WO2023130770A1 - 一种全人源抗新冠病毒广谱中和抗体zw2g10及应用 - Google Patents

一种全人源抗新冠病毒广谱中和抗体zw2g10及应用 Download PDF

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WO2023130770A1
WO2023130770A1 PCT/CN2022/121155 CN2022121155W WO2023130770A1 WO 2023130770 A1 WO2023130770 A1 WO 2023130770A1 CN 2022121155 W CN2022121155 W CN 2022121155W WO 2023130770 A1 WO2023130770 A1 WO 2023130770A1
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monoclonal antibody
cov
seq
fully human
amino acid
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French (fr)
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陈薇
于长明
李建民
迟象阳
孙韩聪
张冠英
张军
范鹏飞
张哲�
陈郑珊
王步森
宰晓东
房婷
付玲
郝勐
陈旖
徐婧含
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中国人民解放军军事科学院军事医学研究院
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Publication of WO2023130770A1 publication Critical patent/WO2023130770A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention discloses an antibody and belongs to the fields of microbiology and immunology.
  • RNA virus The pathogen of new coronary pneumonia (COVID-19) is novel coronavirus-2 (SARS-CoV-2).
  • RNA virus its genome length is about 30 kb.
  • the first 2/3 of the genome is the non-structural gene ORF1a/b, which mainly encodes enzymes related to viral replication (RNA-dependent RNA polymerase, RdRp), and the last 1/3 encodes four structural proteins in turn: spike protein (S), Envelope protein (E), membrane protein (M) and nucleocapsid protein (N).
  • the S protein contains a virus receptor binding region, which can bind to the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of human cells, mediate virus adsorption and entry into cells, and is a key protein for virus invasion of host susceptible cells.
  • ACE2 angiotensin-converting enzyme 2
  • the virus will continuously mutate randomly during the transmission process, some of which will enhance the binding ability of the S protein to the ACE2 receptor and accelerate the spread of the virus among the population, such as K417N, N501Y, E484K, P681R, etc. Therefore, some virus variants carrying key site mutations show stronger infection ability or stronger immune escape ability, which reduces the effectiveness of existing public health interventions or vaccines, as listed by the World Health Organization (WHO).
  • Alpha strain B.1.1.7
  • Beta strain B.1.351
  • Gamma strain P.1
  • Delta strain B.1.617.2
  • Omicron strain B.1.1.529
  • the monoclonal neutralizing antibody targeting the spike protein (S protein) on the surface of the virus has become a potential effective treatment for new coronary pneumonia. It binds to the new coronavirus to inhibit the activity of the virus and protect cells from damage. Compared with small molecule drugs and plasma therapy, monoclonal antibody drugs have a clear mechanism, high selectivity to targets, strong specificity, and few side effects. According to information on the Chineseantibody website, 25 monoclonal antibodies targeting the S protein have entered clinical research, and these monoclonal antibodies are all effective against wild-type novel coronaviruses.
  • LY-CoV555 and LY-CoV016 developed by Lilly, regeneration The REGN10933 developed by Yuan lost its neutralizing activity against the Beta strain, and the antibody drug LY-CoV555-LY-CoV016 cocktail therapy, REGEN-COV cocktail therapy, and AZD7442 cocktail therapy lost its neutralizing activity against the Omicron strain.
  • the development of broad-spectrum monoclonal antibodies with conserved neutralizing epitopes including the development of broad-spectrum neutralizing monoclonal antibodies or a combination of two highly efficient neutralizing monoclonal antibodies, has great clinical application value.
  • single-cell PCR technology has the advantages of full human origin and good natural stability, and has been widely used in the development of new crown neutralizing antibodies.
  • the principle of single-cell PCR technology is that there is a protective monoclonal antibody against the virus in the body of the new coronavirus infection recovery or the new crown vaccine recipient.
  • the gene encoding the antibody is located in a single lymphocyte in the peripheral blood of the human body.
  • Single-cell PCR technology can "fish" for this gene. Then, by means of genetic engineering, the molecule can be prepared on a large scale in vitro.
  • the present invention intends to use flow sorting-single-cell PCR technology to obtain monoclonal antibodies with excellent broad-spectrum neutralizing activity from the peripheral blood of recombinant new coronavirus vaccine recipients, with the purpose of providing a full-body antibody with good protective effect against COVID-19.
  • Human monoclonal therapeutic antibodies to deal with the current epidemic and possible future variants.
  • the present invention screened a monoclonal antibody against SARS-CoV-2 by flow sorting-single-cell PCR technology, and the CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the monoclonal antibody
  • the amino acid sequence of amino acid sequence is shown in SEQ ID NO: 1 No. 26-33, 51-58, 97-117 amino acid sequence respectively;
  • the amino acid sequence of CDR1, CDR2 and CDR3 region of light chain variable region is shown in SEQ ID NO: 5 respectively
  • the 26-34, 52-54, 91-101 amino acid sequences are shown.
  • the monoclonal antibody is named "ZW2G10" in this application.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:1
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO:5.
  • amino acid sequence of the heavy chain constant region of the antibody is shown in SEQ ID NO:3
  • amino acid sequence of the light chain constant region is shown in SEQ ID NO:7.
  • the present invention also provides a polynucleotide encoding the heavy chain and light chain of the above-mentioned monoclonal antibody, the polynucleotide sequence of the heavy chain variable region encoding the antibody is represented by SEQ As shown in ID NO:2, the polynucleotide sequence encoding the light chain variable region of the antibody is shown in SEQ ID NO:6.
  • polynucleotide sequence encoding the heavy chain constant region of the antibody is represented by SEQ ID NO: 4
  • polynucleotide sequence encoding the light chain constant region of the antibody is represented by SEQ ID NO: 8.
  • the present invention also provides a functional element for expressing the above-mentioned polynucleotide encoding the heavy chain and light chain of the monoclonal antibody.
  • This functional element can be a traditional expression vector.
  • the functional element is a linear expression cassette.
  • the functional element is a mammalian expression vector.
  • the present invention also provides a host cell containing the above-mentioned linear expression cassette.
  • the cells are Expi 293F cells.
  • the cells are CHO-K1 or CHO-S cells.
  • CHO-K1 or CHO-S cells can be used to construct stable engineered cell lines to realize industrial production.
  • the present invention also provides the application of the above-mentioned monoclonal antibody in the preparation of a drug for treating COVID-19.
  • the monoclonal antibody provided by the invention is screened by flow sorting-single-cell PCR technology, has a unique CDR partition, and its antigen recognition epitope is located in the RBD region of the S1 protein.
  • the affinity of the antibody to SARS-CoV-2 wild-type S-ECD is 0.243 nM, and the affinity to Alpha strain, Beta strain, Gamma strain, Delta strain, Omicron strain S-ECD is 0.166 nM, 0.518 nM, 0.615 nM respectively , 5.452 nM, 0.765 nM.
  • the EC 50 for the wild type of the new coronavirus was 14.19 ng/mL
  • the EC 50 for neutralizing the Alpha strain was 14.12 ng/mL
  • the EC 50 for neutralizing the Beta strain was 18.41 ng/mL.
