a limitation of the present application.
  • the structure of the right half is A-linker (linker) 1-B-linker (linker) 2-CH2-CH3.
  • the double antibody of this application is divided into two structures: linker 4 and linker 6.
  • linker 4 and linker 6 are shown in Table 25:
  • Heavy chain 1 (scFv-knob chain): Amplify the heavy chain of the antibody (such as 7125V1 antibody) by conventional molecular biology techniques PCR (2 ⁇ Phanta Max Master Mix manufacturer: Vazyme, article number: P515-P1-AA).
  • the antibody (such as 7125V1 antibody) heavy chain variable region gene and light chain variable region gene are linked by linker (such as SEQ ID NO: 32) by overlapping PCR technology
  • linker such as SEQ ID NO: 32
  • the heavy chain (knob chain) constant region sequence of the antibody was linked together by homologous recombination (ClonExpressII rapid cloning kit (Vazyme, product number: C113-02-AB), and finally linked into the vector pCDNA3.4 (Life Technology)
  • the amino acid sequence of the heavy chain variable region of the 7125V1 antibody is SEQ ID NO: 35
  • the amino acid sequence of the light chain variable region of the 7125V1 antibody is SEQ ID NO: 3, see Table 6
  • the antibody heavy chain (knob chain) constant region The sequence is SEQ ID NO:33, as shown in Table 26.
  • the heavy chain 2 (hole chain) PCR amplification of the heavy chain variable region gene of the antibody (such as BA7208) by conventional molecular biology techniques, and then linking with the antibody heavy chain (hole chain) constant region sequence through homologous recombination Together, finally connected into the vector pCDNA3.4 (Life Technology); light chain construction: the light chain variable region gene of the antibody (such as BA7208) is connected into the vector pCDNA3.4 with the nucleic acid sequence of the antibody light chain constant region sequence .
  • the amino acid sequence of the heavy chain variable region of the BA7208 antibody is SEQ ID NO:8, and the amino acid sequence of the light chain variable region of the BA7208 antibody is SEQ ID NO:7, see Table 6.
  • the antibody heavy chain (hole chain) constant region sequence is SEQ ID NO: 34, as shown in Table 26; the antibody light chain constant region sequence is SEQ ID NO: 31, as shown in Table 7.
  • Heavy chain 1 and heavy chain 2 are integrated into a heterodimer by Knob into hole technology.
  • the constant region sequence of the anti-2019-nCoV (or SARS-CoV-2) double antibody molecule is shown in Table 26.
  • Table 26 The constant region sequence of anti-2019-nCoV (or SARS-CoV-2) double antibody molecule
  • biotin-labeled ACE2 protein Novoprotein, C05Y
  • 50 ⁇ L/well 50 ⁇ L/well
  • incubate at 37°C for 1 h
  • add STREP/HRP diluted in PBST 100 ⁇ L/well, and incubate at 37°C for 1 h.
  • 100 ⁇ L TMB 100 ⁇ L to each well for color development
  • 50 ⁇ L 2M H 2 SO 4 to each well after 10 min to stop color development, and read OD450 with a microplate reader.
  • FIG 14A and Figure 14B show that BA7208-7125V1-linker 4 and BA7208-7125V1-linker 6 can block two kinds of Spike RBD proteins (B.1.1.529RBD (Omicron) protein, B.1.621 (Miao) RBD protein) combined with ACE2, IC50 are shown in Table 27 below.
  • the structures of BA7208-7125V1-Linker 4 and BA7208-7125V1-Linker 6 are illustrated in Example 4.
  • BA7208-7125V1-linker 4 and BA7208-7125V1-linker 6 block the binding of two Spike RBD proteins to ACE2
  • the binding kinetics of the antibody to the Spike RBD protein of each mutant strain was measured using a BIAcore 8K instrument based on surface plasmon resonance (surface plasmon resonance, SRP) technology.
  • the Spike RBD protein of each strain was serially diluted 2-fold with HBS-EP + 1 ⁇ (cytiva, BR-1006-69) buffer, starting at 50nM, 2-fold dilution 4 concentration gradients, and set 0 concentration. Startup 3 times.
  • Antibody 2 ⁇ g/ml, injection time 100s, flow rate 10 ⁇ L/min, capture with ProA chip (cytiva, 29127556); antigen protein: binding 120s, flow rate 30 ⁇ L/min, dissociation 600s; regeneration: regeneration with MgCl 2 buffer for 30s , flow rate 30 ⁇ L/min.
  • association constant (ka) and dissociation constant (kd) were calculated using the 1:1binding binding model (BIAcore Evaluation Software version 3.2), and the equilibrium dissociation constant (KD) was calculated as the ratio kd/ka.
  • KD equilibrium dissociation constant
  • VSV vesicular stomatitis virus
  • VSV vesicular stomatitis virus vectors were used to package the pseudoviruses of the S proteins of each strain, and the reagent consumables are shown in Table 13. After 50 ⁇ L of pseudoviruses and 100 ⁇ L of the antibody to be tested were incubated at 37°C for 1 hour, 100 ⁇ L of 293T-ACE2 cells were infected. Quantity: 4E5cells/well, after incubation at 37°C for 20-28 hours, the Luciferase luminescence value RLU was detected by chemiluminescence, and the pseudovirus inhibition rate of the antibody to be tested was calculated according to the RLU reading value. The results of the antibody pseudovirus neutralization activity test are shown in Table 18.
  • the double antibody BA7208-7125V1-Linker 4 has a wide range of pseudovirus neutralizing activity, and can well neutralize the B.1.617.2 (Delta) strain, B.1.1.529 (Omicron) strain and B.1.351 (beta) strain, IC50 were 0.012nM, 0.017nM and 0.010nM, see Table 29.
  • the double antibody can combine the advantages of the two monoclonal antibodies into one body, showing broad-spectrum and excellent neutralizing activity.
  • the experimental method is as follows: Omicron S protein (Omicron Spike Trimer) (40589-V08H26, Sino Biological) or Delta S protein (Delta Spike Trimer) (40589-V08H10, Sino Biological) was mixed with antibody Fab and incubated on ice for 40 minutes , after centrifugation at 16200g, 4°C for 5min, molecular sieve purification was carried out with GE micro Akta and Superose 6 columns, and single peak samples were collected.
  • Figure 16 shows the cryo-electron microscope observation of Delta Spike Trimer with BA7208-Fab and BA7125V1-Fab, and the BA7125-Fab in Figure 16 is the Fab of BA7125V1 described in the above examples.
  • Figure 16a Three BA7208-Fabs (green) and three BA7125V1-Fabs (yellow) in complex with Delta Spike Trimer (dodger blue, plum, and rosy brown).
  • Figure 16b BA7208-Fab (light green, Fab light chain; dark green, Fab heavy chain) in complex with Delta Spike RBD (blue).
  • Figure 16c BA7125V1-Fab (light yellow, Fab light chain; dark yellow, Fab heavy chain) in complex with Delta Spike RBD (blue).
  • Figure 16d Enlarged view of the binding site of Fabs on the Delta RBD, showing the side chains of the hydrogen-bonding residues, the salt bridge and the interaction of a cation.
  • Figure 17 shows the cryo-EM observation of Omicron Spike Trimer with BA7208-Fab.
  • Figure 17a Complexes of three BA7208Fabs (green) with Omicron Spike Trimer (dodger blue, plum, and rosy brown).
  • Figure 17b BA7208-Fab (light green, Fab light chain; dark green, Fab heavy chain) in complex with Omicron Spike RBD in down conformation (dark purple).
  • Figure 17c Complex of BA7208-Fab with Omicron Spike RBD in up conformation (blue).
  • Figure 17d Enlarged view of the binding site of 7208-Fab on the Omicron RBD, showing the side chains of the hydrogen-bonding residues, the salt bridge and the interaction of a cation.
  • the BA7208 monoclonal antibody binds to the T345, R346, and K444 residues on the Delta RBD; the BA7125V1 monoclonal antibody binds to the R403, K417, and Y453, K458, G476, Y489, F490, Y505 residues; BA7208 mAb binds to T345, R346, K440, S443, K444 residues on Omicorn BA.1 RBD.
  • Figure 19A-H shows the structural overlay analysis of BA7208/BA7125V1
  • the BA7125 on Figure 19 is the BA7125V1 described in the above examples.
  • the complex of RBD (cyan) and BA7208-Fab (green) is superimposed on the complex of RBD (cyan) and ACE2 (blue), and it can be seen that the binding sites of BA7208 and ACE2 do not overlap.
  • the complex of RBD (cyan) and BA7125V1-Fab (yellow) is overlaid with the complex of RBD (cyan) and ACE2 (blue) in Figure 19B.
  • the complex of RBD (cyan) and BA7054-Fab (pink) is overlaid with the complex of RBD (cyan) and ACE2 (blue) in Figure 19C.
  • the complexes of RBD (cyan) and BA7208 Fab (green) in Figure 19D-G were overlaid with the complexes of RBD (cyan) and antibodies (blue) REGN10987, VIR-7381, LY-Cov1404 and A23-58.1, respectively.
  • Figure 19D-F shows that BA7208-Fab has similar binding modes to REGN10987, broad-spectrum antibody VIR-7381 and LY-CoV1404.
  • Figure 19H The complex of RBD (cyan) and BA7125V1-Fab (yellow) is overlaid with the complex of RBD (cyan) and A23-58.1 antibody (blue).
  • BA7208-Fab and BA7054-Fab did not directly prevent the binding of ACE2 to Spike-RBD through epitope overlap, while BA7125V1-Fab directly competed with ACE2.
  • BA7208-Fab and BA7054-Fab is inconsistent with their pronounced blocking activity as the IgG form, suggesting bivalent binding of an intact IgG molecule to the 2 RBDs of a Spike or to RBDs coated in plates Bound intact antibody molecules may sterically interfere with ACE2 binding to the RBD.
  • the mutation of the virus on SPIKE-RBD tends to occur in the ACE2 binding region, and the BA7208 epitope outside the ACE2 binding region indicates that the risk of being affected by the mutation will be reduced in the future.
  • each of the four columns represents the binding signal intensity of the chip binding to the omicron RBD, first binding to antibody A, and then binding to antibody B.
  • the first column represents that RBD first reacts with buffer (buffer), and then binds to BA7208, and the signal intensity can reach about 1.2.
  • the second column represents that RBD first binds to BA7208 to saturation, and then binds to BA7208 again.
  • the third column represents the first binding to the buffer (buffer) and then binding to BA7535.
  • the binding signal can reach About 1.2
  • the fourth column represents that RBD first binds to BA7208 to saturation, and then binds to BA7535.
  • the binding signal can still reach about 1, indicating that the binding epitopes of BA7208 and BA7535 are different, and BA7208 does not interfere with the binding of RBD and BA7535, that is, BA7535 , BA7208 will not compete with each other, and has the potential to be used in combination for the treatment of COVID-19.
  • Embodiment 7 BA7208 the application pharmaceutical composition (injection form) preparation
  • BA7208 antibody solution Concentrate the BA7208 antibody solution to 30-70g/L through a 30KD ultrafiltration membrane bag after virus removal and filtration, and then use a dialysis buffer (9.5mM histidine, 10.5mM histidine hydrochloride, pH 5.5-6.5) to deconcentrate the buffer solution. Replacement, dialysis volume 6-8 times, control TMP ⁇ 1.5bar throughout the process, and then concentrate to 70-100g/L, then wash out the BA7208 antibody solution from the ultrafiltration system to ensure that the BA7208 antibody protein concentration is above 55g/L.
  • a dialysis buffer 9mM histidine, 10.5mM histidine hydrochloride, pH 5.5-6.5
  • auxiliary material mother solution (9.5mM histidine, 10.5mM histidine hydrochloride, 32% trehalose, 0.08% polysorbate 80 (II), pH5.5-6.5)
  • dialysis Buffer (9.5mM histidine, 10.5mM histidine hydrochloride, pH 5.5-6.5) dilute the antibody protein solution 40.0 ⁇ 4.0mg/mL
  • the pharmaceutical composition (9.5mM histidine, 10.5mM histidine hydrochloride, 8% trehalose, 0.02% polysorbate 80 (II), and 40.0 ⁇ 4.0 mg/mL of the BA7208 antibody.
  • the prescription composition of the pharmaceutical composition of the present application (which contains 40.0 ⁇ 4.0 mg/mL of antibody) is shown in Table 34:
  • Omicron BA.1 and BA.2 live viruses infected Balb/C wt mice or the hACE2 transgenic mouse K18 model of new coronary pneumonia, and the lungs of the mice were taken the next day after the challenge, and the live viral load in the lungs was detected by FFA method for evaluation Therapeutic or prophylactic protective effects of antibodies.
  • mice The route of injection and the grouping of mice are shown in Table 35 below:
  • BA7208 can effectively prevent and treat Omicron infection through nasal drops and aerosol inhalation
  • K18hACE2 transgenic mice were purchased from Jiangsu Jicui Yaokang, and the Omicron mutant strain BA.2 was isolated and preserved by the Biosafety Level 3 Laboratory of Guangzhou Customs Technology Center. True virus-related experiments were carried out by the Guangzhou Institute of Respiratory Health in a BSL-3 laboratory.
  • BA7208 sample is: 9.5mM histidine, 10.5mM histidine hydrochloride, 8% trehalose, 0.02% polysorbate 80 (PS80), the concentration of antibody BA7208 is 38.905mg/mL.
  • mice were dissected, and the lungs of the mice were put into 1mL PBS buffer for tissue grinding.
  • the ground tissue fluid was centrifuged, and the supernatant was taken to detect the titer of the new coronavirus.
  • the detection method was the FFA method.
  • mice The way of administration by nebulization route and the grouping of mice are shown in Table 37 below:
  • mice The in vivo preventive and therapeutic efficacy of BA7535 and BA7535/BA7208 cocktail (i.e. BA7535 and BA7208 monoclonal antibody combination) against SARS-CoV-2 Omicron BA.5 was evaluated in K18-hACE2-transgenic mice (Jicui Yaokang) .
  • Six to eight-week-old mice were injected intraperitoneally with 2 mg/kg or 10 mg/kg of BA7535 or BA7535+BA7208 monoclonal antibody 24 hours before or 8 hours after infection with 1 ⁇ 10 5 FFU SARS-CoV-2 Omicron BA.5 combination.
  • mice injected with phosphate-buffered saline (PBS) were infected with the same dose of SARS-CoV-2 as a control.
  • virus titers in the lungs and brain were collected using the foci formation assay (FFA) 2 and 4 days after infection.
  • FFA foci formation assay
  • lung tissues were collected and stained for histopathological analysis, and body weight changes were monitored.
  • the SARS-CoV-2 Omicron BA.5 strain was provided by the Guangdong Provincial Center for Disease Control and Prevention, China. Experiments related to the real SARS-CoV-2 virus were carried out in the ABSL-3 laboratory of the Technical Center of Guangzhou Customs District.
  • Example 9 BA7208 is not sensitive to more than 30 mutation sites on the RBD of each mutant strain except R346K
  • the key mutation sites of RBD in 19 SARS-CoV-2 virus variants are shown in Figure 23. Taking antibodies Vir-7381, REGN10933 and REGN10987 as reference, it was evaluated whether four antibodies, BA7208, BA7125V1, BA7054 and BA7134, could effectively inhibit these variants. As shown in Figure 23, light gray indicates that our antibody has neutralizing activity at this site, and black indicates that our antibody has no neutralizing activity. It can be seen that BA7208 is not sensitive to other mutation sites on RBD except R346, and has a broad spectrum.
  • Sample expansion stage expand the sample size according to the candidate dose determined in the first stage, and conduct preliminary drug efficacy research.
  • the test was divided into two groups: the administration group and the placebo group, and the difference in the positive rate of new crown infection between the administration group and the placebo group was observed.
  • Omicron BA.2 Spike protein was mixed with antibody Fab, incubated on ice for 20 minutes, and single peak samples were collected after molecular sieve purification. Place 3.5 ⁇ L sample on a glow discharge grid supported by a thin layer of graphene oxide, blot the moisture with filter paper, and quickly freeze it with liquid ethane with a Thermo scientific Vitrobot Mark IV instrument, and freeze it with a Thermo Fisher Titan Krios G3i electron microscope Electron microscopy imaging. Carry out multiple rounds of 2D and 3D classification and refinement on the imaged particle data, and obtain crystal structure data after further data processing and 3D modeling.
  • the binding epitope of BA.2 RBD and BA7535 includes 7 residues including T415, D420, Y421, A475, N487, Y489 and R493 group (Table 38), forming 6 hydrogen bonds and 1 salt bridge, respectively T415-Y106, D420-Y106, Y421-L103, A475-T28, N487-R98 and Y489-R98 and R493-E50 (in front of RBD amino acid, followed by BA7535 amino acid).
  • the binding epitope of BA7535 can avoid most RBD mutations, and only the mutation at F486, as well as N417T and R493Q mutations may affect the binding of BA7535 to RBD.
  • the binding regions of the two antibodies to RBD were analyzed by PISA, and BA7535 had a larger binding region than REGN10987, and the binding region of BA 7535 was while REGN10987 is Compared with 6 hydrogen bonds and 1 salt bridge in the BA7535 epitope, only 4 hydrogen bonds and 1 salt bridge were formed in the REGN10987 epitope, indicating that BA7535 has a higher affinity for RBD than REGN10987.