  • the EC 50 of Gamma strain was 15.59 ng/mL
  • the neutralizing EC 50 of Delta strain was 36.18 ng/mL
  • the neutralizing EC 50 of Omicron strain was 19.26 ng/mL.
  • the EC 50 for the wild type of the new coronavirus was 1.077 ⁇ g/mL
  • the EC 50 for the neutralization of the Beta strain was 1.423 ⁇ g/mL
  • the EC 50 for the neutralization of the Delta strain was 0.71 ⁇ g/mL, showing that ZW2G10 It has broad-spectrum and high-efficiency neutralizing activity against the current main mutant strains.
  • the monoclonal antibody disclosed in the present invention has the characteristics of high expression, full human origin, and good stability, is suitable for industrial production, and has great clinical application value for coping with outbreaks caused by mutant strains that may occur at present and in the future.
  • FIG. 1 The binding activity diagram of antibody expression supernatant to SARS-CoV-2;
  • Figure 10 The binding kinetics curve of ZW2G10 and WT strain S-ECD protein
  • Figure 15 The binding kinetics curve between ZW2G10 and Omicron strain S-ECD protein.
  • Example 1 Screening and preparation of human anti-SARS-CoV-2 monoclonal antibodies.
  • the collected blood samples were separated from PBMC by Ficoll density gradient centrifugation, and the process was as follows:
  • the bottom of the tube is red blood cells
  • the middle layer is the separation solution
  • the top layer is the plasma/tissue homogenate layer
  • between the plasma layer and the separation solution layer is a thin and dense white membrane, that is: a single nucleus Layer of cells (including lymphocytes and monocytes).
  • FIG. 9 Use a cell sorter (Beckman MofloXDP) to sort SARS-CoV-2 WT (new crown wild strain Genbank number: NC_045512.2) S-ECD-specific single memory B cells.
  • the sorting strategy is: CD3 - /CD19 + / IgG + /CD27 + / SARS-CoV-2 WT S-ECD + , as shown in Figure 1, lymphocytes are circled in Figure 1-A, circled and removed in Figure 1-B Adhesive cells, circled CD3 - / CD19 + B cells in Figure 1-C, circled IgG + /CD27 + memory B cells in Figure 1-D, circled SARS-CoV-2 WT in Figure 1-E S-ECD + memory B cells.
  • a single memory B cell was directly sorted into a 96-well plate, and 20 ⁇ L of RNase-free water and 20 U of RNase inhibitor were pre-added to each well of the 96-well plate, and stored at -80°C.
  • the PCR reaction system contains: 6 ⁇ L of 5 ⁇ buffer, 1.2 ⁇ L of dNTP, 1.2 ⁇ L of reverse transcriptase (Qiagen, 210212), primers as above, single-cell template, and make up to 30 ⁇ L with water.
  • the PCR reaction conditions were: reverse transcription at 50°C for 30 min, pre-denaturation at 95°C for 15 min, followed by 40 cycles of 95°C for 40 s, 55°C for 30 s, 72°C for 1 min, and finally extension at 72°C for 10 min.
  • the PCR reaction system contains: 5 ⁇ L of 10 ⁇ buffer, 4 ⁇ L of 2.5 mM dNTP, 0.5 ⁇ L of DNA polymerase (Quanshijin Biotechnology Co., Ltd., AP141), primers as above, 1 ⁇ L of reverse transcription product as template, water supplement Make up to 50 ⁇ L.
  • the PCR reaction conditions were: pre-denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 57°C for 30 s, 72°C for 45 s, and finally extension at 72°C for 10 min.
  • FIG. 2 is the identification map of capillary electrophoresis after nested PCR amplification of three chain genes of H, ⁇ , ⁇ .
  • FIG. 3 shows the search results of the heavy chain variable region.
  • the V region has the highest homology of 97.57%
  • the J region has the highest homology of 85.48%
  • the D region uses reading frame 2.
  • Figure 3-B shows the search results of the light chain, the highest homology of the V region is 97.92%, and the highest homology of the J region is 89.19%.
  • the linear expression cassette expresses the antibody.
  • the designed linear expression frame contains all the elements for the expression of monoclonal antibodies in mammalian cells.
  • the linear expression frame contains the CMV promoter sequence (Genbank accession number: X03922.1), the coding sequence of the antibody leader peptide, and the antibody variable sequence from the 5' end.
  • the PCR reaction system for amplifying the heavy chain promoter-leader sequence fragment includes: template plasmid pMD-CMVH 10 ng, 10 ⁇ buffer 5 ⁇ L, 2.5 mM dNTP 4 ⁇ L, DNA polymerase 0.5 ⁇ L, primer 5'-CMV- UP (matched with the upstream sequence of the CMV promoter) (5'- GATATACGCGTTGACATTGATTATTGAC -3'), primer 3'- leader-H(HR) (5'- ACACTGAACACCTTTTAAAATTAG -3', for heavy chain fusion, nucleotide sequence of signal peptide sequence:
  • the PCR reaction system for amplifying the light chain promoter-leader sequence fragment includes: template plasmid pMD-CMVL 10 ng, 10 ⁇ buffer 5 ⁇ L, 2.5 mM dNTP 4 ⁇ L, DNA polymerase 0.5 ⁇ L, primer 5'-CMV- UP (5'- GATATACGCGTTGACATTGATTATTGAC -3'), primer 3'- leader-L(HR) (5'- CCCACAGGTACCAGATACCCATAG -3'), used for light chain fusion, the nucleotide sequence of the full-length signal peptide sequence is:
  • the amino acid sequence is MDSQAQVLMLLLLWVSGTCG
  • the signal peptide sequence is derived from the variable region of murine monoclonal antibody), and made up to 50 ⁇ L with water.
  • PCR reaction conditions pre-denaturation at 95°C for 10 min, followed by 30 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 1 min, and finally extension at 72°C for 10 min.
  • the H chain constant region-poly A tail fragment PCR system contains: template plasmid pMD-TKH 10 ng, 10 ⁇ buffer 5 ⁇ L, 2.5 mM dNTP 4 ⁇ L, DNA polymerase 0.5 ⁇ L, primer 5'-CH (5' -ACCAAGGGCCCATCGGTCTTCCCC-3'), primer 3'-TK-POLY(A) (5'- AAGTGTAGCGGTCACGCTGCGCGTAACC -3'), water to make up to 50 ⁇ L.
  • the ⁇ chain constant region-poly A tail fragment PCR system contains: template plasmid pMD-TK ⁇ 10 ng, 10 ⁇ buffer 5 ⁇ L, 2.5 mM dNTP 4 ⁇ L, DNA polymerase 0.5 ⁇ L, primer 5'-C ⁇ (5' - ACTGTGGCTGCACCATCTGTCTTC -3'), primer 3'-TK-POLY(A) (5'- AAGTGTAGCGGTCACGCTGCGCGTAACC -3'), water to make up to 50 ⁇ L.