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Abstract

本发明涉及一种SARS-CoV-2病毒的广谱抗体，所述广谱抗体能结合SARS-CoV-2病毒上的S蛋白，阻断SARS-CoV-2病毒引起的细胞病变或者中和SARS-CoV-2病毒。本发明还涉及编码所述广谱抗体或其抗原结合片段的核酸；含有所述核酸的细胞；含有所述广谱抗体或其抗原结合片段、所述核酸、所述细胞的药物组合物；含有所述广谱抗体或其抗原结合片段、所述核酸、所述药物组合物的试剂盒；以及所述广谱抗体或其抗原结合片段、所述核酸、所述药物组合物在预防、治疗、检测或诊断与SARS-CoV-2病毒相关的疾病的应用。

Description

SARS-CoV-2病毒的广谱抗体及其应用 技术领域
本发明涉及生物医学或生物制药技术领域，尤其涉及一种SARS-CoV-2病毒的广谱抗体及其应用。
背景技术
新型冠状病毒(2019-nCoV，又称SARS-CoV-2)在全球范围内引起的疾病COVID-19(Corona Virus Disease-19)，对人类的生命健康带来了极大的威胁，对全球的经济发展也造成了无法估量的损失。
SARS-CoV-2病毒表面的S蛋白(spike protein)组合成一个三聚体，单个S蛋白约含有1300个氨基酸，属于一类膜融合蛋白，决定病毒的宿主范围和特异性，是宿主中和抗体的重要作用位点。S蛋白含有两个亚基(subunit)S1和S2,S1主要包含有受体结合区(receptor binding domain,RBD)，负责识别细胞的受体,S2含有膜融合过程所需的基本元件。目前的研究报道显示，SARS-CoV-2病毒的S1亚基的RBD区可以通过结合人体细胞表面的ACE2(血管紧张素转换酶2)启动感染(亲和力达15nM)，在S1/S2位点发生酶切后S1亚基脱离，暴露S2酶切位点并进行酶切，发生一系列构象改变后利用FP(融合肽)启动病毒与细胞的膜融合。
新冠病毒(SARS-CoV-2)的突变是病毒的进化方式。在不破坏SARS-CoV-2关键生化表型的前提下，通过抗体靶向位点固定突变，引发抗原表位漂移，以逃避相关抗体的识别。目前已报到的SARS-CoV-2主要突变株包括英国变异株B.1.1.7、南非变异株B.1.351以及巴西变异株P.1。
B.1.1.7的新冠病毒变体是2020年12月英国向世卫组织报告的一种新冠病毒变体，其传播能力要比原始毒株高70％左右，伦敦新冠感染病例超过60％都来自变异病毒。该病毒株最大特点是出现了十几个关键位点的变异，在B.1.1.7突变株谱系中，最值得注意的突变位点是RBD(受体结合区域)中的关键氨基酸突变N501Y。病毒蛋白的结构发生改变后跟人体当中的ACE2受体更容易结合，带来的实际后果就是它的传播力更强。
南非变异株B.1.351出现于2020年8月，截至2020年12月底，南非由B.1.351引起的感染比例已超80％。巴西变异株P.1于2020年12月初在巴西玛瑙斯市出现，到2021年1月中旬已经造成整个城市疫情的大规模爆发。南非变异株B.1.351和巴西变异株P.1除均含有D614G、N501Y以外，还发现存在E484K变异，该突变具有减弱抗病毒中和抗体的效果，也可能导致病毒逃过免疫系统的识别。
缪(B.1.621)首先在哥伦比亚发现,具有T95I,Y144S，Y145N，R346K，E484K,N501Y,D614G，P681H,D950N突变，具有免疫逃逸的潜在特性，该变异毒株同时表现出较强传染性，与此前分别在英国和南非发现的两种变异毒株存在类似特征。
德尔塔变异株B.1.617.2最早于2020年10月在印度发现，其发生了13个突变位点。除了与Alpha、Beta和Gamma突变株一样有D614G突变，Delta突变株还有一些独有的突变，尤其是L4524，使得新冠病毒棘突蛋白与人体细胞ACE2受体有更强的亲和力；其P681R，则可通过促进前棘突蛋白裂解为活性S1/S2构型从而增强突变株感染细胞能力。
奥米克戎变异株B.1.1.529于2021年11月最先于南非等地发现，同时具有前4个VOC 变异株Alpha(阿尔法)、Beta(贝塔)、Gamma(伽玛)和Delta(德尔塔)刺突蛋白的重要氨基酸突变位点，同时存在“K417N+E484A+N501Y”三重突变，包括增强细胞受体亲和力和病毒复制能力的突变位点。此外，奥密克戎变异株还存在其他多个可能降低部分单克隆抗体中和活性的突变流行病学和实验室监测数据显示南非感染奥密克戎变异株病例数激增以及部分取代Delta变异株，传播力有待进一步监测研究。
针对上述新型冠状病毒，开发特异性抗体阻断病毒S蛋白对宿主细胞的感染对于新冠肺炎的预防和治疗具有重要的意义。
发明内容
本发明提供一种广谱抗体或其抗原结合片段,能结合SARS-CoV-2病毒上的S蛋白，阻断SARS-CoV-2病毒引起的细胞病变或者中和SARS-CoV-2病毒。本发明还提供了衍生自所述广谱抗体或其抗原结合片段的多特异性抗体、双特异性抗体、抗体组合，以及编码所述广谱抗体或其抗原结合片段、所述多特异性抗体、所述双特异性抗体的核酸，或核酸组合；含有所述核酸、所述核酸组合的细胞；含有所述广谱抗体或其抗原结合片段、所述多特异性抗体、所述双特异性抗体、所述抗体组合、所述核酸、所述核酸组合、所述细胞的药物组合物；含有所述广谱抗体或其抗原结合片段、所述多特异性抗体、所述双特异性抗体、所述抗体组合、所述核酸、所述核酸组合、所述药物组合物的试剂盒；以及所述广谱抗体或其抗原结合片段、所述多特异性抗体，所述双特异性抗体，所述抗体组合、所述核酸、所述核酸组合、所述药物组合物在预防、治疗、检测或诊断与SARS-CoV-2病毒相关的疾病的应用。
本发明的一个方面提供一种广谱抗体或其抗原结合片段，所述抗体结合新型冠状病毒(即SARS-CoV-2,又称2019-nCoV)上的S蛋白，阻断SARS-CoV-2病毒引起的细胞病变或者中和SARS-CoV-2病毒。
在本申请的方案中，所述新型冠状病毒，即SARS-CoV-2或2019-nCoV为2019年首次发现的新冠病毒原始毒株与后续出现的新冠病毒突变株的统称。进一步的，所述SARS-CoV-2病毒VOCs毒株，VOIs毒株，VUMs毒株，以及野生型毒株中的一种或多种；更优选的，所述VOCs毒株包括B.1.1.7毒株、B.1.351毒株、p.1毒株、B.1.617.2毒株、BA.1毒株、BA.1.1毒株、BA.2毒株、BA.2.12.1毒株、BA.2.13毒株、BA.2.75毒株、BA.3毒株、BA.4毒株和BA.5毒株中的一种或多种；所述VOIs毒株包括C.37毒株、B.1.621毒株中的一种或多种；所述VUMs毒株包括B.1.617.1毒株，B.1.640毒株，C.1.2毒株，B.1.630毒株中的一种或多种；所述野生型毒株为Wuhan-Hu-1毒株。
在本申请的方案中，进一步的，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,AZ.5毒株,以及野生型毒株中的一种或多种；优选的，所述野生型毒株为Wuhan-Hu-1毒株；优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB毒株中的一种或多种。
进一步的，所述S蛋白为SARS-CoV-2病毒表面的S蛋白(spike protein)，所述S蛋白含有两个亚基(subunit)S1和S2。更进一步的，所述抗体能结合所述SARS-CoV-2病毒上 的S蛋白指的是结合S蛋白的S1和S2亚基中的一个或多个，或结合S1亚基的RBD蛋白。
进一步的，本申请中所述抗体或抗原结合片段能阻断SARS-CoV-2病毒引起的表达ACE2细胞的病变，或阻断SARS-CoV-2病毒对表达ACE2的细胞的感染、入侵等。更进一步的，所述细胞包括天然表达ACE2的细胞或人工表达ACE2的细胞。更进一步的，所述细胞为哺乳动物细胞。更进一步的，所述哺乳动物包括人、以及非人动物如鼠或猴等。
在本发明的一个具体实施方式中，所述抗体或其抗原结合片段在50nM、40nM、30nM、20nM、10nM、5nM、1nM或0.1nM浓度以下时仍阻断SARS-CoV-2病毒引起的细胞病变，或者中和SARS-CoV-2病毒。
在本发明的一个具体实施方式中，本发明提供包含下述3个轻链互补决定区和/或3个重链互补决定区，可结合SARS-CoV-2病毒S蛋白的广谱抗体或其抗原结合片段，其中，
所述广谱抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:38所示的LCDR1、SEQ ID NO:39所示的LCDR2和SEQ ID NO:40所示的LCDR3，和/或所述广谱抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:41所示的HCDR1、SEQ ID NO:42所示的HCDR2和SEQ ID NO:43所示的HCDR3；
所述广谱抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:21所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:23所示的LCDR3，和/或所述广谱抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:27所示的HCDR1、SEQ ID NO:28所示的HCDR2和SEQ ID NO:29所示的HCDR3；
所述广谱抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:9所示的LCDR1、SEQ ID NO:10所示的LCDR2和SEQ ID NO:11所示的LCDR3，和/或所述广谱抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:12所示的HCDR1、SEQ ID NO:13所示的HCDR2和SEQ ID NO:14所示的HCDR3；
所述广谱抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:15所示的LCDR1、SEQ ID NO:16所示的LCDR2和SEQ ID NO:17所示的LCDR3，和/或所述广谱抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:18所示的HCDR1、SEQ ID NO:19所示的HCDR2和SEQ ID NO:20所示的HCDR3；或者
所述广谱抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:21所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:23所示的LCDR3，和/或所述广谱抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:24所示的HCDR1、SEQ ID NO:25所示的HCDR2和SEQ ID NO:26所示的HCDR3。
在本申请方案中，关于VL(轻链可变区)、VH(重链可变区)、LCDR(轻链互补决定区)、HCDR(重链互补决定区)、LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3的各个实施方式可以各自单独实施，也可以任意组合实施。
在本发明的一个具体实施方式中，所述抗体或其抗原结合片段包含：
(1)SEQ ID NO:36所示的轻链可变区，和/或SEQ ID NO:37所示的重链可变区；所述抗体或其抗原结合片段结合SARS-CoV-2病毒S蛋白；优选的，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,AZ.5毒株,以及野生型毒株中的一种或多种；优选的，所述野生型毒株为Wuhan-Hu-1毒株；优选的，所述Omicron毒株包括 BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB毒株中的一种或多种；
(2)SEQ ID NO:7所示的轻链可变区，和/或SEQ ID NO:8所示的重链可变区；所述抗体或其抗原结合片段结合SARS-CoV-2病毒S蛋白；优选的，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,AZ.5毒株,以及野生型毒株中的一种或多种；优选的，所述野生型毒株为Wuhan-Hu-1毒株；优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB毒株中的一种或多种；
(3)SEQ ID NO:1所示的轻链可变区，和/或SEQ ID NO:2所示的重链可变区；所述抗体或其抗原结合片段结合SARS-CoV-2病毒S蛋白；优选的，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,AZ.5毒株,以及野生型毒株中的一种或多种；优选的，所述野生型毒株为Wuhan-Hu-1毒株；优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB毒株中的一种或多种；
(4)SEQ ID NO:3所示的轻链可变区，和/或SEQ ID NO:4所示的重链可变区；所述抗体或其抗原结合片段结合SARS-CoV-2病毒S蛋白；优选的，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,AZ.5毒株,以及野生型毒株中的一种或多种；优选的，所述野生型毒株为Wuhan-Hu-1毒株；优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB毒株中的一种或多种；
(5)SEQ ID NO:5所示的轻链可变区，和/或SEQ ID NO:6所示的重链可变区；所述抗体或其抗原结合片段结合SARS-CoV-2病毒S蛋白；优选的，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,AZ.5毒株,以及野生型毒株中的一种或多种；优选的，所述野生型毒株为Wuhan-Hu-1毒株；优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB毒株中的一种或多种；或者
(6)SEQ ID NO:3所示的轻链可变区，和/或SEQ ID NO:35所示的重链可变区；所述抗体 或其抗原结合片段结合SARS-CoV-2病毒S蛋白；优选的，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,AZ.5毒株,以及野生型毒株中的一种或多种；优选的，所述野生型毒株为Wuhan-Hu-1毒株；优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB毒株中的一种或多种。
在本发明的一个具体实施方式中，所述抗体或其抗原结合片段的重链恒定区的序列为SEQ ID NO:30。
进一步的，所述抗体或其抗原结合片段的轻链恒定区的序列为SEQ ID NO:31。
在本发明的一个具体实施方式中，提供了下述结合SARS-CoV-2病毒S蛋白的抗体或其抗原结合片段：
在本发明的方案中，本发明的所述广谱抗体或其抗原结合片段包括单克隆抗体、多克隆抗体、嵌合抗体、人源化抗体、Fab、Fab’、F(ab’)2、Fv、scFv或dsFv片段等。
在本发明的一个具体实施方式中，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,AZ.5毒株,以及野生型毒株中的一种或多种；优选的，所述野生型毒株为Wuhan-Hu-1毒株；优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB毒株中的一种或多种；
更优选的，所述抗体或其抗原结合片段结合SARS-CoV-2病毒的RBD的T345,R346,K444,R403,K417,Y453,K458,G476,Y489,F490,Y505,K440,S443，T415、D420、Y421、A475、N487和R493残基中的一个或多个；
更优选的，所述抗体或其抗原结合片段结合Delta RBD上的T345,R346和K444残基；所述抗体或其抗原结合片段结合Omicorn BA.1 RBD上的T345,R346,K440,S443和K444残基；所述抗体或其抗原结合片段结合Delta RBD上的R403,K417,Y453,K458,G476,Y489,F490和Y505残基；或所述抗体或其抗原结合片段结合Omicron BA.2 RBD上的T415、D420、Y421、A475、N487、Y489和R493残基；
更优选的，所述抗原结合片段为Fab、Fab’、F(ab’)2、Fv、scFv或dsFv片段；
更优选的，BA7208或其抗原结合片段结合Delta RBD上的T345,R346和K444残基；BA7208或其抗原结合片段结合Omicorn BA.1 RBD上的T345,R346,K440,S443和K444残基； BA7125V1或其抗原结合片段结合Delta RBD上的R403,K417,Y453,K458,G476,Y489,F490和Y505残基；或BA7535或其抗原结合片段结合Omicron BA.2 RBD上的T415、D420、Y421、A475、N487、Y489和R493残基。
本发明的第二方面提供了一种多特异性抗体，所述多特异性抗体衍生自所述第一方面的广谱抗体或抗原结合片段；优选的，所述多特异性抗体包括双特异性抗体；更优选的，所述多特异性抗体衍生自BA7054，BA7125，BA7134，BA7208，BA7125V1，BA7535中的一个或多个。
本发明的第三方面提供一种双特异性抗体，包括与SARS-CoV-2病毒上的S蛋白结合的第一抗体或抗原结合片段，以及与SARS-CoV-2病毒上的S蛋白结合的第二抗体或抗原结合片段，其中，所述第一抗体或抗原结合片段为所述第一方面的广谱抗体或抗原结合片段，和/或所述第二抗体或抗原结合片段为所述第一方面的广谱抗体或抗原结合片段。
进一步的，所述第一抗体或抗原结合片段与所述第二抗体或抗原结合片段相同或不同；优选的，所述第一抗体或抗原结合片段与所述第二抗体或抗原结合片段结合相同或不同种SARS-CoV-2病毒的S蛋白；更优选的，所述第一抗体或抗原结合片段与所述第二抗体或抗原结合片段结合S蛋白上的相同或不同表位。
优选的，所述双特异性抗体结合SARS-CoV-2病毒的RBD的T345,R346,K444,R403,K417,Y453,K458,G476,Y489,F490,Y505,K440,S443，T415、D420、Y421、A475、N487和R493残基中的一个或多个。
优选的，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,AZ.5毒株,以及野生型毒株中的一种或多种；优选的，所述野生型毒株为Wuhan-Hu-1毒株；优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB毒株中的一种或多种。