  • the ⁇ chain constant region-poly A tail fragment PCR system contains: template plasmid pMD-TK ⁇ 10 ng, 10 ⁇ buffer 5 ⁇ L, 2.5 mM dNTP 4 ⁇ L, DNA polymerase 0.5 ⁇ L, primer 5'-C ⁇ (CTACGTCAGCCCAAGGCTGCCCCC) , Primer 3'-TK-POLY(A) (5'- AAGTGTAGCGGTCACGCTGCGCGTAACC -3'), water to make up to 50 ⁇ L.
  • the PCR reaction conditions were: pre-denaturation at 95°C for 10 min, followed by 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 2 min, and finally extension at 72°C for 10 min.
  • the single underlined part is used for fusion with upstream fragments, and the underlined bold part is used for fusion with downstream fragments.
  • the PCR reaction system contains: 5 ⁇ L of 10 ⁇ buffer, 4 ⁇ L of 2.5 mM dNTP, 0.5 ⁇ L of DNA polymerase (Quanshijin Biotechnology Co., Ltd., AP141), primers as above, 1 ⁇ L of nested PCR product as template, water supplement Make up to 50 ⁇ L.
  • the PCR reaction conditions were: pre-denaturation at 95°C for 4 min, followed by 40 cycles of 95°C for 30 s, 57°C for 30 s, 72°C for 45 s, and finally extension at 72°C for 10 min.
  • the PCR reaction system includes:
  • Template Purified promoter-leader sequence fragment 10 ng, heavy chain/light chain variable region fragment 10 ng, heavy chain/light chain constant region-poly A tail fragment 10 ng, 10 ⁇ buffer 5 ⁇ L, 12.5 mM dNTP 4 ⁇ L, DNA polymerase (Quanshijin Biotechnology Co., Ltd., AP151-13) 0.5 ⁇ L, primer 5'-CMV-UP (5'- GATATACGCGTTGACATTGATTATTGAC-3') and 3'-TK-POLY(A) (5'- AAGTGTAGCGGTCACGCTGCGCGTAACC -3', make up to 50 ⁇ L with water.
  • the PCR reaction conditions were: pre-denaturation at 95°C for 10 min, followed by 30 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 3 min, and finally extension at 72°C for 10 min.
  • PCR reaction product was directly recovered with the recovery kit of OMEGA Company.
  • DNA quantification PCR recovery products were quantified using Nano (GE Healthcare).
  • Blocking Wash 3 times with a plate washer (BIO-TEK, 405_LS) on the day of the experiment, add 100 ⁇ L of blocking solution to each well, and incubate at 37°C for 1 hour.
  • the light and heavy chain recombinant expression plasmids were constructed for ZW2G10, and the monoclonal antibody was expressed and prepared.
  • the heavy chain was amplified, and the 1.4kb heavy chain fragment was recovered by gel cutting.
  • the expression vector pCDNA3.4 (ThermoFisher Scientific, A14697) was digested with EcoR I/BamH I and recovered.
  • the heavy chain and vector fragments by homologous recombination (NEBuilder HiFi DNA Assembly Master Mix, E2621L) method for ligation, transformed TOP10 and picked clones for sequencing identification, and successfully constructed the heavy chain expression vector pCDNA3.4-ZW2G10-H.
  • the light chain was amplified, the light chain fragment of about 0.7kb was recovered from the gel, the light chain and the carrier fragment were connected by homologous recombination, transformed into TOP10, and the clones were selected for sequencing and identification, and the successful light chain was constructed Expression vector pCDNA3.4-ZW2G10- ⁇ .
  • Expi293 expression system mix 15 ⁇ g of the heavy chain and 15 ⁇ g of the light chain and transfect into Expi 293F cells, operate according to the instructions (ThermoFisher Scientific, A14635), harvest the culture medium after 5-6 days, and centrifuge about 30 mL of the supernatant, Use a prepacked Protein A affinity chromatography column with a volume of 5 mL, equilibrate with 20 mM PBS before loading the sample, inject the sample after the conductance shows the baseline, and wash the column with 20 mM PBS until the baseline is stable after loading the sample. Use 0.1 M glycine buffer with pH 3.0 to elute the target protein. When the OD 280 is close to the baseline, stop collecting.
  • the protein sample can be reduced to two fragments of 50 kDa and 25 kDa in size by mercaptoethanol, which correspond to the theoretical molecular weights of the heavy chain and light chain of the antibody respectively. It is expected that the Marker molecular weights from large to small are: 250, 130, 100, 70, 55, 55, 35, 25, 15, 10 kDa.
  • Blocking Wash 3 times with a plate washer (BIO-TEK, 405_LS) on the day of the experiment, add 100 ⁇ L of blocking solution to each well, and incubate at 37°C for 1 hour.
  • ZW2G10 specifically binds to the S-ECD, S1 and RBD proteins of SARS-CoV-2 WT (Genebank number: NC_045512.2). There is a dose-response relationship, but it does not bind to NTD protein and S2 protein. The results showed that the epitope recognized by monoclonal antibody ZW2G10 was located in the RBD region of S1 protein.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region are shown in SEQ ID NO: 1 26-33, 51-58, 97- Shown in the 117th amino acid sequence, the polynucleotide sequence encoding the heavy chain variable region is represented by SEQ ID NO: 2; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5, and the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the light chain variable region are shown in SEQ ID NO: 5, respectively.
  • ID NO: 5 shown in the 26-34, 52-54, 91-101 amino acid sequence the polynucleotide sequence encoding the light chain variable region is represented by SEQ ID NO: 6.
  • ZW2G10 specifically binds to the S-ECD protein of Alpha strain, Beta strain, Gamma strain, Delta strain, and Omicron strain, and exhibits a dose-response relationship.
  • monoclonal antibody ZW2G10 could cross-link the S-ECD protein of Alpha strain, Beta strain, Gamma strain, Delta strain and Omicron strain.
  • the purified monoclonal antibody was serially diluted 3 times from the initial concentration (ZW2G10 monoclonal antibody initial concentration 3.7 ⁇ g/ml) with medium DMEM+10% FBS, added to a 96-well culture plate, and set up 3 replicate wells with a volume of 50 ⁇ L/ml.
  • the results are shown in Figure 8.
  • the EC 50 of the monoclonal antibody ZW2G10 disclosed by the present invention against the wild-type pseudovirus of the new coronavirus is 14.19 ng/mL
  • the EC 50 of neutralizing the Alpha strain pseudovirus is 14.12 ng/mL
  • neutralizing the Beta strain pseudovirus The EC 50 for neutralizing the pseudovirus of the Gamma strain was 15.59 ng/mL
  • the EC 50 for neutralizing the pseudovirus of the Delta strain was 36.18 ng/mL
  • the EC 50 for neutralizing the pseudovirus of the Omicron strain was 19.26 ng /mL.
  • the results show that ZW2G10 has a broad-spectrum and high-efficiency neutralizing activity against the pseudoviruses of the current main mutant strains.
  • Example 5 Identification of euvirus neutralizing activity of antibody ZW2G10.
  • the purified monoclonal antibody was added to a 96-well culture plate with a volume of 120 ⁇ L/well from the initial concentration (the initial concentration of ZW2G10 monoclonal antibody was 100 ⁇ g/ml, 3-fold serial dilution) in the culture medium DMEM+2% FBS; Add 120 ⁇ L of COVID-19 virus suspension (dilute the virus with DMEM+2% FBS, add 100 TCID 50 /well), mix well, and incubate in a cell culture incubator for 1 h.
  • the results are shown in Figure 9.
  • the EC 50 of the monoclonal antibody ZW2G10 disclosed in the present invention against the wild type of the new coronavirus is 1.077 ⁇ g/mL
  • the EC 50 of the neutralizing Beta strain is 1.423 ⁇ g/mL
  • the EC 50 of the neutralizing Delta strain is 0.710 ⁇ g /mL. It shows that ZW2G10 has high-efficiency neutralizing activity against the true viruses of the wild type, Beta and Delta variants of SARS-CoV-2.
  • SPR Surface plasmon resonance
  • Protein exchange Use a desalting column to exchange the antibody and antigen protein into HBS-EP+ buffer, place the desalting column in an empty collection tube, loosen the cap of the desalting column, and centrifuge at 1500 g for 1 min to remove the original protein in the column. Liquid, add 300 ⁇ L HBS-EP+ buffer, centrifuge at 1500 g for 1 min, repeat 4 times, put the desalting column in a new collection tube, add 100 ⁇ L protein solution to the column, centrifuge at 1500 g for 2 min, collect the filtered liquid , Determination of protein concentration using NanoVue.
  • Sample preparation Dilute the ligand (antibody) to 0.5 ⁇ g/mL with HBS-EP+ buffer, and dilute the analyte (antigen) to 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM.
  • Figures 10-15 are the determination diagrams of the affinity constants of ZW2G10 and S-ECD of WT, Alpha, Beta, Gamma, Delta, and Omicron strains, respectively, and their KDs are 0.243 nM, 0.166 nM, 0.518 nM, 0.615 nM, and 5.452 nM and 0.765 nM.
  • the results show that the neutralizing antibody has a good affinity with the wild type of SARS-CoV-2 and the S antigen of the current main mutant strain, making it possible to develop a specific drug for new coronary pneumonia.
  • the invention provides an anti-new coronavirus monoclonal antibody and application thereof.
  • the antibody is easy for industrial production and drug preparation, and has industrial applicability.
  • tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

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Abstract

提供了一种抗SARS-CoV-2的全人源单克隆抗体，其识别的抗原表位位于S1蛋白的RBD区，该抗体对主要新冠病毒变异株具有广谱中和活性。

Description

一种全人源抗新冠病毒广谱中和抗体ZW2G10及应用 技术领域
本发明公开了一种抗体，属于微生物学和免疫学领域。
 背景技术
新冠肺炎（COVID-19）的病原体是新型冠状病毒-2（SARS-CoV-2），SARS-CoV-2属于冠状病毒科的β-冠状病毒属，是一类有囊膜的单股正链RNA病毒，其基因组长度约为30 kb。基因组的前2/3是非结构基因ORF1a/b，主要编码与病毒复制相关的酶（RNA依赖的RNA聚合酶，RdRp），后1/3依次编码四种结构蛋白：刺突蛋白（S），包膜蛋白（E），膜蛋白（M）和核衣壳蛋白（N）。其中，S蛋白含有病毒受体结合区，可与人细胞表面的血管紧张素转换酶2（ACE2）受体结合，介导病毒吸附和进入细胞，是病毒入侵宿主易感细胞的关键蛋白。
病毒在传播过程中会不断地随机产生突变，其中有些突变会增强S蛋白与ACE2受体的结合能力，加快病毒在人群中的传播，如K417N，N501Y，E484K，P681R等。因而一些携带关键位点突变的病毒变异株表现出更强的感染能力或更强的免疫逃逸能力，使现有的公共卫生干预措施或疫苗的有效性降低，如被世界卫生组织（WHO）列为“关注变异株”（variants of concern, VOC）的Alpha 株(B.1.1.7)、Beta株 (B.1.351)、Gamma株 (P.1)、Delta株 (B.1.617.2) 和Omicron株 (B.1.1.529)等，这些变异株的出现对疫情防控提出了新的严峻挑战。
 技术问题