更进一步的，所述第一抗原结合片段为Fab，所述第二抗原结合片段为scfv；
更优选的，所述双特异性抗体具有knob-hole Fc区；
更优选的，所述第一抗原结合片段连接的重链恒定区为具有hole的重链恒定区,所述第二抗原结合片段连接的重链恒定区为具有knob的重链恒定区；
更优选的，所述第二抗原结合片段通过VL或VH与具有knob的重链恒定区连接；更优选，所述VL或VH与具有knob的重链恒定区通过连接物连接；
更优选的，所述双特异性抗体还具有轻链恒定区；
更优选的，所述第一抗体或抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，所述3个轻链互补决定区包含SEQ ID NO:21所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:23所示的LCDR3，和/或3个重链互补决定区包含SEQ ID NO:27所示的HCDR1、SEQ ID NO:28所示的HCDR2和SEQ ID NO:29所示的HCDR3；所述第二抗体或抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，所述3个轻链互补决定区包含SEQ ID NO:15所示的LCDR1、SEQ ID NO:16所示的LCDR2和SEQ ID NO:17所示的LCDR3，和/或3个重链互补决定区包含SEQ ID NO:18所示的HCDR1、SEQ ID NO:19所示的HCDR2和SEQ ID NO:20所示的HCDR3；
更优选的，所述第一抗体或抗原结合片段包含SEQ ID NO:7所示的轻链可变区，和/或SEQ ID NO:8所示的重链可变区；所述第二抗体或抗原结合片段包含SEQ ID NO:3所示的轻链可变区，和/或SEQ ID NO:35所示的重链可变区；
更优选的，所述第一抗体或抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，所述3个轻链互补决定区包含SEQ ID NO:21所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:23所示的LCDR3，和/或所述广谱抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:27所示的HCDR1、SEQ ID NO:28所示的HCDR2和SEQ ID NO:29所示的HCDR3；所述第二抗体或抗原结合片段包含3个轻链互补决定区包含SEQ ID NO:38所示的LCDR1、SEQ ID NO:39所示的LCDR2和SEQ ID NO:40所示的LCDR3，和/或所述广谱抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:41所示的HCDR1、SEQ ID NO:42所示的HCDR2和SEQ ID NO:43所示的HCDR3；
更优选的，所述第一抗体或抗原结合片段包含SEQ ID NO:7所示的轻链可变区，和/或SEQ ID NO:8所示的重链可变区；所述第二抗体或抗原结合片段包含SEQ ID NO:36所示的轻链可变区，和/或SEQ ID NO:37所示的重链可变区；
更优选的，所述第一抗体或抗原结合片段为BA7208 Fab，所述第二抗体或抗原结合片段为BA7125V1scfv；或者所述第一抗体或抗原结合片段为BA7208 Fab，所述第二抗体或抗原结合片段为BA7535scfv。
更进一步的，所述VL或VH与具有knob的重链恒定区之间的连接物为连接多肽，优选的，序列可以为AA。更进一步的，所述scfv中VL与VH的连接序列可以为SEQ ID NO:32。
更进一步的，所述双特异性抗体中，抗体重链(knob链)恒定区序列可以为SEQ ID NO:33；抗体重链(hole链)恒定区序列可以为SEQ ID NO:34。
本发明的第三方面还提供了一种抗体组合，其包含结合SARS-CoV-2病毒上的S蛋白的两种或多种抗体或抗原结合片段的组合；
优选的，所述抗体组合为两种抗体或抗原结合片段的组合；其中，所述第一抗体或抗原结合片段为所述第一方面的广谱抗体或抗原结合片段，和/或所述第二抗体或抗原结合片段为所述第一方面的广谱抗体或抗原结合片段；更优选的，所述第一抗体或抗原结合片段与所述第二抗体或抗原结合片段相同或不同；更优选的，所述第一抗体或抗原结合片段与所述第二抗体或抗原结合片段结合相同或不同种SARS-CoV-2病毒的S蛋白；更优选的，所述第一抗体或抗原结合片段与所述第二抗体或抗原结合片段结合S蛋白上的相同或不同表位；
更优选的，所述第一抗体或抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，所述3个轻链互补决定区包含SEQ ID NO:21所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:23所示的LCDR3，和/或3个重链互补决定区包含SEQ ID NO:27所示的HCDR1、SEQ ID NO:28所示的HCDR2和SEQ ID NO:29所示的HCDR3；所述第二抗体或抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，所述3个轻链互补决定区包含SEQ ID NO:15所示的LCDR1、SEQ ID NO:16所示的LCDR2和SEQ ID NO:17所示的LCDR3，和/或3个重链互补决定区包含SEQ ID NO:18所示的HCDR1、SEQ ID NO:19所示的HCDR2和SEQ ID NO:20所示的HCDR3；
更优选的，所述第一抗体或抗原结合片段包含SEQ ID NO:7所示的轻链可变区，和/或SEQ ID NO:8所示的重链可变区的第一抗体或抗原结合片段；所述第二抗体或抗原结合片段包含SEQ ID NO:3所示的轻链可变区，和/或SEQ ID NO:35所示的重链可变区；
更优选的，所述第一抗体或抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，所述3个轻链互补决定区包含SEQ ID NO:21所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:23所示的LCDR3，和/或所述广谱抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:27所示的HCDR1、SEQ ID NO:28所示的HCDR2和SEQ ID NO:29所示的HCDR3；所述第二抗体或抗原结合片段包含3个轻链互补决定区包含SEQ ID NO:38所示的LCDR1、SEQ ID NO:39所示的LCDR2和SEQ ID NO:40所示的LCDR3，和/或所述广谱抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:41所示的HCDR1、SEQ ID NO:42所示的HCDR2和SEQ ID NO:43所示的HCDR3；
更优选的，所述第一抗体或抗原结合片段包含SEQ ID NO:7所示的轻链可变区，和/或SEQ ID NO:8所示的重链可变区；所述第二抗体或抗原结合片段包含SEQ ID NO:36所示的轻链可变区，和/或SEQ ID NO:37所示的重链可变区；
更优选的，所述第一抗体或抗原结合片段为BA7208抗体或抗原结合片段，所述第二抗体或抗原结合片段为BA7125V1抗体或抗原结合片段；或者所述第一抗体或抗原结合片段为BA7208抗体或抗原结合片段，所述第二抗体或抗原结合片段为BA7535抗体或抗原结合片段；
更优选的，所述抗体组合结合SARS-CoV-2病毒的RBD的T345,R346,K444,R403,K417,Y453,K458,G476,Y489,F490,Y505,K440,S443，T415、D420、Y421、A475、N487和R493残基中的一个或多个；
优选的，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,,以及野生型毒株中的一种或多种；优选的，所述野生型毒株为Wuhan-Hu-1毒株；优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB中的一种或多种。
在本申请的方案中，BA7208或其抗原结合片段结合Delta RBD上的T345,R346和K444残基；BA7208或其抗原结合片段结合Omicorn BA.1 RBD上的T345,R346,K440,S443和K444残基；BA7125V1或其抗原结合片段结合Delta RBD上的R403,K417,Y453,K458,G476,Y489,F490和Y505残基；或BA7535或其抗原结合片段结合Omicron BA.2 RBD上的T415、D420、Y421、A475、N487、Y489和R493残基。在本申请的方案中，通过冷冻电镜分析本申请抗体或抗原结合片段的表位。
本发明的第四方面提供了一种核酸，编码所述广谱抗体或其抗原结合片段，或所述多特异性抗体，所述双特异性抗体，或所述抗体组合。本发明第四方面还提供一种核酸组合，其包括编码第三方面所述抗体组合中各抗体的核酸的组合。
本发明的第五方面提供了一种载体，其包含编码所述广谱抗体或其抗原结合片段，或所述多特异性抗体，或所述双特异性抗体的核酸；或包含编码所述抗体组合中各抗体的核酸的组合。所述载体可用于表达所述广谱抗体或其抗原结合片段，或所述多特异性抗体，或所述双特异性抗体，或所述抗体组合。优选地，所述载体可以是病毒载体；优选地，所述病毒载体包含但不限于慢病毒载体、腺病毒载体、腺相关病毒载体或逆转录病毒载体等；优选地，所述载体可以是非病毒载体；优选地，所述载体可以是哺乳细胞表达载体；优选地，所述表达载体可以是细菌表达载体；优选地，所述表达载体可以是真菌表达载体。
本发明的第六方面提供了一种细胞，所述细胞包括所述核酸、核酸组合或所述载体，所述细胞可表达所述广谱抗体或其抗原结合片段，或所述多特异性抗体，或所述双特异性抗体，或所述抗体组合。优选地，所述细胞为细菌细胞；优选地，所述细菌细胞为大肠杆菌细胞等；优选地，所述细胞为真菌细胞；优选地，所述真菌细胞为酵母细胞；优选地，所述酵母细胞为毕赤酵母细胞等；优选地，所述细胞为哺乳动物细胞；优选地，所述哺乳动物细胞为中国仓鼠卵巢细胞(CHO)、人胚胎肾细胞(293)、B细胞、T细胞、DC细胞或NK细胞等。
本发明的第七方面提供了一种药物组合物，其包所述的广谱抗体或其抗原结合片段、所述多特异性抗体、所述双特异性抗体、所述抗体组合、核酸、核酸组合、载体或细胞，优选地，所述药物组合物还包含药学上可接受的载体，优选地，所述药学上可接受的载体包括以下中的一种或多种：药学上可接受的溶剂、分散剂、附加剂、塑形剂、药物辅料。
进一步的，所述药物组合物为鼻喷、鼻滴、雾化或注射制剂；优选的，所述注射制剂为静脉注射制剂；更优选的，所述药物组合物含治疗有效量的，优选的，约30mg至约2400mg，优选的，约1200mg或约2400mg的所述抗体或抗原结合片段、所述的多特异性抗体，所述的双特异性抗体，或所述的抗体组合。在本申请方案中，所述抗体组合包括BA7208和BA7535单抗的组合，或者BA7208和BA7125V1单抗的组合；优选的，在抗体组合中，BA7208和BA7535摩尔比为1：1；优选的，在所述抗体组合中，BA7208和BA7125V1摩尔比为1：1；更优选的，所述抗体组合中两个抗体混合后一起给药。
进一步的，所述药物组合物为单位制剂，所述单位制剂为鼻喷、鼻滴、雾化或注射制剂，并且该单位制剂含有治疗有效量的，优选的，30毫克至2400毫克，优选的，约1200mg或约2400mg的所述的抗体或其抗原结合片段、所述多特异性抗体，所述的双特异性抗体或所述的抗体组合；优选的，所述药物组合物含有选自所述抗体或其抗原结合片段、所述多特异性抗体，所述双特异性抗体和所述抗体组合中的一种，以及缓冲液；更优选的，所述缓冲液包括海藻糖和聚山梨酯80中的一种或多种；更优选的，所述药物组合物pH为5.5-6.5；更优选的，所述缓冲液还包括盐酸组氨酸和组氨酸中的一种或多种；更优选的，所述盐酸组氨酸和组氨酸的摩尔比为10.5：9.5；更优选的，基于所述药物组合物的总体积，所述药物组合物包括0.04-0.1g/mL海藻糖,0.0001-0.0003g/mL聚山梨酯80，以及10-50mg/mL的选自所述抗体或其抗原结合片段、所述多特异性抗体，所述双特异性抗体和所述抗体组合中的一种；更优选的，基于所述药物组合物的总体积，所述药物组合物包括10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，以及40±4mg/mL的选自所述抗体或其抗原结合片段、所述多特异性抗体，所述双特异性抗体和所述抗体组合中的一种。更优选的，基于所述药物组合物的总体积，所述药物组合物包括10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，以及40±4mg/mL的所述抗体或其抗原结合片段。
更进一步的，所述药物组合物的给药对象为治疗受试者或预防受试者；所述治疗受试者包括感染SARS-CoV-2病毒的无症状、轻型、普通型、重型或危重型患者；优选的，所述治疗受试者为发病时间≤7天且为在72小时之内按照《新型冠状病毒肺炎诊疗方案(试行第九版)》确诊感染SARS-CoV-2病毒的无症状、轻型、普通型、重型或危重型患者；
所述治疗受试者包括感染SARS-CoV-2病毒的无症状、轻度、中度、重度及危患者；优选的，所述治疗受试者为72小时内实验室检查(如RT-PCR检查)确认感染SARS-CoV-2，且按NIH指南确诊的无症状、轻度、中度、重度及危重COVID-19的患者；或者
所述预防受试者包括暴露前预防受试者、或暴露后预防受试者；优选的，所述暴露前预 防受试者包括新冠病毒暴露高风险人群、健康受试者、或者其他不适合接种疫苗的受试者；所述暴露后预防受试者包括新冠肺炎确诊患者和/或无症状感染者的密切接触者。
本发明的第八方面提供了一种试剂盒，其包含本发明所述广谱抗体或其抗原结合片段，或所述多特异性抗体，或所述双特异性抗体，或所述抗体组合，或包含编码广谱抗体或其抗原结合片段或所述多特异性抗体，或所述双特异性抗体的核酸，或所述抗体组合中各抗体的核酸的组合，或所述细胞，或所述药物组合物。进一步的，所述试剂盒还包括装有适当缓冲试剂的容器或说明书。
本发明的第九方面提供了所述广谱抗体或其抗原结合片段、所述多特异性抗体、所述双特异性抗体、所述抗体组合、核酸、核酸组合、载体或细胞在制备治疗或预防疾病的药物中的应用。
本发明的第十方面提供了所述广谱抗体或其抗原结合片段、所述多特异性抗体、所述双特异性抗体、所述抗体组合、核酸、或核酸组合在制备诊断、检测试剂盒中的应用。
本发明的第十一方面提供了一种治疗或预防疾病的方法，包括向有需要的受试者施用本发明的所述广谱抗体或抗原结合片段、所述多特异性抗体、所述双特异性抗体、所述抗体组合、核酸、核酸组合、载体、细胞或药物组合物。
进一步的，所述方法包括向有需要的受试者施用含有效量的所述抗体或抗原结合片段的药物组合物；优选的，向有需要的受试者施用含约30mg至约2400mg，优选的，约1200mg或约2400mg的所述抗体或抗原结合片段的药物组合物、所述多特异性抗体，所述双特异性抗体或所述抗体组合；更优选的，所述药物组合物为鼻喷、鼻滴、雾化或注射制剂；优选的，所述注射制剂为静脉注射制剂；更优选的，所述药物组合物为单位制剂，所述单位制剂为鼻喷、鼻滴、雾化或注射制剂，并且该单位制剂含有约30mg至约2400mg，优选的，约1200mg或约2400mg的所述抗体或抗原结合片段、所述多特异性抗体，所述双特异性抗体或所述抗体组合；更优选的，所述药物组合物为鼻喷、鼻滴、雾化或注射制剂，优选的，所述注射制剂为静脉注射制剂。
本发明的第十二方面提供了一种诊断、检测的方法，包括将本发明的所述广谱抗体或抗原结合片段、所述多特异性抗体、所述双特异性抗体、所述抗体组合、核酸、核酸组合、试剂盒或药物组合物给予有需要的受试者或样本。
本发明的第十三方面提供了所述的广谱抗体或其抗原结合片段、所述多特异性抗体、所述双特异性抗体、所述抗体组合、核酸、核酸组合、载体、细胞或药物组合物用于治疗、预防疾病的用途。
本发明的第十四方面提供了所述的广谱抗体或其抗原结合片段、所述多特异性抗体、所述双特异性抗体、所述抗体组合、核酸、核酸组合、试剂盒、或药物组合物用于检测、诊断的用途。
本发明的第十五方面提供了所述广谱抗体或其抗原结合片段，所述多特异性抗体、所述双特异性抗体、所述抗体组合、所述的核酸，所述核酸组合、或所述的药物组合物用于预防、治疗、检测或诊断与SARS-CoV-2病毒相关的疾病的应用。
在本发明的方案中，所述疾病是COVID-19肺炎及其他相关并发症。进一步的，所述广谱抗体或抗原结合片段、所述多特异性抗体、所述双特异性抗体、所述抗体组合、能阻断SARS-CoV-2病毒或其假病毒对细胞的感染、入侵等，或者中和SARS-CoV-2病毒或其假病毒。更进一步的，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、 Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,AZ.5毒株,以及野生型毒株中的一种或多种；优选的，所述野生型毒株为Wuhan-Hu-1毒株；优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB毒株中的一种或多种。
本发明还提供了BA7054，BA7125，BA7134，BA7208，BA7125V1，BA7535，BA7208-7125V1-连接子4，BA7208-7125V1-连接子6抗体，BA7208-7535-连接子4，BA7208与BA7125V1抗体组合，BA7535与BA7208抗体组合在用于预防、治疗、检测或诊断与SARS-CoV-2病毒相关的疾病的应用；更进一步的，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,AZ.5毒株,以及野生型毒株中的一种或多种；优选的，所述野生型毒株为Wuhan-Hu-1毒株；优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB毒株中的一种或多种。
本发明还提供了BA7054，BA7125，BA7134，BA7208，BA7125V1，BA7535，BA7208-7125V1-连接子4，BA7208-7125V1-连接子6抗体，BA7208-7535-连接子4，BA7208与BA7125V1抗体组合，BA7535与BA7208抗体组合用于预防、治疗、检测或诊断与SARS-CoV-2病毒相关的疾病的应用；更进一步的，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,AZ.5毒株,以及野生型毒株中的一种或多种；优选的，所述野生型毒株为Wuhan-Hu-1毒株；优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB毒株中的一种或多种。
表1显示了上述SARS-CoV-2病毒主要毒株的名称，出现地以及主要的突变情况。