靶向病毒表面刺突蛋白（S蛋白）的单克隆中和抗体成为潜在的有效治疗新冠肺炎的手段，它通过与新冠病毒结合，抑制病毒的活性，保护细胞免受侵害。相比小分子药物和血浆疗法，单抗药物机理清晰，对靶点的选择性高、特异性强、副作用小。根据chineseantibody网站信息，已有25项靶向S蛋白的单抗进入临床研究，这些单抗对于应对野生型新型冠状病毒都是有效的。目前国外已有四种靶向S蛋白的单抗获得了美国FDA的紧急使用授权，包括再生元的单抗REGN10933和REGN10987联用，礼来的LY-COV555和LY-COV016，Vir公司的VIR-7831和阿斯利康的AZD7442；国内，腾盛华创公司的抗新冠病毒中和抗体BRII-196和BRII-198联合疗法于2021年12月8日获中国国家药品监督管理局（NMPA）应急批准。然而，随着SARS-CoV-2的不断进化和变异，研发中的大部分单抗对一种或多种变异株失去中和活性，其中，礼来研发的LY-CoV555和LY-CoV016、再生元研发的REGN10933对Beta株病毒失去中和活性, 且抗体药物LY-CoV555-LY-CoV016鸡尾酒疗法、REGEN-COV鸡尾酒疗法、AZD7442鸡尾酒疗法对Omicron株失去中和活性。为应对病毒免疫逃逸，研发具有保守中和表位的广谱单抗，包括研发广谱的中和单抗或两种高效中和单抗的组合物，具有重大临床应用价值。
当前，中和单抗可以通过杂交瘤技术，人源化转基因小鼠，噬菌体文库筛选以及单细胞PCR技术制备。单细胞PCR技术具有全人源，天然稳定性好等优点，被广泛用于新冠中和抗体的研发。单细胞PCR技术的原理是新型冠状病毒感染恢复者或新冠疫苗接种者体内存在对抗病毒的保护性单克隆抗体，编码抗体的基因位于人体外周血单个淋巴细胞内，通过流式细胞仪分选和单细胞PCR技术可以“钓取”此基因。然后通过基因工程手段，可实现体外规模化制备此分子。
本发明拟采用流式分选-单细胞PCR技术从重组新型冠状病毒疫苗接种者的外周血中获得具有优异广谱中和活性的单抗，目的是提供针对COVID-19具有良好保护效果的全人源单克隆治疗性抗体，以应对当前流行以及将来可能出现的变异株。
 技术解决方案
基于上述发明目的，本发明通过流式分选-单细胞PCR技术筛选到了一种抗SARS-CoV-2的单克隆抗体，所述单克隆抗体的重链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:1第26-33、51-58、97-117位氨基酸序列所示；轻链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:5第26-34、52-54、91-101位氨基酸序列所示。所述单克隆抗体在本申请中被命名为“ZW2G10”。
在一个优选的实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，轻链可变区的氨基酸序列如SEQ ID NO:5所示。
在一个更为优选的实施方案中，所述抗体的重链恒定区的氨基酸序列如SEQ ID NO:3所示，所述轻链恒定区的氨基酸序列如SEQ ID NO:7所示。
第二，本发明还提供了一种编码上述单克隆抗体重链和轻链的多核苷酸，编码所述抗体的重链可变区的多核苷酸序列由SEQ ID NO:2所示，编码所述抗体的轻链可变区的多核苷酸序列由SEQ ID NO:6所示。
在一个优选的实施方案中，编码所述抗体的重链恒定区的多核苷酸序列由SEQ ID NO:4所示，编码所述抗体的轻链恒定区的多核苷酸序列由SEQ ID NO:8所示。
第三，本发明还提供了一种表达上述编码单克隆抗体重链和轻链的多核苷酸的功能元件，这种功能元件可以是传统的表达载体。
在一个优选的实施方案中，所述功能元件为线性表达框。
在另一个优选的实施方案中，所述功能元件为哺乳动物表达载体。
第四，本发明还提供了一种含有上述线性表达框的宿主细胞。
在一个优选的实施方案中，所述细胞为Expi 293F细胞。
在另一个优选的实施方案中，所述细胞为CHO-K1或CHO-S细胞，本发明可以使用CHO-K1或CHO-S细胞构建稳转工程细胞株，实现产业化生产。
最后，本发明还提供了上述单克隆抗体在制备COVID-19治疗药物中的应用。
 有益效果
本发明提供的单克隆抗体通过流式分选-单细胞PCR技术筛选获得，具有独特的CDR分区，其抗原识别表位位于S1蛋白的RBD区。所述抗体与SARS-CoV-2野生型S-ECD的亲和力为0.243 nM, 与Alpha株、Beta株、Gamma株、Delta株、Omicron株S-ECD的亲和力分别为0.166 nM、0.518 nM、0.615 nM、5.452 nM、0.765 nM。在假病毒中和实验中，对新冠病毒野生型的EC 50是14.19 ng/mL，中和Alpha株的EC 50是14.12 ng/mL，中和Beta株的EC 50是18.41 ng/mL，中和Gamma株的EC 50是15.59 ng/mL，中和Delta株的EC 50是36.18 ng/mL，中和Omicron株的EC 50是19.26 ng/mL。在真病毒中和实验中，对新冠病毒野生型的EC 50是1.077 μg/mL，中和Beta株的EC 50是1.423 μg/mL，中和Delta株的EC 50是0.71 μg/mL，显示ZW2G10对目前的主要变异株具有广谱高效中和活性。本发明公开的单克隆抗体具有高表达、全人源、稳定性好的特点，适合产业化生产，对于应对目前及将来可能出现的变异株导致的爆发流行具有重大临床应用价值。
附图说明
图1. 流式细胞仪单细胞分选图；
图2. H、κ、λ三种链基因的巢式PCR扩增后毛细管电泳的鉴定图谱；
图3. 单克隆抗体可变区序列的检索结果输出图；
图4. 抗体表达上清与SARS-CoV-2的结合活性图；
图5. 亲和层析纯化后的单抗SDS-PAGE检测图谱；
图6. ELISA检测单抗ZW2G10与S蛋白、S1蛋白、RBD蛋白、NTD蛋白、S2蛋白的结合活性随浓度变化的曲线图；
图7. ELISA检测ZW2G10的交叉结合活性；
图8. ZW2G10对新冠假病毒的广谱中和活性；
图9. ZW2G10在细胞模型上对真病毒的EC 50测定曲线图；
图10. ZW2G10与WT株 S-ECD蛋白的结合动力学曲线图；
图11. ZW2G10与Alpha株S-ECD蛋白的结合动力学曲线图；
图12. ZW2G10与Beta 株S-ECD蛋白的结合动力学曲线图；
图13. ZW2G10与Gamma株S-ECD蛋白的结合动力学曲线图；
图14. ZW2G10与Delta株S-ECD蛋白的结合动力学曲线图；
图15. ZW2G10与Omicron株S-ECD蛋白的结合动力学曲线图。
 本发明的实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的权利要求所限定的保护范围构成任何限制。
实施例1 人源抗SARS-CoV-2单克隆抗体的筛选和制备。
1. 血液样品的采集。
在获得知情同意书后，采集重组新型冠状病毒疫苗免疫接种者第二次免疫14天后血液样品20 mL，用于后续实验。
2. 流式分选记忆B细胞。
将采集的血样利用Ficoll密度梯度离心法分离PBMC，过程如下：
1）取新鲜抗凝全血，EDTA抗凝。
2）在离心管中加入与血液样本等体积的分离液，将血样平铺到分离液液面上方，保持两液面界面清晰。
3）配平，室温，水平转子800 g，加减速度3，离心30 min。
4）离心结束后，管底是红细胞，中间层是分离液，最上层是血浆/组织匀浆层，血浆层与分离液层之间是一层薄且较致密的白膜，即：单个核细胞（包括淋巴细胞和单核细胞）层。
5）将白膜层小心吸取到新的50 mL离心管中，用PBS稀释3倍，颠倒混匀。室温，水平转子600 g，离心10 min，弃上清。重复洗涤2 次。
6）用PBS将淋巴细胞重悬备用。
7）将用来分选的细胞计数，按照下表推荐用量，先加入除Anti-His tag和Anti-FLAG tag抗体以外的所有抗体以及抗原，4℃孵育1 h，随后PBS+2% FBS清洗两次，再加入Anti-His tag和Anti-FLAG tag抗体，用PBS+2% FBS补足反应体系，4℃孵育1 h。
表1. 流式分选荧光抗体/抗原
Figure 233792dest_path_image001
。
  8）使用含2% FBS的PBS重复洗涤2-3次，1 mL FPBS重悬，用40 μm细胞筛去除细胞团，4℃避光保存供分选。
9）使用细胞分选仪（Beckman MofloXDP）分选SARS-CoV-2 WT （新冠野生株Genbank编号：NC_045512.2）S-ECD特异的单个记忆B细胞。分选策略为： CD3 -/CD19 +/ IgG +/CD27 +/ SARS-CoV-2 WT S-ECD +，如图1，图1-A中圈出淋巴细胞，图1-B中圈出去除粘连的细胞，图1-C中圈出CD3 -/ CD19 +的B细胞，图1-D中圈出IgG +/CD27 +的记忆B细胞，图1-E中圈出SARS-CoV-2 WT S-ECD +的记忆B细胞。直接将单个记忆B细胞分选至96孔板中，96孔板中每孔预先加入20 μL去RNA酶水和20 U RNA酶抑制剂，-80℃保存。
结果：分选获得253个SARS-CoV-2 S-ECD +的记忆B细胞。
3. 利用单细胞-PCR技术扩增全人源单抗可变区基因。
1）反转录PCR
参考说明书（QIAGEN，210212），程序简单介绍如下：
通过流式细胞仪分选了456个单细胞。向每个反应体系中同时加入以下全部的针对重链（heavy chain，H）、Kappa轻链( kappa chain，κ)、Lamda轻链(Lamda chain，λ)各亚型的特异引物（引物序列见表2）。
引物：
H：5′ L-VH 1、5′ L-VH 3、5′ L-VH 4/6，5′L-VH 5、HuIgG-const-anti、3′ Cm CH1；
κ：5′ L Vκ 1/2、5′ L Vκ 3、5′ L Vκ 4、3′ Cκ 543–566；
λ：5′ L Vλ 1、5′ L Vλ 2、5′ L Vλ 3、5′ L Vλ 4/5、5′ L Vλ 6、5′ L Vλ 7、5′ L Vλ 8、3′ Cλ。
表2 反转录PCR引物 
Figure 432823dest_path_image003
。
PCR反应体系中包含：5×缓冲液6 μL、dNTP 1.2 μL、反转录酶（Qiagen，210212）1.2 μL、引物如上、模板为单细胞，水补齐至30 μL。
PCR反应条件为：50℃反转录30 min，95℃预变性15 min，接着95℃ 40 s，55℃ 30 s，72℃ 1 min，40个循环，最后72℃延伸10 min。
2）巢式PCR
取反转录产物1μL为模板，进行巢式PCR反应扩增H、κ、λ的可变区，扩增重链可变区、κ轻链可变区和λ轻链可变区的引物如下表3所示。
表3. 巢式PCR引物
Figure 212560dest_path_image004
。
PCR反应体系中包含：10×缓冲液5 μL、2.5 mM dNTP 4 μL、DNA聚合酶（全式金生物技术有限公司，AP141）0.5 μL、引物如上、模板为反转录产物1 μL、水补齐至50 μL。PCR反应条件为：95 ℃预变性10 min，接着，95 ℃ 30 s，57 ℃ 30 s，72 ℃ 45 s，40个循环，最后72 ℃延伸10 min。
3）毛细管电泳
对巢式PCR扩增产物用QIAGEN DNA Fast Analysis Cartridge （Qiagen，929008）进行毛细管电泳，一个单细胞中重链和轻链基因均扩增成功的克隆，被认为是配对成功的克隆。图2是对H、κ、λ三种链基因的巢式PCR扩增后毛细管电泳的鉴定图谱。
4）序列分析
经PCR鉴定阳性的克隆进行DNA序列测定和分析，登录IMGT网站（http://www.imgt.org/IMGT_vquest/analysis）进行可变区检索，为典型的抗体序列，符合预期。检索结果如图3所示，图3-A显示重链可变区的检索结果，V区同源性最高为97.57%，J区同源性最高为85.48%，D区使用读框2。图3-B显示轻链的检索结果，V区同源性最高为97.92%，J区同源性最高为89.19%。
4. 线性表达框表达抗体。
相比传统的表达载体构建方法，构建线性表达框更为快速。设计的线性表达框含有单抗在哺乳细胞内表达的所有元件，线性表达框从5’端依次含有CMV启动子序列（Genbank登记号：X03922.1）、抗体前导肽的编码序列、抗体可变区（从单细胞中扩增获得）、抗体恒定区（生工生物合成，重链恒定区序列由SEQ ID NO:3所示，DNA编码序列由SEQ ID NO:4所示，Lamda型轻链恒定区序列由SEQ ID NO:7所示，DNA编码序列由SEQ ID NO:8所示）、多聚A尾（Genbank登记号：X03896.1 ）连接起来，将该线性形式的DNA转染入细胞中进行抗体表达。
具体过程是通过体外重叠延伸PCR技术将各个PCR片段连接构建：
1）扩增启动子-前导序列
以pMD-CMVH和pMD-CMVL为模板，分别扩增重链和轻链的启动子-前导序列片段。扩增重链启动子-前导序列片段的PCR反应体系中包括：模板质粒pMD-CMVH 10 ng，10×缓冲液5 μL、2.5 mM dNTP 4 μL、DNA聚合酶 0.5 μL、引物5'-CMV-UP (与CMV启动子上游序列匹配) （5'- GATATACGCGTTGACATTGATTATTGAC -3'）、引物3'- leader-H(HR)（5'- ACACTGAACACCTTTTAAAATTAG -3', 用于重链的融合, 信号肽序列的核苷酸序列：
5'-ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTAATTTTAAAA G GTGT C-3'），编码的氨基酸序列为MNFGLSLIFLVLILKGV。扩增轻链启动子-前导序列片段的PCR反应体系中包括：模板质粒pMD-CMVL 10 ng，10×缓冲液5 μL、2.5 mM dNTP 4 μL、DNA聚合酶 0.5 μL、引物5'-CMV-UP（5'- GATATACGCGTTGACATTGATTATTGAC -3'）、引物3'- leader-L(HR) (5'- CCCACAGGTACCAGATACCCATAG -3')，用于轻链的融合，全长信号肽序列核苷酸序列为：
5'-ATGGATTCACAGGCCCAGGTTCTTATGTTACTGCTGCTATGGGTATC TGGTACCTGTGGG，氨基酸序列为MDSQAQVLMLLLLWVSGTCG，信号肽序列来源鼠源单抗可变区）、水补齐至50 μL。
PCR反应条件：95 ℃预变性10 min，接着95 ℃ 30 s，60 ℃ 30 s，72 ℃ 1 min，30个循环，最后72℃延伸10 min。
2）扩增抗体恒定区-多聚A尾片段