本发明提供的广谱抗体或其抗原结合片段具有以下的一种或多种优势：
1、对SARS-CoV-2病毒的S蛋白或S1蛋白或S2蛋白有亲和力；
2、对SARS-CoV-2病毒的Spike RBD蛋白与ACE2的结合有阻断能力；
3、阻断SARS-CoV-2病毒的假病毒对细胞的感染；
4、阻断SARS-CoV-2病毒的真病毒对细胞的感染；
5、具有良好的代谢稳定性，并且副作用和毒性小，安全性高；
6、小鼠体内实验表明，本发明的广谱抗体具有良好的预防或治疗效果。
附图说明
图1示出了S蛋白免疫小鼠血清滴度；
图2示出了ELISA检测候选抗体BA7054、BA7125、BA7134、BA7208阻断野生型、B.1.351和B.1.617.2毒株的RBD蛋白与hACE2的结合；
图3A-3C示出了抗体BA7054、BA7134、BA7208、BA7125V1阻断B.1.351、B.1.617.2和B.1.1.529.1毒株的RBD，以及3个对照抗体与hACE2的结合；图3D示出了抗体BA7535阻断11种毒株蛋白与hACE2的结合；图3E-3M示出了抗体BA7535阻断9种毒株蛋白与hACE2的结合。
图4A-4I示出了双抗BA7208/7125V1及其亲代单抗对B.1.617.2、B.1.529.1和B.1.621变体的RBD的结合动力学的图。图4J-4K示出了BA7535、BA7208对BA.1(B.1.1.529.1)RBD的结合动力学的图。
图5A-5H示出了表13中BA7208，BA7125V1，BA7054，BA7134，BA7208/BA7125V1抗体以及3个对照抗体对多种SARS-CoV-2变体的假病毒中和曲线，数据收集自两个生物重复，以平均值±SD表示。图5I示出了BA7535对2种SARS-CoV-2变体的假病毒中和曲线。
图6示出了BA7208抗体对多个假病毒的中和活性。
图7示出了BA7208对Omicron各突变株具有广谱且优异的中和活性。
图8示出了BA7535具有广谱且优异的中和活性。
图9A-9D示出了抗体BA7535、BA7208、BA7535+BA7208组合、以及LY-COV1404(礼来上市抗体)在假病毒系统中对30多个以前和新出现的变体的中和活性。
图10A-10G示出了抗SARS-CoV-2抗体在FRNT试验中对SARS-CoV-2变体的活病毒中和作用。
图11A-11B示出了BA7208和BA7208/7125V1对SARS-CoV-2变种Omicron BA.1和BA.2的中和活性。图11C-11E示出了BA7535对Omicron BA.1，BA.2，BA.5变异株的真病毒中和曲线，进行了三次生物重复。
图12A示出了Balb/c小鼠单次静脉注射抗体药物BA7208、BA7125V1、BA7054、BA7134、BA7208/7125V1双抗注射液药代动力学曲线；图12B示出了大鼠多次给药BA7208的药代动力学曲线；图12C示出了BA7535单次静脉注射药代动力学曲线。图12D-12F示出了BA7208小鼠单次给药毒性试验结果。图12G示出了双抗BA7208/7535-连接子4(BA7208 Fab,7535scfv)的小鼠PK数据-中和曲线。
图12H-图12L示出了BA7208的介导ADCC,ADCP的实验结果，其中图12H示出了以表达SARS-CoV-2 Spike(野生型)的CHO-K1细胞为靶细胞，BA7208的ADCC活性。图12I示出了以表达SARS-CoV-2 Spike(野生型)的CHO-K1细胞为靶细胞，BA7208的ADCP活性。图12J示出了以表达SARS-CoV-2 BA.1 Spike的HEK293T细胞为靶细胞，BA7208的ADCP活性。图12K示出了以表达SARS-CoV-2 BA.1 Spike的HEK293T细胞为靶细胞，BA7208的ADCC活性。使用具有相同恒定区域的不相关的mAb作为同型对照(未显示)，实验一式两份。数据以平均值±SD表示。
图13示出了抗2019-nCoV(或SARS-CoV-2)双特异性抗体的结构示意图。
图14A示出了ELISA检测双抗BA7208-7125V1-连接子4和BA7208-7125V1-连接子6阻断B.1.1.529(又称奥米克戎(Omicron)毒株)RBD蛋白与hACE2的结合。
图14B示出了ELISA检测双抗BA7208-7125V1-连接子4和BA7208-7125V1-连接子6阻断B.1.621(又称缪(Mu)毒株)RBD蛋白与hACE2的结合。
图15示出了双抗BA7208/7535-连接子4(BA7208 Fab,7535scfv)的假病毒的中和曲线。
图16示出了用BA7208-Fab/BA7125V1-Fab对Delta Spike Trimer进行冷冻电镜观察图(图上的BA7125-Fab为实施例中描述的BA7125V1的Fab)。
图17示出了用BA7208-Fab对Omicron Spike Trimer进行冷冻电镜观察图。
图18示出了Omicorn RBD突变位点与BA7208 Fab重链的相互作用。
图19A-19H示出了BA7208，BA7125V1，BA7254结构叠加分析图(图19 A-19H上的BA7125为实施例中描述的BA7125V1)。
图20示出了基于生物层干涉仪(BLI)的竞争性结合试验比较了BA7535、BA7208抗体的竞争性结合结果。
图21A-21B示出了BA7208注射给予小鼠后，预防组和治疗组肺部活病毒滴度。图21C-21D示出了BA7208滴鼻和雾化给予小鼠后，预防组和治疗组肺部活病毒滴度。
图22A示出了BA7535和BA7535/BA7208组合在hACE2转基因小鼠中的预防和治疗试验路线。图22B示出了监测体重变化(n＝4)，符号代表平均值±SEM。图22C示出了在2和4dpi时，通过病灶形成法(FFA)分析肺部和大脑的病毒载量(Viral burden)，虚线代表检测限(LOD)。图22D示出了预防组和治疗组在4dpi收集的H&E染色的肺部切片的病理变化。与PBS对照组相比，预防组以及高剂量和低剂量的治疗组没有观察到明显的肺部病变。图片显示低倍(向上；比例尺，500μm)和高倍放大(向下；比例尺，100μm)。每组n＝4的代表图像。
图23显示了各种SARS-CoV-2变异株RBD上的突变位点。
图24A显示了BA.2 Spike三聚体与BA7535-Fab复合物的晶体结构。图24B显示了BA.2 RBD和BA7535-Fab复合物的晶体结构。
图25显示了BA7535与RBD的结合表位和ACE2的结合表位有部分重合。
图26显示了RBD/BA7535-Fab与REGN10987(PDB ID:6XDG)的晶体结构的比较。
具体实施方式
下面结合具体实施例，进一步阐述本发明。所描述的实施例是本发明一部分实施例，而不是全部的实施例。应理解，举出以下实施例是为了向本发明所属技术领域的一般专业人员就如何利用本发明之方法和组合物提供一个完整的公开和说明，并非用于限制本发明的范围。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1.抗2019-nCoV(或SARS-CoV-2)单克隆抗体的产生
1.1各蛋白及所用小鼠如表2所示。
表2各S蛋白来源及所用小鼠
转基因小鼠免疫方式如表3所示。
表3转基因小鼠免疫方式

共有5种突变株的Spike RBD抗原蛋白，为B.1.617.1 RBD,B.1.617.2 RBD,B.1.351 RBD和p.1 RBD蛋白，B.1.1.529.1 RBD，以及2种突变株的Spike蛋白，为B.1.617.2 Spike和B.1.351 Spike蛋白，按照表3方式对各小鼠分别进行免疫。免疫采用腹部皮下注射，5种突变株的Spike RBD抗原蛋白(即B.1.617.1 RBD,B.1.617.2 RBD,B.1.351 RBD，p.1 RBD蛋白和B.1.1.529.1 RBD)免疫剂量为40μg/只，2种突变株的Spike蛋白(即B.1.617.2 Spike和B.1.351 Spike蛋白)免疫剂量为35μg/只。首免使用弗氏完全佐剂乳化抗原，二免-四免使用弗氏不完全佐剂乳化抗原。上述蛋白按照表3免疫小鼠后，检测小鼠血清滴度结果如图1，2500X，12500X，以及62500X代表血清稀释比例。
1.2噬菌体库的建立
处死小鼠，解剖取出脾脏，把脾脏用注射器胶塞研磨破碎并用滤网过滤，把滤过的脾细胞冷冻备，提取RNA后获得cDNA，噬菌体库的建立依据通常方法进行。构建库的库容数据如表4所示。
表4各毒株免疫小鼠构建噬菌体库库容
1.3以两种方法进行筛选
1.3.1平板筛选，用B.1.617.2的Spike RBD蛋白(即B.1.617.2 RBD)(义翘神州，40592-V08H90)或B.1.351的Spike RBD蛋白(即B.1.351 RBD)(义翘神州，40592-V08H85)或B.1.617.1的Spike RBD蛋白(即B.1.617.1 RBD)(义翘神州，40592-V08H88)或p.1的Spike RBD蛋白(即p.1 RBD)(义翘神州，40592-V08H86)或B.1.617.2的Spike蛋白(B.1.617.2 Spike)(义翘神州，40589-V08H10)或B.1.351的Spike蛋白(即B.1.351 Spike)(义翘神州，40589-V08H13)，B.1.1.529.1 RBD(北京百普赛斯生物科技股份有限公司)包被平板。第二天加入噬菌体库孵育2h，洗涤4-10次后用洗脱缓冲液洗脱特异性结合的噬菌体。
1.3.2磁珠筛选，将B.1.617.2或B.1.351或B.1.617.1或p.1的Spike RBD蛋白或B.1.1.529.1RBD蛋白按照试剂盒步骤进行生物素化，再与Thermo的磁珠结合，经BSA封闭后与噬菌体库孵育2h，洗涤4-10次后用Elution Buffer洗脱特异性结合的噬菌体。筛选获得抗体克隆及来源见表5。
表5筛选策略以及得到抗体来源

实施例2.完整抗体的分子构建与生产
构建177个阳性克隆IgG1并测序，其中4个lead抗体可变区氨基酸序列如下表6：(CDR区以下划线标识，分析系统为IMGT系统),BA7125V1为BA7125重链可变区点突变获得的抗体。
表6.活性克隆氨基酸序列的可变区序列