H链恒定区-多聚A尾片段PCR体系中包含：模板质粒pMD-TKH 10 ng、10×缓冲液5 μL、2.5 mM dNTP 4 μL、DNA聚合酶 0.5 μL、引物5'-CH （5'-ACCAAGGGCCCATCGGTCTTCCCC-3'）、引物3'-TK-POLY(A) （5'- AAGTGTAGCGGTCACGCTGCGCGTAACC -3'）、水补齐至50 μL。
κ链恒定区-多聚A尾片段PCR体系中包含：模板质粒pMD-TKκ 10 ng、10×缓冲液5 μL、2.5 mM dNTP 4 μL、DNA聚合酶 0.5 μL、引物5'-Cκ （5'- ACTGTGGCTGCACCATCTGTCTTC -3'）、引物3'-TK-POLY(A) （5'- AAGTGTAGCGGTCACGCTGCGCGTAACC -3'）、水补齐至50 μL。
λ链恒定区-多聚A尾片段PCR体系中包含：模板质粒pMD-TKλ 10 ng、10×缓冲液5 μL、2.5 mM dNTP 4 μL、DNA聚合酶 0.5 μL、引物5'-Cλ （CTACGTCAGCCCAAGGCTGCCCCC）、引物3'-TK-POLY(A) （5'- AAGTGTAGCGGTCACGCTGCGCGTAACC -3'）、水补齐至50 μL。
PCR反应条件为：95℃预变性10 min，接着95℃ 30 s，60℃ 30 s，72℃ 2 min，30个循环，最后72℃延伸10 min。
3）扩增抗体可变区
取巢式PCR产物1μL为模板，使用TransStart Taq DNA polymerase并按照产品说明书，分别使用对应的混合引物对抗体的H链、κ链、λ链进行扩增，对应引物如下表4所示。
表4.  PCR引物
Figure 138928dest_path_image005
Figure 953300dest_path_image006
※   单独划线部分用于与上游片段融合，划线黑体部分用于与下游片段的融合。
PCR反应体系中包含：10×缓冲液5 μL、2.5 mM dNTP 4 μL、DNA聚合酶（全式金生物技术有限公司，AP141）0.5 μL、引物如上、模板为巢式PCR产物1 μL、水补齐至50 μL。
PCR反应条件为：95 ℃预变性4 min，接着，95 ℃ 30 s，57 ℃ 30 s，72 ℃ 45 s，40个循环，最后72 ℃延伸10 min。
4）分别扩增重链和轻链的线性表达框
PCR反应体系中包括：
模板：纯化后的启动子-前导序列片段10 ng、重链/轻链可变区片段10 ng、重链/轻链恒定区-多聚A尾片段10 ng，10×缓冲液5 μL、12.5 mM dNTP 4 μL、DNA聚合酶（全式金生物技术有限公司，AP151-13 ）0.5 μL、引物5'-CMV-UP （5'- GATATACGCGTTGACATTGATTATTGAC -3'）和3'-TK-POLY(A) （5'- AAGTGTAGCGGTCACGCTGCGCGTAACC -3'、水补齐至50 μL。
PCR反应条件为：95℃预变性10 min，接着95℃ 30 s，60℃ 30 s，72℃ 3 min，30个循环，最后72℃延伸10 min。
5）PCR产物回收纯化和定量
PCR反应产物直接用OMEGA公司回收试剂盒回收。DNA定量：用Nano（GE Healthcare）对PCR回收产物进行定量。
6）细胞接种：将293T细胞以2×10 5/mL接种于96孔细胞培养板中，在含有5% CO 2的细胞温箱中，37℃培养过夜。
7）细胞共转染：次日，96孔板每孔加入20 μL无血清的Opti-MEM培养基中，构建成功的重链和轻链线性表达框PCR产物各0.1 μg，混匀后加入0.4 μL转染试剂Turbofect（Thermo Scientific, R0531），共同孵育15-20 min后逐滴加至过夜培养的293T细胞培养孔中。在含有5% CO 2的细胞温箱中，37℃培养48 h后收细胞培养上清备用。
5. ELISA筛选具有结合活性的抗体。
1) 包被：实验前一天96孔酶联板，取重组的新冠野生株SARS-CoV-2 S-ECD抗原及羊抗人IgG（H&L）抗体（Abcam，ab97221）用包被液稀释至浓度2 μg/mL，包被酶标板，每孔100 μL，4℃包被过夜。
2) 封闭：实验当天用洗板机（BIO-TEK，405_LS）洗3次，每孔加入100 µL封闭液，37℃ 孵育1小时。
3) 样品孵育：洗板3次，加入50 µL的转染细胞培养上清和50 µL稀释液，37℃ 孵育1小时。
4) 二抗孵育：洗板3次，将HPR标记的羊抗人IgG二抗（Abcam，ab97225）以1:10000用稀释液进行稀释，每孔100 µL加入到ELISA板对应孔中，37℃ 孵育1小时。
5) 显色：洗板3次，每孔加入100 µL的TMB单组份显色液，显色6 min，室温避光，之后每孔加入50 µL终止液终止反应。
6) 用酶标仪上检测450-630nm处的OD值，以未加待测样品的孔为阴性对照，OD 450-630＞阴性对照2.1倍以上的孔为阳性。
结果：将34株单抗进行表达，并对SARS-CoV-2 S-ECD的结合活性进行鉴定。结果显示有3株单抗与SARS-CoV-2 S-ECD能够特异性结合,如图4所示。
6.表达载体的构建和酶切鉴定。
对ZW2G10构建轻、重链重组表达质粒，进行单抗的表达制备。
1) pCDNA3.4-ZW2G10-H表达质粒构建：
以线性表达框为模板，扩增重链，切胶回收1.4kb大小的重链片段，表达载体pCDNA3.4（ThermoFisher Scientific，A14697）使用EcoR I/BamH I酶切后回收，将重链和载体片段通过同源重组（NEBuilder HiFi DNA Assembly Master Mix, E2621L）方法进行连接，转化TOP10挑取克隆进行测序鉴定，构建成功重链的表达载体pCDNA3.4-ZW2G10-H。
2) pCDNA3.4-ZW2G10-λ表达质粒构建：
以轻链表达框为模板，扩增轻链，胶回收约0.7kb的轻链片段，将轻链和载体片段通过同源重组方法连接，转化TOP10挑取克隆进行测序鉴定，构建成功轻链的表达载体pCDNA3.4-ZW2G10-λ。
3) 单抗的瞬时表达和亲和层析纯化
使用Expi293 表达系统，取15 μg重链和15 μg轻链混合后转染Expi 293F细胞，按照说明书进行操作（ThermoFisher Scientific，A14635），5-6天后收获培养液，离心后上清约30 mL，使用体积为5 mL的预装Protein A亲和层析柱，上样前使用20 mM PBS平衡，待电导显示到基线后进样，上样结束后，使用20 mM PBS洗涤色谱柱至基线平稳，使用0.1 M pH 3.0的甘氨酸缓冲液洗脱目的蛋白，待OD 280近基线后，停止收集，使用至少3个柱体积的20 mM的PBS洗涤色谱柱，至基线平稳后，用20%的乙醇洗涤色谱柱。亲和层析纯化后的单抗SDS-PAGE检测结果见图5 ：蛋白样品可被巯基乙醇还原为50 kDa、25 kDa 大小的两个片段，分别对应抗体重链和轻链的理论分子量，符合预期， Marker分子量从大到小依次为：250，130，100，70，55，55，35，25，15，10 kDa。
实施例2. 抗体ZW2G10识别表位分析。
1) 包被：实验前一天96孔酶联板，取重组的SARS-CoV-2 WT S-ECD抗原、S1抗原、RBD抗原和S2抗原用包被液稀释至浓度2 μg/mL，包被酶标板，每孔100 μL，4℃包被过夜。