通过常规的分子生物学技术PCR(2×Phanta Max Master Mix厂家：Vazyme货号：P515-P1-AA批号：7E351H9)扩增抗体可变区基因，通过同源重组分别将抗体重链可变区基因连接入带有抗体重链恒定区序列的核酸序列的载体pCDNA3.4(Life Technology)，将抗体轻链可变区基因连接入带有抗体轻链恒定区序列的核酸序列的载体pCDNA3.4。
本申请实施例中各抗体的可变区序列参见表7，重链和轻链恒定区序列见表7。
表7
将测序后的阳性克隆提取质粒后共转染进入HEK293细胞在37℃\8％CO2\125rpm摇床中培养，瞬时表达7天后上清通过Protein A亲和层析，纯化获得抗体，并通过UV280结合理论消光系数确定抗体浓度。
实施例3.Anti-2019-nCoV单克隆抗体分子的表征
3.1 ELISA检测抗体阻断野生型、Beta(B.1.351)和Delta(B.1.617.2)毒株的RBD蛋白与hACE2的结合
用pH 9.6 CBS分别稀释3种Spike RBD蛋白至0.125μg/mL(野生型RBD即WT RBD)、 0.25μg/mL(B.1.351RBD)和0.125μg/mL(B.1.617.2 RBD)，包被酶标板,100μL/孔，4℃孵育过夜；洗板后用脱脂奶粉封闭。洗板后每孔加入PBST稀释好的抗体50μL，抗体终浓度为(4μg/mL、1μg/mL、0.25μg/mL)。然后加入生物素标记的ACE2蛋白(Novoprotein，C05Y)，50μL/孔，37℃孵育1h；洗板后加入PBST稀释的STREP/HRP，100μL/孔，37℃孵育1h。洗板后每孔加入100μL TMB显色，10min后每孔加入50μL 2M H2SO4终止显色，用酶标仪读取OD450，表格8-10内除抗体浓度外的数值代表OD450值。图2和表8-10结果显示BA7054、BA7125、BA7134、BA7208均能阻断3种Spike RBD蛋白与ACE2的结合。
表8检测抗体阻断野生型Spike RBD与hACE2的蛋白结合灵敏度
表9检测抗体药物阻断B.1.351(又称贝塔(Beta)毒株)Spike RBD与hACE2的蛋白结合灵敏度
表10检测抗体药物阻断B.1.617.2(又称德尔塔(Delta)毒株)Spike RBD与hACE2的蛋白结合灵敏度
3.2 ELISA检测抗体阻断各毒株蛋白与hACE2的结合
3.2.1抗体BA7054、BA7134、BA7208、BA7125V1阻断B.1.351(又称贝塔(Beta)毒株)、B.1.617.2(又称德尔塔(Delta)毒株)和B.1.1.529.1(又称奥米克戎(Omicron)BA.1毒株)的RBD与hACE2的结合
用pH 9.6 CBS分别稀释3种Spike RBD蛋白(即B.1.351、B.1.617.2和B.1.1.529.1毒株 的RBD蛋白)至0.125μg/mL，包被酶标板,100μL/孔，4℃孵育过夜；洗板后用脱脂奶粉封闭。洗板后每孔加入PBST稀释好的抗体50μL，抗体终浓度为(4μg/mL、1μg/mL、0.25μg/mL、0.0625μg/mL、0.015625μg/mL、0.00390625μg/mL、0.0009765625μg/mL、0.000244140625μg/mL)。然后加入生物素标记的ACE2蛋白(Novoprotein，C05Y)，50μL/孔，37℃孵育1h；洗板后加入PBST稀释的STREP/HRP，100μL/孔，37℃孵育1h。洗板后每孔加入100μL TMB显色，10min后每孔加入50μL 2M H2SO4终止显色，用酶标仪读取OD450，按照以下公式计算抑制率，抑制率％＝(PBST的OD450–抗体的OD450)/PBST的OD450*100％。结果见图3A-3C，通过ELISA分析四种候选抗体和三种对照抗体对B.1.351、B.1.617.2和B.1.1.529.1与hACE2结合的RBD的阻断活性。实验平行进行3次，数值＝平均值±SD。由图3A-3C可以看出，各抗体能阻断B.1.351(又称贝塔(Beta)毒株)RBD蛋白和B.1.617.2(又称德尔塔(Delta)毒株)RBD蛋白与ACE2的结合，但对B.1.1.529.1(又称奥米克戎(Omicron)BA.1毒株)RBD蛋白而言，只有BA7208和BA7134能阻断其与ACE2的结合，其他抗体无阻断活性。
3.2.2抗体BA7535阻断11种毒株蛋白与hACE2的结合
检测方法同3.2.1，不同之处在于Spike RBD蛋白包括Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Omicron B.1.1.529.1(即BA.1毒株)、Lambda(C.37)毒株、Mu(B.1.621)，Kappa(B.1.617.1)毒株，C.12毒株，B.1.630毒株，B.1.640毒株，野生型毒株(即Wuhan-Hu-1毒株或Original WT)，Beta(B.1.351)的RBD蛋白，各RBD蛋白购自北京义翘神州，检测结果参见图3D，由图3D可以看出，抗体BA7535能阻断各毒株蛋白与hACE2的结合。
BA7535阻断各突变株RBD与ACE2结合的IC50(μg/mL)见表10-1。
表10-1
3.2.3 ELISA检测抗体BA7535阻断P.1、B.1.617.2、C.37、B.1.621、B.1.617.1、C.1.2、B.1.630、B.1.640、BA.1毒株的RBD蛋白与hACE2的结合
检测方法同3.2.1，不同之处在于Spike RBD蛋白包括P.1、B.1.617.2、C.37、B.1.621、B.1.617.1、C.1.2、B.1.630、B.1.640、BA.1毒株的RBD蛋白，各RBD蛋白购自北京义翘神州，检测结果参见图3E-3M，图3E-3M可以看出，抗体BA7535对9个SARS-CoV-2变体(P.1、B.1.617.2、C.37、B.1.621、B.1.617.1、C.1.2、B.1.630、B.1.640、BA.1)的RBD均表现出广泛的阻断活性。
3.3检测抗体与各突变毒株RBD蛋白的亲和力
3.3.1抗体BA7054、BA7134、BA7208、BA7125V1，双抗BA7208-7125V1与各突变株Spike RBD蛋白的结合动力学
使用基于表面等离振子共振(surface plasmon resonance，SRP)技术的BIAcore 8K仪器测量。
对于动力学测量，将各毒株Spike RBD蛋白用HBS-EP+1×(cytiva，BR-1006-69)缓冲液2倍连续稀释，50nM起始，2倍稀释4个浓度梯度，并设置0浓度。Startup 3次。抗体：2μg/ml，进样时间100s，流速10μL/min，用ProA芯片(cytiva，29127556)捕获；抗原蛋白：结合120s，流速30μL/min，解离600s；再生：用MgCl2缓冲液再生30s，流速30μL/min。使用1:1binding结合模型(BIAcore Evaluation Software version 3.2)计算结合常数(ka)和解离常数(kd)，平衡解离常数(KD)以比率kd/ka计算。亲和力数据分别见表11，表12。
表11 Biacore 8K检测抗体亲和力
从表11可以看出，尽管各抗体与不同突变株RBD亲和力具有区别，总体上我们的抗体结合比较广谱；但BA7125V1和BA7054对Omicron毒株BA.1(B.1.1.529.1)无结合，BA7208结合最好，比较广谱，且亲和力较高，只对Mu(B.1.621)结合稍弱，对关键的BA.1(B.1.1.529.1)亲和力最好。
表12 Biacore 8K检测抗体亲和力
表12显示了单抗与双抗亲和力的比较，双抗BA7208-7125V1序列和结构信息参见以下实施例4-5，双抗BA7208/7125V1及其亲代单抗BA7208，BA7125V1对B.1.617.2、B.1.529.1和B.1.621变体的RBD的结合动力学，结果参见图4A-4I。从表12和图4A-4I可以看出双抗的亲和力优于2种母本单抗，并且将两种母本单抗的优势合并，拥有最好的广谱性。
3.3.2抗体BA7535、BA7208与突变株BA.1(B.1.1.529.1)RBD蛋白的结合动力学
检测方法同3.3.1,结果参见图4J-4K,可以看出，BA7535对BA.1 RBD的亲和力高于BA7208，测量的平衡常数(KD)分别为0.10±0.02nM和1.81±0.26nM。
3.4抗体在假病毒上的中和活性
3.4.1抗体BA7208，BA7125V1，BA7054和BA7134对假病毒的中和活性
采用VSV(水疱性口炎病毒)载体包装各毒株S蛋白的假病毒，试剂耗材见表12。50μL假病毒与100μL待检抗体37℃孵育1h后侵染100μL 293T-ACE2细胞,每孔细胞数：4E5cells/孔，37℃孵育20-28h后采用化学发光法检测Luciferase发光值RLU，根据RLU读值计算待检抗体的假病毒抑制率。抗体的缓冲液体系为pH7.4，0.01M PBS缓冲液，试剂耗材见表13，各抗体假病毒中和活性检测结果见表14。
表13
表14抗体BA7208，BA7125V1，BA7054和BA7134对假病毒的中和活性
注：IC50值越低，代表中和活性越高
从表14，以及图5A-5H可以看出，BA7208，BA7125V1，BA7054，和BA7134具有广泛的假病毒中和活性。BA7208和BA7125V1具有广谱且互补的中和活性。除了B.1.640和Mu，BA7208可以中和13种突变株中的11种，IC50在2.81ng/mL到6.22ng/mL之间，并 且对BA.1和BA.2有最好的中和活性，IC50分别为3.52ng/mL和3.43ng/mL。BA7125V1可以中和除了2种Omicorn突变株之外的11种突变株，IC50在11.36ng/mL到42.31ng/mL之间。BA7054可以中和除了Mu，Omicorn和B.1.640之外的9种突变株，IC50在4.47ng/mL到10.11ng/mL之间。BA7134可以中和除了B.1.640和Mu之外的11种突变株，IC50在3.81ng/mL到147.60ng/mL之间。BA7208、BA7125V1和BA7054具有明显高于VIR-7381、REGN10933和REGN10987的中和活性。
进一步还可以看出，双抗BA7208-7125V1最广谱，可以中和所有的13种突变株，IC50在2.33ng/mL到116.10ng/mL之间；双抗BA7208-7125V1中和活性大部分毒株中略低于BA7208和BA7125V1中最好单抗的活性(10.8ng/mL的活性也是很好)；在关键毒株Omcicron上双抗BA7208-7125V1比单抗BA7208的活性低3倍。BA7125V1+BA7208联用与双抗BA7208-7125V1类似，也具有最广谱的中和活性，可以弥补2种单抗各自的劣势。
3.4.2抗体BA7535对BA.1.1以及BA.2的SARS-Cov-2假病毒中和活性
图5I显示了抗体BA7535对BA.1.1以及BA.2的SARS-Cov-2假病毒中和活性,可以看出，BA7535对BA.1.1，以及BA.2的SARS-Cov-2假病毒均具有良好的中和活性。
表15显示了抗体BA7535对BA.1.1以及BA.2的SARS-Cov-2假病毒IC50(μg/mL)
3.4.3 BA7208抗体的假病毒中和活性数据补充-1
假病毒中和活性实验步骤：50μL各新冠假病毒(北京三药)与100μL梯度稀释的BA7208抗体混合后37℃孵育1h，以50μL+100μL DMEM作为阴性对照，以150μL DMEM作为作为空白，然后加入100μL过表达人ACE2的293T细胞悬液(4×105cells/mL)，37℃孵育20-28h后，吸取150μL上清舍弃，加入100μL britelite plus(PerkinElmer)作为底物，酶标仪读取荧光值，计算抑制率＝1-(样品平均RLU值-空白平均RLU值)/(阴性对照平均RLU值-空白平均RLU值)*100％)。以样品浓度和抑制率作图，用GraphPad Prism软件拟合计算IC50。表16显示了BA7208抗体的假病毒中和活性数据。
表16
从图6和表16可以看出，BA7208具有广谱且优异的中和活性，对检测的毒株中的15个中和活性IC50<10ng/mL。
3.4.4BA7208抗体的假病毒中和活性数据补充-2
假病毒中和活性实验步骤参见3.4.2，各新冠假病毒购自北京三药，结果见图7和表17。
表17

从图7和表17可以看出，BA7208对Omicron各突变株具有广谱且优异的中和活性，对检测的毒株中的BA.2.75的中和活性IC50<10ng/mL。
3.4.5BA7535抗体的假病毒中和活性数据补充
假病毒中和活性实验步骤：50μL各新冠假病毒(购自北京三药)与100μL梯度稀释的BA7208抗体混合后37℃孵育1h，以50μL+100μL DMEM作为阴性对照，以150μL DMEM作为作为空白，然后加入100μL过表达人ACE2的293T细胞悬液(4×105cells/mL)，37℃孵育20-28h后，吸取150μL上清舍弃，加入100μL britelite plus(PerkinElmer)作为底物，酶标仪读取荧光值，计算抑制率＝1-(样品平均RLU值-空白平均RLU值)/(阴性对照平均RLU值-空白平均RLU值)*100％)。以样品浓度和抑制率作图，用GraphPad Prism软件拟合计算IC50。BA7535抗体的假病毒中和活性数据见表18，其中BA.4/5代表BA.4和BA.4，BA.4和BA.5的RBD突变一样，因此BA.4和BA.5的假病毒一样。
表18

从图8和表18可以看出，BA7535具有广谱且优异的中和活性，对检测的毒株中的19个中和活性IC50<10ng/mL。
3.4.6抗体BA7535、BA7208及其组合、以及LY-COV1404(礼来上市抗体)在假病毒系统中对30多个以前和新出现的变体的中和活性
假病毒中和活性实验步骤参见3.4.3，各假病毒购自北京三药，实验结果参见图9A-9D，以及表19。
表19
从图9A-9D以及表19可以看出，BA7535单独可以中和所有测试的以前和新出现的SARS-CoV-2变体；D614G、Alpha、BQ.1.1和XBB只是削弱了BA7535的效力，但没有一个变体可以逃脱其中和作用。BA7535和BA7208的组合(在抗体组合中，BA7208和BA7535摩尔比为1：1；所述抗体组合中两个抗体混合后一起给药)提供了更广泛的中和效力和更高的抵抗力；LY-Cov1404对以前流通的Omicron变体仍然有效，但被BA.2.38.1(K444N)、BQ.1(K444T)、BQ.1.1(K444T)和XBB(V445P+G446S)逃逸。
3.5抗体在真病毒上的中和活性
3.5.1 BA7208，BA7125V1，BA7054，BA7208/7125V1在真病毒上的中和活性
梯度稀释的50μL抗体与50μL SARS-CoV-2病毒(180FFU，病灶形成单位)在96微孔板中混合，于37℃孵育1h，转移至铺有Vero E6细胞(ATCC)的96孔板中，37℃孵育1h使病毒侵染。去除培养液加入覆盖介质(100μL MEM含1.2％羧甲基纤维素)，将板子37℃孵育24h，去除覆盖物，向细胞加入4％多聚甲醛后孵育30分钟，将细胞用0.2％Triton X-100透化，与兔抗SARS-CoV-N IgG(义翘神州，Cat 40143-R001)的抗体室温下交叉反应1h，然后加入HRP标记的羊抗兔IgG(H+L)抗体(1:4000稀释)(Jackson ImmunoResearch)室 温孵育。加入TrueBlue过氧化物酶底物(KPL)进行反应，用EliSpot reader(Cellular Technology Ltd)对SARS-CoV-2斑进行计数。各真病毒来自广州医科大学。
结果参见表20，以及图10A-10G(基于FRNT方法进行的真病毒中和曲线)。
表20真病毒中和实验IC50
由表20，以及图10A-10G可以看出：我们的4个抗体对野生型病毒和B.1.351真病毒都具有较好的中和活性，BA7208对BA.1.1真病毒有最好的中和活性。
3.5.2 BA7208对Omicron BA.1，BA.2变异株的真病毒中和活性优异
真病毒实验过程参见3.5.1，各真病毒来自广州医科大学，实验结果见图11A-11B和表21-1。
表21-1
图11A-11B可以看出，BA7208和双抗BA7208/7125V1对SARS-CoV-2变种Omicron BA.1和BA.2有很强的中和作用，IC50值在0.0329至0.316微克/毫升之间。由表21-1可以看出，BA7208抗体对Omciron BA.1和BA.2变异株真病毒中和的IC50值分别为：53.2、18.17ng/mL。
3.5.3 BA7535对Omicron BA.1，BA.2，BA.5变异株的真病毒中和活性优异
病毒实验过程参见3.5.1，各真病毒来自广州医科大学，实验结果见图11C-11E和表21-2。可以看出，BA7535有效地中和了BA.1、BA.2和BA.5活病毒，IC50分别为0.38ng/mL、1.1ng/mL和2.2ng/mL(图11C-11E)。
表21-2

3.6药代动力学
3.6.1 BA7208、BA7125V1,BA7054,BA7134和BA7208/7125V1双抗单次静脉注射药代动力学
采用Balb/c小鼠(体重25±3g)，按10mg/kg分别单次静脉注射5种抗体(BA7208、BA7125V1,BA7054,BA7134和BA7208/7125V1双抗)，并于给药结束5分钟、30分钟、1小时、4小时、8小时、1(24h)、3(72h)、5(120h)、7(168h)、10(240h)、14(336h)天采集血样，以ELISA法测定血清中药物浓度，以Phoenix WinNonlin 6.4计算药动学参数。各抗体药代动力学参数见表22，药代动力学曲线见图12A。结果显示，除BA7054外，各抗体在小鼠体内均具备良好的代谢稳定性。
表22 Balb/c小鼠单次静脉注射抗体药物注射液药代动力学参数
3.6.2 BA7208大鼠多次给药的药代动力学
药代动力学实验过程实验参数如下表23：
表23
BA7208药代动力学实验过程实验参数
图12B示出了大鼠多次给药BA7208的药代动力学试验结果。根据LY-CovMab的小鼠T1/2为9.5天，人体T1/2为28天；BA7208的大鼠T1/2为8.4～11.2天，预测其人体T1/2可达28天。因BA7208针对BA.2真病毒的IC50为18.17ng/mL(有效浓度)，根据人体T1/2预测在人体预防给药的半年后血液浓度仍高于IC50，预测单次给药对人体的保护作用可维持半年以上。
3.6.3BA7535单次静脉注射药代动力学
采用Balb/c小鼠(体重25±3g)，按10mg/kg分别单次静脉注射抗体BA7535，并于给药结束5分钟、1小时、6小时、1(24h)天、3(72h)天、5(120h)天、7(168h)天、10(240h)天、14(336h)天采集血样，以ELISA法测定血清中药物浓度，以Phoenix WinNonlin6.4计算药动学参数。
药代动力学曲线见图12C，可以看出，BA7535抗体在小鼠体内具备良好的代谢稳定性。
3.6.4 Balb/c小鼠单次静脉内给予BA7535的PK参数
BA7535显示出令人满意的半衰期和AUC(0-t)，终末半衰期(t1/2，λz)约为95小时，AUC(0-t)约为12785小时*μg/mL。结果参见下表24-1。
表24-1
3.7 BA7208小鼠单次给药毒性试验
BA7208的小鼠单次给药毒性试验结果参见图12D-12F，可以看出14天动物存活率、精神状态、体重和摄食量均未见明显异常，解剖未发现脏器异常，整体较耐受。
3.8双抗BA7208/7535-连接子4(BA7208 Fab,7535scfv)的小鼠PK实验
双抗BA7208/7535-连接子4的小鼠PK实验过程同3.6.3BA7535单抗的小鼠PK，尾静脉注射给药，结果参见图12G，可以看出双抗在小鼠体内较稳定，终末半衰期(t1/2，λz)约为92.3小时，AUC(0-t)约为12108小时*μg/mL，与单抗类似。
双抗BA7208/7535-连接子4的PK参数如下表24-2：
表24-2
3.9 BA7208介导ADCC,ADCP的能力
ADCC实验过程：
以CHO-K1-Spike细胞(Genscript)为靶细胞，Jurkat细胞(G7011，Promega)为效应细胞进行ADCC报告生物测定。靶细胞、效应细胞和连续稀释的抗体在白色96孔板中混合，并在细胞培养箱(37℃，5％CO2)中培养6小时。然后加入Bio-Lite显色溶液，在室温(RT)下孵育15分钟。用Tecan微孔板读数器读取平板。实验一式两份，数值＝平均值±SD。
ADCP实验过程：
对于ADCP，CHO-K1-Spike细胞(Genscript)被用作靶细胞，Jurkat-FcγRIIA-H131细胞(Vazyme)为效应细胞。将靶细胞、效应细胞和连续稀释的抗体加入白色96孔板中混合，并在细胞培养箱(37℃，5％CO2)中培养6小时。然后加入Bio-Lite显色溶液，在RT孵育2-5分钟。用Tecan微孔板读数器读取平板。实验一式两份，数值＝平均值±SD。
实验结果见图12H-图12L，可以看出BA7208显示了强的介导ADCC,ADCP的能力。
实施例4抗2019-nCoV(或SARS-CoV-2)双抗分子构建
本申请抗2019-nCoV(或SARS-CoV-2)双抗的结构示意图如图13所示，图13仅用作示例，不应理解为对本申请的限制。其中，右半部分结构为A-连接子(linker)1-B-连接子(linker)2-CH2-CH3。本申请双抗分为连接子4和连接子6两种结构。
连接子4与连接子6的结构和序列如下表25：
表25

重链1(scFv-knob链)的构建：通过常规的分子生物学技术PCR(2×Phanta Max Master Mix厂家：Vazyme，货号：P515-P1-AA)分别扩增抗体(例如7125V1抗体)的重链可变区基因和轻链可变区基因，然后通过overlap PCR技术将抗体(例如7125V1抗体)重链可变区基因和轻链可变区基因通过linker(例如SEQ ID NO:32)连接在一起，再与抗体重链(knob链)恒定区序列通过同源重组(ClonExpressII快速克隆试剂盒(Vazyme，货号：C113-02-AB)连接在一起，最后连接入载体pCDNA3.4(Life Technology)。7125V1抗体的重链可变区的氨基酸序列为SEQ ID NO:35，7125V1抗体的轻链可变区的氨基酸序列为SEQ ID NO:3，参见表6；抗体重链(knob链)恒定区序列为SEQ ID NO:33，如表26所示。
重链2(hole链)的构建：通过常规的分子生物学技术PCR扩增抗体(例如BA7208)的重链可变区基因，再与抗体重链(hole链)恒定区序列通过同源重组连接在一起，最后连接入载体pCDNA3.4(Life Technology)；轻链的构建：将抗体(例如BA7208)轻链可变区基因连接入带有抗体轻链恒定区序列的核酸序列的载体pCDNA3.4。BA7208抗体的重链可变区的氨基酸序列为SEQ ID NO:8，BA 7208抗体的轻链可变区的氨基酸序列为SEQ ID NO:7，参见表6。抗体重链(hole链)恒定区序列为SEQ ID NO:34，如表26所示；抗体轻链恒定区序列为SEQ ID NO:31，如表7所示。
重链1和重链2通过Knob into hole技术整合成异源二聚体。
抗2019-nCoV(或SARS-CoV-2)双抗分子的恒定区序列见表26。
表26抗2019-nCoV(或SARS-CoV-2)双抗分子的恒定区序列
实施例5抗2019-nCoV(或SARS-CoV-2)双抗分子或抗体组合的表征
5.1 ELISA检测双抗BA7208-7125V1-连接子4和BA7208-7125V1-连接子6阻断B.1.1.529(又称奥米克戎(Omicron)毒株)和B.1.621(又称缪(Mu))毒株的RBD蛋白与hACE2的结合
用pH 9.6 CBS分别稀释2种Spike RBD蛋白至0.125μg/mL包被酶标板,100μL/孔，4℃孵育过夜；洗板后用脱脂奶粉封闭。洗板后每孔加入PBST稀释好的抗体50μL，抗体终浓度为(4μg/mL、1μg/mL、0.25μg/mL、0.0625μg/mL、0.015625μg/mL、0.00390625μg/mL、0.0009765625μg/mL、0.000244140625μg/mL，抗体分子量125KD左右)。然后加入生物素标记的ACE2蛋白(Novoprotein，C05Y)，50μL/孔，37℃孵育1h；洗板后加入PBST稀释的STREP/HRP，100μL/孔，37℃孵育1h。洗板后每孔加入100μL TMB显色，10min后每孔加入50μL 2M H2SO4终止显色，用酶标仪读取OD450。图14A和图14B分别显示BA7208-7125V1-连接子4和BA7208-7125V1-连接子6能够阻断2种Spike RBD蛋白(B.1.1.529RBD(奥米克戎)蛋白、B.1.621(缪)RBD蛋白)与ACE2的结合，IC50分别如下表27所示。BA7208-7125V1-连接子4和BA7208-7125V1-连接子6的结构说明见实施例4。
表27 BA7208-7125V1-连接子4和BA7208-7125V1-连接子6阻断2种Spike RBD蛋白与ACE2的结合
5.2检测双抗BA7208-7125V1-连接子6与B.1.1.529(奥米克戎)、B.1.621(缪)和B.1.617.2(德尔塔)毒株RBD蛋白的亲和力
抗体与各突变株Spike RBD蛋白的结合动力学使用基于表面等离振子共振(surface plasmon resonance，SRP)技术的BIAcore 8K仪器测量。
对于动力学测量，将各毒株Spike RBD蛋白用HBS-EP+1×(cytiva，BR-1006-69)缓冲液2倍连续稀释，50nM起始，2倍稀释4个浓度梯度，并设置0浓度。Startup 3次。抗体：2μg/ml，进样时间100s，流速10μL/min，用ProA芯片(cytiva，29127556)捕获；抗原蛋白：结合120s，流速30μL/min，解离600s；再生：用MgCl2缓冲液再生30s，流速30μL/min。使用1:1binding结合模型(BIAcore Evaluation Software version 3.2)计算结合常数(ka)和解离常数(kd)，平衡解离常数(KD)以比率kd/ka计算。结果显示各抗体药物均能与相应抗原结合，亲和力数据见表28。
表28各双抗与各抗原亲和力

5.3双抗在假病毒上的中和活性
5.3.1双抗BA7208-7125V1-连接子4在假病毒上的中和活性。
采用VSV(水疱性口炎病毒)载体包装各毒株S蛋白的假病毒，试剂耗材见表13。50μL假病毒与100μL待检抗体37℃孵育1h后侵染100μL 293T-ACE2细胞,每孔细胞数：4E5cells/孔，37℃孵育20-28h后采用化学发光法检测Luciferase发光值RLU，根据RLU读值计算待检抗体的假病毒抑制率，抗体假病毒中和活性检测结果见表18。双抗BA7208-7125V1-连接子4具有广泛的假病毒中和活性，可以很好的中和B.1.617.2(德尔塔)毒株、B.1.1.529(奥米克戎)毒株和B.1.351(贝塔)毒株，IC50分别为0.012nM、0.017nM和0.010nM，参见表29。
表29双抗BA7208-7125V1-连接子4在假病毒上的中和活性
5.3.2 BA7535和BA7208的单抗组合在假病毒上的中和活性参见3.4.6。
5.3.3双抗BA7208/7535-连接子4(BA7208 Fab,7535scfv)的假病毒的中和活性
假病毒实验过程参见3.4.3，各假病毒购自北京三药，实验结果见图15和表29-1。
表29-1双抗BA7208/7535-连接子4在假病毒上的中和活性
补充：双抗BA7208/7535-连接子4假病毒数据-IC50
从以上结果可以看出，双抗可以将2种单抗的优势结合于一体，表现出广谱且优异的中和活性。
5.5双抗BA7208/7125V1在真病毒上的中和活性参见3.5.1。
实施例6抗体表位分析
6.1.通过冷冻电镜分析本申请抗体或抗原结合片段的表位
实验方法如下：Omicron S蛋白(即Omicron Spike Trimer)(40589-V08H26,义翘神州)或Delta S蛋白(即Delta Spike Trimer)(40589-V08H10，义翘神州)与抗体Fab混合，冰上孵育40min，16200g,4℃离心5min后，用GE micro Akta和Superose 6柱子进行分子筛纯化，收集单峰样品。将4μL样品置于由氧化石墨烯薄层支撑的300目辉光放电网格(Quantifoil,R1.2/1.3)上，用滤纸吸干水分，经Thermo scientific Vitrobot Mark IV仪器用液态乙烷快速冷冻，用搭载K3相机和BioQuantum成像滤波器的Thermo Fisher Titan Krios G3i电子显微镜进行冷冻电镜成像。对成像的颗粒数据进行多轮2D和3D分类与细化，经进一步的数据处理和3D建模，获得晶体结构数据。实验结果如图16和表30-31所示，以及图17和表32所示。
图16显示了用BA7208-Fab，BA7125V1-Fab对Delta Spike Trimer进行冷冻电镜观察图,图16上的BA7125-Fab为以上实施例中描述的BA7125V1的Fab。图16a.三个BA7208-Fab(绿色)和三个BA7125V1-Fab(黄色)与Delta Spike Trimer(蓝色(dodger blue)、深紫色(plum),以及棕色(rosy brown))的复合物。图16b.BA7208-Fab(浅绿色，Fab轻链；深绿色，Fab重链)与Delta Spike RBD(蓝色)的复合物。图16c.BA7125V1-Fab(浅黄色，Fab轻链；深黄色，Fab重链)与Delta Spike RBD(蓝色)的复合物。图16d.Fabs在Delta RBD上的结合点的放大图，显示了形成氢键的残基的侧链，盐桥和一个阳离子的相互作用。
表30.Delta RBD与BA7208 Fab的蛋白相互作用位点.
斜体残基位于BA7208 Fab轻链，下划线残基位于BA7208 Fab重链
表31.Delta RBD与BA7125V1Fab之间的蛋白相互作用位点

斜体残基位于BA7125V1 Fab轻链，下划线残基位于BA7125V1 Fab重链
图17显示了用BA7208-Fab对Omicron Spike Trimer进行冷冻电镜观察图。图17a.三个BA7208Fab(绿色)与Omicron Spike Trimer(蓝色(dodger blue)、深紫色(plum),以及棕色(rosy brown))的复合物。图17b.BA7208-Fab(浅绿色，Fab轻链；深绿色，Fab重链)与Omicron Spike RBD在向下构象中的复合物(深紫色)。图17c.BA7208-Fab与Omicron Spike RBD在向上构象中的复合物(蓝色)。图17d.7208-Fab在Omicron RBD上的结合点的放大图，显示了形成氢键的残基的侧链，盐桥和一个阳离子的相互作用。
表32.Omicorn BA.1 RBD与BA7208 Fab之间的蛋白相互作用位点
斜体残基位于BA7208 Fab轻链，下划线残基位于BA7208 Fab重链
由图16和表30-31所示，以及图17和表32结果可以看出，BA7208单抗结合Delta RBD上的T345,R346,K444残基；BA7125V1单抗结合Delta RBD上的R403,K417,Y453,K458,G476,Y489,F490,Y505残基；BA7208单抗结合Omicorn BA.1 RBD上的T345,R346,K440,S443,K444残基。
进一步结构解析发现(参见图18)，Omicorn BA.1 RBD的15个突变位点的14个不直接与BA7208-Fab结合，只有N440K与BA7208重链Fab的W34和D56形成氢键，Omicorn BA.2的另外3个RBD突变位点(T376A,D405N和R408S)并不位于BA7208的结合位点。图18可以看出，BA7208与Omicron RBD结合位点避开了BA.1和BA.2的18个突变位点中的17个。
图19A-H显示了BA7208/BA7125V1结构叠加分析,图19上的BA7125为以上实施例中描述的BA7125V1。图19A中RBD(青色)和BA7208-Fab(绿色)的复合体与RBD(青色)和ACE2(蓝色)的复合体叠加，可以看出，BA7208与ACE2结合位点不重叠。图19B中RBD(青色)和BA7125V1-Fab(黄色)的复合物与RBD(青色)和ACE2(蓝色)的复合物叠加。图19C中RBD(青色)和BA7054-Fab(粉色)的复合物与RBD(青色)和ACE2(蓝色)的复合物叠加。图19D-G中RBD(青色)和BA7208 Fab(绿色)的复合物分别与RBD(青色)和抗体(蓝色)REGN10987、VIR-7381、LY-Cov1404和A23-58.1的复合物叠加。图19D-F可以看出，BA7208-Fab与REGN10987、广谱抗体VIR-7381和LY-CoV1404有相似的结合模式。图19H RBD(青色)和BA7125V1-Fab(黄色)的复合物与RBD(青色)和A23-58.1抗体(蓝色)的复合物叠加。
如图19A-19C所示，BA7208-Fab和BA7054-Fab并不通过表位重叠直接阻止ACE2与Spike-RBD的结合，而BA7125V1-Fab则直接与ACE2发生竞争。BA7208-Fab和BA7054-Fab的这一结果与它们作为IgG形式的显著阻断活性不一致，这表明一个完整的IgG分子与一个Spike的2个RBD的双价结合或与包被于平板中的RBD结合的完整抗体分子可能会立体地干扰ACE2与RBD的结合。
如图19D-19H所示，BA7208-Fab和REGN10987、VIR 7381和LY-COV1404的结合模型类似，与A23-58.1的不同，BA7125V1与A23-58.1的结合模型类似。
综上，病毒在SPIKE-RBD上的变异更倾向于与发生在ACE2结合区域，BA7208表位在ACE2结合区域之外预示未来受到变异影响的风险降低。
6.2.我们使用基于生物层干涉仪(BLI)的竞争性结合试验比较了它们与Delta RBD的初步结合表位与Vir-7381、REGN10933和REGN10987。
表33数据显示BA7054、BA7208、Vir-7381和REGN10987相互竞争，表明它们的表位相互接近。BA7054和BA7208都与Vir-7381和REGN10987竞争，表明BA7054和BA7208也可能像Vir-7381和REGN10987一样在ACE2结合区的边缘结合一个表位。BA7208和BA7125V1没有相互竞争，表明它们的表位没有重叠。
表33
6.3基于生物层干涉仪(BLI)的竞争性结合试验比较了BA7535、BA7208抗体的竞争性结合
基于生物层干涉仪(BLI)的竞争性结合试验过程：
抗体的竞争性结合是在ForteBio Octet Red 96系统(Pall Forte BioCorporation,Menlo Park,CA)上使用串联格式分选法进行的。生物素化的Omicron BA.1变体的RBD蛋白(Sino Biological,Cat.40592-V08H121)被加载到SA传感器(Fortebio,Cat.18-5019)。然后将传感器暴露在30μg/mL的第一抗体(如抗体A)或PBST中100秒，然后暴露在30μg/mL的第二抗体(如抗体B)中100秒。使用ForteBio的数据分析软件9.0处理数据。
BLI实验结果参见图20，图20中(mock为没有抗体的buffer，即PBST)4根柱子每根代表芯片结合omicron RBD以后，先结合抗体A，再结合抗体B的结合信号强度。从左向右，第一个柱子代表RBD先与缓冲液(buffer)反应，再结合BA7208，信号强度可达1.2左右，第二根柱子代表RBD先结合BA7208至饱和后，再次结合BA7208，可以再有0.15左右的结合信号，很低，即先结合的BA7208占据RBD标位后，再结合BA7208基本结合不上了，第三根柱子代表先结合缓冲液(buffer)再结合BA7535，结合信号可达1.2左右，第四根柱子代表RBD先结合BA7208至饱和后，再结合BA7535，结合信号仍然能达到1左右，说明BA7208和BA7535的结合表位不一样，BA7208不干扰RBD与BA7535的结合，即BA7535、BA7208不会相互竞争，具有联合应用于治疗COVID-19的潜力。
实施例7 BA7208本申请药物组合物(注射液形式的)配制
将除病毒过滤后BA7208抗体溶液通过30KD超滤膜包浓缩至30-70g/L，然后用透析缓冲液(9.5mM组氨酸,10.5mM盐酸组氨酸,pH 5.5-6.5)进行缓冲液的置换，透析体积6-8倍，整个过程控制TMP≤1.5bar，再浓缩至70-100g/L后将BA7208抗体溶液从超滤系统中冲洗出来，保证BA7208抗体蛋白浓度在55g/L以上。然后向BA7208抗体蛋白溶液中加入辅料母液(9.5mM组氨酸，10.5mM盐酸组氨酸，32％海藻糖，0.08％聚山梨酯80(Ⅱ),pH5.5-6.5)，然后再用透析缓冲液(9.5mM组氨酸,10.5mM盐酸组氨酸,pH 5.5-6.5)将抗体蛋白溶液稀释至 40.0±4.0mg/mL，除菌过滤后得到所述药物组合物(9.5mM组氨酸，10.5mM盐酸组氨酸，8％海藻糖，0.02％聚山梨酯80(Ⅱ)，以及40.0±4.0mg/mL的所述BA7208抗体。
本申请药物组合物(其中含抗体40.0±4.0mg/mL)处方组成如表34所示：
表34
pH 6.0(5.5-6.5)
实施例8各抗体在体内的预防和治疗效力
8.1 BA7208以注射途径给药能有效预防和治疗Omicron感染
Omicron BA.1和BA.2活病毒感染Balb/C wt小鼠或新冠肺炎hACE2转基因小鼠K18模型，攻毒后第二天取小鼠肺脏，采用FFA方法检测肺部活病毒载量，评估抗体的治疗或预防的保护效果。
注射途径给药方式和小鼠的分组如下表35所示：
表35
从图21A-21B中可看出，预防组和治疗组肺部活病毒滴度均显著降低，肺部病毒基本清除，BA7208对小鼠保护作用优异。
8.2 BA7208以滴鼻和雾化吸入途径给药均能有效预防和治疗Omicron感染
K18hACE2转基因小鼠购买自江苏集萃药康，Omicron变异株BA.2由广州海关技术中心生物安全三级实验室分离并保存。真病毒相关试验由广州呼吸健康研究院于BSL-3级试验室开展。
BA7208样品配方为：9.5mM组氨酸，10.5mM盐酸组氨酸，8％海藻糖，0.02％聚山梨酯80(PS80)，抗体BA7208浓度38.905mg/mL。
将Omicron BA.2活病毒采用滴鼻的方式感染K18hACE2转基因小鼠，采用滴鼻50μL途径给药(给药剂量1mg/kg，稀释缓冲液为PBS)或雾化吸入10min途径给药(给药剂量3mg/kg，稀释缓冲液为PBS)，于感染后4h(治疗组)或感染前24h(预防组)进行给药，攻毒24h后(1dpi)将小鼠断颈处死。
对小鼠进行解剖，取小鼠肺部放入1mL PBS缓冲液中进行组织研磨，将研磨好的组织液离心，取上清进行新冠病毒滴度检测，检测方法为FFA法。
滴鼻途径给药方式和小鼠的分组如下表36所示：
表36
雾化途径给药方式和小鼠的分组如下表37所示：
表37
从图21C-21D中可看出，攻毒后的小鼠，经过滴鼻和雾化给予BA7208后，预防组和治疗组肺部活病毒滴度均显著降低，低至检测限，表明肺部病毒基本清除，BA7208对小鼠保护作用优异。
8.3 BA7535、BA7208和它们的组合在体内的中和效力
我们评估了BA7535单独或联合BA7208使用对K18-hACE2转基因小鼠模型的Omicron BA.5变体感染的预防和治疗活性。
实验过程：在K18-hACE2-转基因小鼠(集萃药康)中评估了BA7535和BA7535/BA7208鸡尾酒(即BA7535和BA7208单抗组合)对SARS-CoV-2 Omicron BA.5的体内预防和治疗效力。在感染1×105FFU SARS-CoV-2 Omicron BA.5之前24小时或之后8小时，对六至八周大的小鼠腹腔注射2mg/kg或10mg/kg的BA7535或BA7535+BA7208单抗组合。注射磷酸盐缓冲盐水(PBS)的小鼠受到相同剂量的SARS-CoV-2的感染，作为对照。为了研究SARS-CoV-2在肺部和大脑中的存在，在感染2天和4天后用病灶形成法(FFA)采集肺部和大脑的病毒滴度。同时收集肺部组织并进行染色，进行组织病理学分析，监测体重变化。SARS-CoV-2 Omicron BA.5菌株由中国广东省疾病预防控制中心提供。与真SARS-CoV-2病毒有关的实验在广州海关区技术中心ABSL-3实验室进行。
实验结果分析：感染2天和4天后收集肺部和大脑，分别对复制的病毒进行量化。与对照组相比，2和10mg/kg的BA7535的用量使肺部病毒滴度降低了约2.5个数量级。在肺部和大脑中都观察到病毒复制的完全消除(abrogated)(图22A和22C)。同样，BA7535与BA7208(每种mAb均为1mg/kg或5mg/kg)联合使用，完全消除(abrogated)了BA.5病毒在肺部和大脑中的复制，接受mAb(BA7535/BA7208)鸡尾酒的动物似乎受益于BA7208mAb的额外贡献，因为mAb(BA7535/BA7208)鸡尾酒治疗的动物保持了相对稳定的体重，而那些对照动物的体重则急剧下降(图22B)。对感染SARS-CoV-2的K18-hACE2转基因小鼠进行苏木精和伊红染色的肺部切片分析(图22D)，对照组(PBS)显示肺部病变，血管和分支周围的炎症细胞增多，炎症细胞浸润突出。与对照组(PBS)相比，预防组，以及高剂量BA7535治疗组(BA7535-T-10mg/kg)和低剂量BA7535治疗组(BA7535-T-2mg/kg)没有观察到明显的肺部病变。总之，无论是单独使用还是与BA7208联合使用，BA7535都能保护K18-hACE2转基因小鼠免受SARS-CoV-2 BA.5感染。
实施例9 BA7208对各变异株RBD上除R346K外的其它30余个突变位点均不敏感
19种SARS-CoV-2病毒变体中RBD的关键突变位点如图23所示。以抗体Vir-7381、REGN10933和REGN10987为参考，评估了BA7208，BA7125V1，BA7054和BA7134四种抗体是否能有效地抑制这些变体。如图23所示，浅灰色代表该位点我们抗体有中和活性，黑色代表我们抗体没有中和活性。可以看出，BA7208对RBD上除R346之外的其他突变位点不敏感，比较广谱。
实施例10 BA7208鼻喷制剂II T临床研究
1.研究目的：探究BA7208鼻喷制剂对新冠易感人群感染的预防效果
2.应用人群及场景：集中隔离点，高风险地区出差人员，医护人员，新冠患者密切接触者等。
3.研究设计：
·设计为单臂、开放标签、剂量递增的探索性临床试验，旨在评价BA7208鼻喷制剂作为预防药物在新冠感染易感人群中的药代动力学、初步安全性
和初步药效研究。
·计划入组18-65岁新冠感染易感人员100-300名，经必要健康筛查后接受BA7208鼻喷制剂给药。
·研究分两阶段进行：
IIT临床a
·(1)SAD，4个剂量组，每组10人，经不同浓度BA7208鼻喷制剂给药；评价药代动力学及初步安全性，选择候选剂量开展多次给药剂量试验。
·(2)MAD,3个剂量组，每组20人，经不同浓度BA7208鼻喷制剂给药；评价药代动力学及初步安全性，选择候选剂量开展剂量扩增试验。
IIT临床b
·(1)样本扩大阶段：根据第一阶段确定的候选剂量扩大样本量，进行初步药效研究。试验分为两组:给药组和安慰剂组，观察:给药组和安慰剂组新冠感染阳性率差异。
实施例11 BA7535抗体表位研究
Omicron BA.2 Spike蛋白与抗体Fab混合，冰上孵育20min，分子筛纯化后收集单峰样品。将3.5μL样品置于由氧化石墨烯薄层支撑的辉光放电网格，用滤纸吸干水分，经Thermo scientific Vitrobot Mark IV仪器用液态乙烷快速冷冻，用Thermo Fisher Titan Krios G3i电子显微镜进行冷冻电镜成像。对成像的颗粒数据进行多轮2D和3D分类与细化，经进一步的数据处理和3D建模，获得晶体结构数据。
BA.2 Spike三聚体与BA7535-Fab复合物的晶体结构如图24A，Spike三聚体的3个RBD均处于“向上”构象，1个Spike蛋白可以结合3个BA7535-Fab，并且结合区域位于RBD的顶端。BA.2 RBD和BA7535-Fab复合物的晶体结构(图24B)中可看到，BA.2的RBD与BA7535结合表位包括T415,D420,Y421,A475,N487,Y489和R493共7个残基(表38)，形成6个氢键和1个盐桥，分别为T415-Y106,D420-Y106,Y421-L103,A475-T28,N487-R98和Y489-R98以及R493-E50(前面为RBD氨基酸，后面为BA7535氨基酸)。BA7535的结合表位可避开绝大部分RBD突变，仅在F486位点的突变，以及N417T和R493Q突变可能会影响到BA7535与RBD的结合。
将BA.2 RBD/BA7535-Fab复合物的晶体结构与Spike-RBD/ACE2复合物晶体结构(PDB ID:6VW1)叠加后，如图25所示，BA7535与RBD的结合表位和ACE2的结合表位有部分重合，说明BA7535的中和机制是通过结合于RBD，占据了ACE2的结合位点，从而直接阻断了RBD与ACE2的结合。
比较了RBD/BA7535-Fab与REGN10987(PDB ID:6XDG)的晶体结构，如图26所示，两个抗体的结合表位并不重合，结合模式也有区别。尽管BA7535结合于RBD的顶端，却避开了RBD上大多数突变位点，可大大提高BA7535的广谱性。将两种抗体与RBD的结合区域用PISA进行分析，BA7535比REGN10987有更大的结合区域，BA 7535结合区域面积为而REGN10987的为相比于BA7535表位中6个氢键和1个盐桥，REGN10987表位中仅形成4个氢键和1个盐桥，表明BA7535对RBD比REGN10987有更高的亲和力。BA7535重链与RBD结合的溶剂化自由能ΔG＝-6.3kcal/mol，低于REGN10987的ΔG＝-3.8kcal/mol，同样表明BA7535相比REGN10987对RBD有更高的亲和力。
表38

Claims (17)

  1. 一种抗体或其抗原结合片段，所述抗体或其抗原结合片段结合SARS-CoV-2病毒上的S蛋白，其特征在于，所述抗体或其抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，其中
    所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:38所示的LCDR1、SEQ ID NO:39所示的LCDR2和SEQ ID NO:40所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:41所示的HCDR1、SEQ ID NO:42所示的HCDR2和SEQ ID NO:43所示的HCDR3；
    所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:21所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:23所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:27所示的HCDR1、SEQ ID NO:28所示的HCDR2和SEQ ID NO:29所示的HCDR3；
    所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:9所示的LCDR1、SEQ ID NO:10所示的LCDR2和SEQ ID NO:11所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:12所示的HCDR1、SEQ ID NO:13所示的HCDR2和SEQ ID NO:14所示的HCDR3；
    所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:15所示的LCDR1、SEQ ID NO:16所示的LCDR2和SEQ ID NO:17所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:18所示的HCDR1、SEQ ID NO:19所示的HCDR2和SEQ ID NO:20所示的HCDR3；或者
    所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:21所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:23所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:24所示的HCDR1、SEQ ID NO:25所示的HCDR2和SEQ ID NO:26所示的HCDR3。
  2. 根据权利要求1所述抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含轻链可变区，和/或重链可变区；
    所述轻链可变区包含与SEQ ID NO:36所示的序列具有至少95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列，和/或所述重链可变区包含与SEQ ID NO:37所示的序列具有至少95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列；
    所述轻链可变区包含与SEQ ID NO:7所示的序列具有至少95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列，和/或所述重链可变区包含与SEQ ID NO:8所示的序列具有至少95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列；
    所述轻链可变区包含与SEQ ID NO:1所示的序列具有至少95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列，和/或所述重链可变区包含与SEQ ID NO:2所示的序列具有至少95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列；
    所述轻链可变区包含与SEQ ID NO:3所示的序列具有至少95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列，和/或所述重链可变区包含与SEQ ID NO:4所示的序列具有至少95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列；
    所述轻链可变区包含与SEQ ID NO:5所示的序列具有至少95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列，和/或所述重链可变区包含与SEQ ID NO:6所示的序列具有至少95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列；或者
    所述轻链可变区包含与SEQ ID NO:3所示的序列具有至少95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列，和/或所述重链可变区包含与SEQ ID NO:35所示的序列具有至少95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列。
  3. 根据权利要求1所述抗体或其抗原结合片段，其特征在于，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,AZ.5毒株,以及野生型毒株中的一种或多种；
    优选的，所述野生型毒株为Wuhan-Hu-1毒株；
    优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB毒株中的一种或多种；
    更优选的，所述抗体或其抗原结合片段结合SARS-CoV-2病毒的RBD的T345,R346,K444,R403,K417,Y453,K458,G476,Y489,F490,Y505,K440,S443，T415、D420、Y421、A475、N487和R493残基中的一个或多个；
    更优选的，所述抗体或其抗原结合片段结合Delta RBD上的T345,R346和K444残基；所述抗体或其抗原结合片段结合Omicorn BA.1 RBD上的T345,R346,K440,S443和K444残基；所述抗体或其抗原结合片段结合Delta RBD上的R403,K417,Y453,K458,G476,Y489,F490和Y505残基；或所述抗体或其抗原结合片段结合Omicron BA.2 RBD上的T415、D420、Y421、A475、N487、Y489和R493残基；
    更优选的，所述抗原结合片段为Fab、Fab’、F(ab’)2、Fv、scFv或dsFv片段；
    更优选的，BA7208或其抗原结合片段结合Delta RBD上的T345,R346和K444残基；BA7208或其抗原结合片段结合Omicorn BA.1 RBD上的T345,R346,K440,S443和K444残基；BA7125V1或其抗原结合片段结合Delta RBD上的R403,K417,Y453,K458,G476,Y489,F490和Y505残基；或BA7535或其抗原结合片段结合Omicron BA.2 RBD上的T415、D420、Y421、A475、N487、Y489和R493残基。
  4. 根据权利要求1-3任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体包含SEQ ID NO:30所示的重链恒定区，或者包含SEQ ID NO:31所示的轻链恒定区。
  5. 一种多特异性抗体，所述多特异性抗体衍生自权利要求1-4任一项所述的抗体或其抗原结合片段；优选的，所述多特异性抗体包括双特异性抗体。
  6. 一种双特异性抗体，包括与SARS-CoV-2病毒上的S蛋白结合的第一抗体或抗原结合片段，以及与SARS-CoV-2病毒上的S蛋白结合的第二抗体或抗原结合片段，其中，所述第一抗体或抗原结合片段为根据权利要求1-4任一项所述的抗体或抗原结合片段，和/或所述第二抗体或抗原结合片段为根据权利要求1-4任一项所述的抗体或抗原结合片段；
    优选的，所述第一抗体或抗原结合片段与所述第二抗体或抗原结合片段相同或不同；
    优选的，所述第一抗体或抗原结合片段与所述第二抗体或抗原结合片段结合相同或不同种SARS-CoV-2病毒的S蛋白；更优选的，所述第一抗体或抗原结合片段与所述第二抗体或抗原结合片段结合S蛋白上的相同或不同表位；
    更优选的，所述双特异性抗体结合SARS-CoV-2病毒的RBD的T345,R346,K444,R403,K417,Y453,K458,G476,Y489,F490,Y505,K440,S443，T415、D420、Y421、 A475、N487和R493残基中的一个或多个；
    优选的，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,,以及野生型毒株中的一种或多种；优选的，所述野生型毒株为Wuhan-Hu-1毒株；优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB中的一种或多种。
  7. 根据权利要求6所述的双特异性抗体，所述第一抗原结合片段为Fab，所述第二抗原结合片段为scfv；
    更优选的，所述双特异性抗体具有knob-hole Fc区；
    更优选的，所述第一抗原结合片段连接的重链恒定区为具有hole的重链恒定区,所述第二抗原结合片段连接的重链恒定区为具有knob的重链恒定区；
    更优选的，所述第二抗原结合片段通过VL或VH与具有knob的重链恒定区连接；更优选，所述VL或VH与具有knob的重链恒定区通过连接物连接；
    更优选的，所述双特异性抗体还具有轻链恒定区；
    更优选的，所述第一抗体或抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，所述3个轻链互补决定区包含SEQ ID NO:21所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:23所示的LCDR3，和/或3个重链互补决定区包含SEQ ID NO:27所示的HCDR1、SEQ ID NO:28所示的HCDR2和SEQ ID NO:29所示的HCDR3；所述第二抗体或抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，所述3个轻链互补决定区包含SEQ ID NO:15所示的LCDR1、SEQ ID NO:16所示的LCDR2和SEQ ID NO:17所示的LCDR3，和/或3个重链互补决定区包含SEQ ID NO:18所示的HCDR1、SEQ ID NO:19所示的HCDR2和SEQ ID NO:20所示的HCDR3；
    更优选的，所述第一抗体或抗原结合片段包含SEQ ID NO:7所示的轻链可变区，和/或SEQ ID NO:8所示的重链可变区；所述第二抗体或抗原结合片段包含SEQ ID NO:3所示的轻链可变区，和/或SEQ ID NO:35所示的重链可变区；
    更优选的，所述第一抗体或抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，所述3个轻链互补决定区包含SEQ ID NO:21所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:23所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:27所示的HCDR1、SEQ ID NO:28所示的HCDR2和SEQ ID NO:29所示的HCDR3；所述第二抗体或抗原结合片段包含3个轻链互补决定区包含SEQ ID NO:38所示的LCDR1、SEQ ID NO:39所示的LCDR2和SEQ ID NO:40所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:41所示的HCDR1、SEQ ID NO:42所示的HCDR2和SEQ ID NO:43所示的HCDR3；
    更优选的，所述第一抗体或抗原结合片段包含SEQ ID NO:7所示的轻链可变区，和/或SEQ ID NO:8所示的重链可变区；所述第二抗体或抗原结合片段包含SEQ ID NO:36所示的轻链可变区，和/或SEQ ID NO:37所示的重链可变区；
    更优选的，所述第一抗体或抗原结合片段为BA7208 Fab，所述第二抗体或抗原结合片段为BA7125V1scfv；或者所述第一抗体或抗原结合片段为BA7208 Fab，所述第二抗体或抗原 结合片段为BA7535 scfv。
  8. 一种抗体组合，其包含结合SARS-CoV-2病毒上的S蛋白的两种或多种抗体或抗原结合片段的组合；
    优选的，所述抗体组合为两种抗体或抗原结合片段的组合，其中，第一抗体或抗原结合片段为根据权利要求1-4任一项所述的抗体或抗原结合片段，和/或第二抗体或抗原结合片段为根据权利要求1-4任一项所述的抗体或抗原结合片段；
    更优选的，所述第一抗体或抗原结合片段与所述第二抗体或抗原结合片段相同或不同；更优选的，所述第一抗体或抗原结合片段与所述第二抗体或抗原结合片段结合相同或不同种SARS-CoV-2病毒的S蛋白；更优选的，所述第一抗体或抗原结合片段与所述第二抗体或抗原结合片段结合S蛋白上的相同或不同表位；
    更优选的，所述第一抗体或抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，所述3个轻链互补决定区包含SEQ ID NO:21所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:23所示的LCDR3，和/或3个重链互补决定区包含SEQ ID NO:27所示的HCDR1、SEQ ID NO:28所示的HCDR2和SEQ ID NO:29所示的HCDR3；所述第二抗体或抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，所述3个轻链互补决定区包含SEQ ID NO:15所示的LCDR1、SEQ ID NO:16所示的LCDR2和SEQ ID NO:17所示的LCDR3，和/或3个重链互补决定区包含SEQ ID NO:18所示的HCDR1、SEQ ID NO:19所示的HCDR2和SEQ ID NO:20所示的HCDR3；
    更优选的，所述第一抗体或抗原结合片段包含SEQ ID NO:7所示的轻链可变区，和/或SEQ ID NO:8所示的重链可变区的第一抗体或抗原结合片段；所述第二抗体或抗原结合片段包含SEQ ID NO:3所示的轻链可变区，和/或SEQ ID NO:35所示的重链可变区；
    更优选的，所述第一抗体或抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，所述3个轻链互补决定区包含SEQ ID NO:21所示的LCDR1、SEQ ID NO:22所示的LCDR2和SEQ ID NO:23所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:27所示的HCDR1、SEQ ID NO:28所示的HCDR2和SEQ ID NO:29所示的HCDR3；所述第二抗体或抗原结合片段包含3个轻链互补决定区包含SEQ ID NO:38所示的LCDR1、SEQ ID NO:39所示的LCDR2和SEQ ID NO:40所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:41所示的HCDR1、SEQ ID NO:42所示的HCDR2和SEQ ID NO:43所示的HCDR3；
    更优选的，所述第一抗体或抗原结合片段包含SEQ ID NO:7所示的轻链可变区，和/或SEQ ID NO:8所示的重链可变区；所述第二抗体或抗原结合片段包含SEQ ID NO:36所示的轻链可变区，和/或SEQ ID NO:37所示的重链可变区；
    更优选的，所述第一抗体或抗原结合片段为BA7208抗体或抗原结合片段，所述第二抗体或抗原结合片段为BA7125V1抗体或抗原结合片段；或者所述第一抗体或抗原结合片段为BA7208抗体或抗原结合片段，所述第二抗体或抗原结合片段为BA7535抗体或抗原结合片段；
    更优选的，所述抗体组合结合SARS-CoV-2病毒的RBD的T345,R346,K444,R403,K417,Y453,K458,G476,Y489,F490,Y505,K440,S443，T415、D420、Y421、A475、N487和R493残基中的一个或多个；
    优选的，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron 毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,,以及野生型毒株中的一种或多种；优选的，所述野生型毒株为Wuhan-Hu-1毒株；优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB中的一种或多种。
  9. 一种核酸，其编码权利要求1-4任一项所述的抗体或其抗原结合片段，或权利要求5所述的多特异性抗体，或权利要求6-7任一项所述的双特异性抗体。
  10. 一种核酸组合，其包括编码权利要求8所述抗体组合中各抗体的核酸的组合。
  11. 一种细胞，其包含权利要求9的核酸或权利要求10所述的核酸组合。
  12. 一种药物组合物，其含有权利要求1-4任一项所述的抗体或其抗原结合片段，或权利要求5所述的多特异性抗体，或权利要求6-7任一项所述的双特异性抗体，或权利要求8所述的抗体组合，或权利要求9所述的核酸，权利要求10所述的核酸组合，或权利要求11所述的细胞。
  13. 根据权利要求12所述的药物组合物，所述药物组合物为鼻喷、鼻滴、雾化或注射制剂；优选的，所述注射制剂为静脉注射制剂；更优选的，所述药物组合物含治疗有效量的，优选的，约30mg至约2400mg，优选的，约1200mg或约2400mg的权利要求1至4中任一所述抗体或抗原结合片段、或权利要求5所述的多特异性抗体，或权利要求6-7任一项所述的双特异性抗体，或权利要求8所述的抗体组合；
    更优选的，所述药物组合物为单位制剂，所述单位制剂为鼻喷、鼻滴、雾化或注射制剂，并且该单位制剂含有治疗有效量的，优选的，30毫克至2400毫克，优选的，约1200mg或约2400mg的权利要求1-4中任一项所述的抗体或其抗原结合片段、或权利要求5所述的多特异性抗体，或权利要求6-7任一项所述的双特异性抗体，或权利要求8所述的抗体组合；优选的，所述药物组合物含有选自所述抗体或其抗原结合片段、所述多特异性抗体，所述双特异性抗体和所述抗体组合中的一种，以及缓冲液；更优选的，所述缓冲液包括海藻糖和聚山梨酯80中的一种或多种；更优选的，所述药物组合物pH为5.5-6.5；更优选的，所述缓冲液还包括盐酸组氨酸和组氨酸中的一种或多种；更优选的，所述盐酸组氨酸和组氨酸的摩尔比为10.5：9.5；更优选的，基于所述药物组合物的总体积，所述药物组合物包括0.04-0.1g/mL海藻糖,0.0001-0.0003g/mL聚山梨酯80，以及10-50mg/mL的选自所述抗体或其抗原结合片段、所述多特异性抗体，所述双特异性抗体和所述抗体组合中的一种；更优选的，基于所述药物组合物的总体积，所述药物组合物包括10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，以及40±4mg/mL的选自所述抗体或其抗原结合片段、所述多特异性抗体，所述双特异性抗体和所述抗体组合中的一种。
  14. 一种试剂盒，其含有权利要求1-4任一项所述的抗体或其抗原结合片段，或权利要求5所述的多特异性抗体，或权利要求6-7任一项所述的双特异性抗体，或权利要求8所述的抗体组合，或权利要求9所述的核酸，或权利要求10所述的核酸组合，或权利要求11所述的细胞，或权利要求12-13任一项所述的药物组合物。
  15. 权利要求1-4任一项所述的抗体或其抗原结合片段，权利要求5所述的多特异性抗体，权利要求6-7任一项所述的双特异性抗体，权利要求8所述的抗体组合，权利要求9所述的核酸，或权利要求10所述的核酸组合，权利要求11所述的细胞，或权利要求14所述的试剂盒在制备用于预防、治疗、检测或诊断与SARS-CoV-2病毒相关的疾病的药物中的应用；
    优选的，所述SARS-CoV-2病毒包括Alpha(B.1.1.7)毒株、Beta(B.1.351)毒株、Gamma(p.1)毒株、Delta(B.1.617.2)毒株、Lambda(C.37)毒株、Mu(B.1.621)毒株、Omicron毒株，Kappa(B.1.617.1)毒株,C.1.2毒株,B.1.630毒株,B.1.640毒株,B.1.526毒株,B.1.525毒株,AZ.5毒株,以及野生型毒株中的一种或多种；
    优选的，所述野生型毒株为Wuhan-Hu-1毒株；
    优选的，所述Omicron毒株包括BA.1(B.1.1.529.1)、BA.1.1、BA.2、BA.2.12.1、BA.2.13、BA.2.38、BA.2.38.1、BA.2.74、BA.2.75、BA.2.76、BA.2.77、BA.2.79、BA.2.80、BA.3、BA.4、BA.5、BA.4.6、BA.4.7、BA.5.5.1、BQ.1、BQ.1.1、XBB毒株中的一种或多种。
  16. 根据权利要求15所述的应用，所述药物为鼻喷、鼻滴、雾化或注射制剂；优选的，所述注射制剂为静脉注射制剂；更优选的，所述药物含治疗有效量的，优选的，约30mg至约2400mg，优选的，约1200mg或约2400mg的权利要求1至4中任一所述抗体或抗原结合片段、或权利要求5所述的多特异性抗体，或权利要求6-7任一项所述的双特异性抗体，或权利要求8所述的抗体组合；
    优选的，所述药物为单位制剂，所述单位制剂为鼻喷、鼻滴、雾化或注射制剂，并且该单位制剂含有治疗有效量的，优选的，30毫克至2400毫克，优选的，约1200mg或约2400mg的权利要求1-4中任一项所述的抗体或其抗原结合片段、或权利要求5所述的多特异性抗体，或权利要求6-7任一项所述的双特异性抗体，或权利要求8所述的抗体组合；优选的，所述药物含有选自所述抗体或其抗原结合片段、所述多特异性抗体，所述双特异性抗体和所述抗体组合中的一种，以及缓冲液；更优选的，所述缓冲液包括海藻糖和聚山梨酯80中的一种或多种；更优选的，所述药物pH为5.5-6.5；更优选的，所述缓冲液还包括盐酸组氨酸和组氨酸中的一种或多种；更优选的，所述盐酸组氨酸和组氨酸的摩尔比为10.5：9.5；更优选的，基于所述药物的总体积，所述药物包括0.04-0.1g/mL海藻糖,0.0001-0.0003g/mL聚山梨酯80，以及10-50mg/mL的选自所述抗体或其抗原结合片段、所述多特异性抗体，所述双特异性抗体和所述抗体组合中的一种；更优选的，基于所述药物的总体积，所述药物包括10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，以及40±4mg/mL的选自所述抗体或其抗原结合片段、所述多特异性抗体，所述双特异性抗体和所述抗体组合中的一种。
  17. 一种治疗或预防SARS-CoV-2病毒引起的疾病的方法，包括向有需要的受试者施用所述权利要求1-4任一项所述的抗体或其抗原结合片段，权利要求5所述的多特异性抗体，权利要求6-7任一项所述的双特异性抗体，权利要求8所述的抗体组合，权利要求12-13任一项所述的药物组合物；
    所述有需要的受试者包括治疗受试者或预防受试者；
    所述治疗受试者包括感染SARS-CoV-2病毒的无症状、轻型、普通型、重型或危重型患者；优选的，所述治疗受试者为发病时间≤7天且为在72小时之内按照《新型冠状病毒肺炎诊疗方案(试行第九版)》确诊感染SARS-CoV-2病毒的无症状、轻型、普通型、重型或危重型患者；
    所述治疗受试者包括感染SARS-CoV-2病毒的无症状、轻度、中度、重度及危患者；优选的，所述治疗受试者为72小时内实验室检查(如RT-PCR检查)确认感染SARS-CoV-2，且按NIH指南确诊的无症状、轻度、中度、重度及危重COVID-19的患者；或者
    所述预防受试者包括暴露前预防受试者、或暴露后预防受试者；优选的，所述暴露前预防受试者包括新冠病毒暴露高风险人群、健康受试者、或者其他不适合接种疫苗的受试者；所述暴露后预防受试者包括新冠肺炎确诊患者和/或无症状感染者的密切接触者。
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