2) 封闭：实验当天用洗板机（BIO-TEK，405_LS）洗3次，每孔加入100 µL封闭液，37℃ 孵育1小时。
3) 样品孵育：洗板3次，除首孔外，每孔加入100 μL稀释液，将抗体稀释至首孔1 μg/mL，4倍梯度稀释，100 μL/孔，每个抗体设置三个复孔，在37℃孵育1 h。
4) 二抗孵育：洗板3次，将HPR标记的羊抗人IgG二抗（Abcam，ab97225）以1:10000用稀释液进行稀释，每孔100 µL加入到ELISA板对应孔中，37℃ 孵育1小时。
5) 显色：洗板3次，每孔加入100 µL的TMB单组份显色液，显色6 min，室温避光，之后每孔加入50 µL终止液终止反应。
6) 用酶标仪上检测450-630nm处的OD值，以未加待测样品的孔为阴性对照，OD 450-630＞阴性对照2.1倍以上的孔为阳性。
结果：检测ZW2G10与不同抗原表位的结合活性，具体见图6， ZW2G10与SARS-CoV-2 WT（新冠野生株Genebank编号：NC_045512.2）的S-ECD、S1和RBD蛋白均特异结合，呈现剂量反应关系，而与NTD蛋白和S2蛋白均不结合。结果表明，单抗ZW2G10识别的表位位于S1蛋白的RBD区。
单抗ZW2G10的序列分析结果如下：
重链可变区的氨基酸序列如SEQ ID NO:1所示，重链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:1第26-33、51-58、97-117位氨基酸序列所示，编码重链可变区的多核苷酸序列由SEQ ID NO:2所示；轻链可变区的氨基酸序列如SEQ ID NO: 5所示，轻链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:5第26-34、52-54、91-101位氨基酸序列所示，编码轻链可变区的多核苷酸序列由SEQ ID NO:6所示。
实施例3：抗体ZW2G10的交叉结合活性鉴定。
对ZW2G10与SARS-CoV-2的受关注变异株（Variants of concern）S蛋白的交叉结合活性进行鉴定，方法同上，结果如图7所示。ZW2G10与Alpha株、Beta株、Gamma株、Delta株、Omicron株的S-ECD蛋白均特异结合，且呈现剂量反应关系。结果表明，单抗ZW2G10能够交叉结合Alpha株、Beta株、Gamma株、Delta株、Omicron株的S-ECD蛋白。
实施例4. 抗体ZW2G10的假病毒中和活性鉴定。
1) 将纯化单抗用培养基 DMEM+10% FBS 自初始浓度（ZW2G10单抗初始浓度3.7 μg/ml）, 3倍系列稀释，加入 96 孔培养板，设置3个复孔，体积50 μL/孔；随即每孔加入50 μL新冠病毒野生型（新冠野生株Genbank编号：NC_045512.2）或突变株的假病毒悬液（用DMEM+10% FBS稀释病毒至合适滴度），充分混匀，另设置存活对照（不加病毒和抗体）和死亡对照（只加病毒），置37℃ 5% CO 2细胞培养箱孵育 1 h。
2) 将HEK293T细胞用0.25%的胰酶消化后，用培养基（DMEM+10% FBS）稀释至2.5×10 5 cells/mL 浓度，接种到 96 孔细胞培养板中，接种体积 100 μL/孔，置 37℃ 5% CO 2 细胞培养箱培养过夜。
3) 48 h后弃100 μL细胞培养上清，加入100 μL显色底物，避光孵育2 min。
4) 吸取150 μL转移到96孔白色微孔板，利用Tecan Spark多功能微孔板检测仪读取Luciferase信号值；用（Luc样本孔-Luc死亡对照） /（ Luc存活对照-Luc死亡对照）计算细胞活率，用 GraphPad Prism 8 拟合曲线，计算抗体EC 50 值。
结果见图8，本发明公开的单抗ZW2G10对新冠病毒野生型假病毒的EC 50是14.19 ng/mL，中和Alpha株假病毒的EC 50是14.12 ng/mL，中和Beta株假病毒的EC 50是18.41 ng/mL，中和Gamma株假病毒的EC 50是15.59 ng/mL，中和Delta株假病毒的EC 50是36.18 ng/mL，中和Omicron株假病毒的EC 50是19.26 ng/mL。结果显示ZW2G10对目前的主要变异株的假病毒具有广谱高效中和活性。
实施例5. 抗体ZW2G10的真病毒中和活性鉴定。
1) 将Vero E6细胞用0.25%的胰酶消化后，用培养基（DMEM+10% FBS）稀释至3×10 5 cells/mL 浓度， 接种到 96 孔细胞培养板中，接种体积 100 μL/孔， 置 37℃ 5% CO 2 细胞培养箱培养过夜。
2) 实验当天，将纯化单抗用培养基 DMEM+2% FBS 自初始浓度（ZW2G10单抗初始浓度100 μg/ml, 3倍系列稀释，加入 96 孔培养板，体积 120 μL/孔；随即每孔加入 120 μL COVID-19病毒悬液（用DMEM+2% FBS稀释病毒， 加入100 TCID 50 /孔），充分混匀，置细胞培养箱孵育 1 h。
3) 弃去 96 孔板中细胞培养上清，每孔加入 200 μL 共孵育后的病毒-抗体混合悬液；另设置存活对照（不加病毒和抗体）和死亡对照（只加病毒），置 37℃ 5% CO 2 细胞培养箱培养 72 h。
4) 72 h 后弃去细胞培养上清，加入 50 μL 结晶紫染色液室温染色 30 min，弃去染液，加入200 μL/孔纯水，重复洗涤 6 次。
5) 弃尽洗液，用吸水纸拍干板孔中水分，加入100 μL脱色液充分溶解，以 OD 620 为参考，用酶标仪测 OD 570 值；用（OD样本孔-OD死亡对照） /（OD 存活对照-OD死亡对照）计算细胞活率，用 GraphPad Prism 8 拟合曲线，计算抗体EC 50 值。
结果见图9，本发明公开的单抗ZW2G10的对新冠病毒野生型的EC 50是1.077 μg/mL，中和Beta株的EC 50是1.423 μg/mL，中和Delta株的EC 50是0.710 μg/mL。说明ZW2G10对SARS-CoV-2的野生型、Beta和Delta变异株的真病毒均具有高效中和活性。
实施例6. 表面等离子共振法（SPR）测定单抗与S抗原的亲和力。
1) 配置缓冲液：量取50mL 10×HBS-EP+缓冲液、450mL去离子水，混匀后放入500mL缓冲液瓶。
2) 蛋白换液：使用脱盐柱将抗体和抗原蛋白换液至HBS-EP+缓冲液中，将脱盐柱置于空的收集管中，拧松脱盐柱盖子，1500 g离心1 min去除柱中原有液体，加入300 μL HBS-EP+缓冲液，1500 g离心1 min，重复4次，将脱盐柱置于新的收集管，将100 μL蛋白溶液加入柱中，1500 g离心2 min，收集滤下液体，使用NanoVue测定蛋白浓度。
3) 打开Biocore T200机器，将进液管A插入HBS-EP+缓冲液瓶中，放入ProteinA芯片，运行Prime程序。
4) 样品准备：用HBS-EP+缓冲液将配体（抗体）稀释至0.5 μg/mL，将分析物（抗原）稀释至100 nM、50 nM、25 nM、12.5 nM、6.25 nM、3.125 nM、1.5625 nM、0.78125 nM。
5) 使用多循环检测方法进行样品检测：打开Run/Wizard中的Kinetics/Affinity，Flow path选择2-1或4-3，Chip type选择Protein A，选择Ligand capture，点击下一步；在Setup界面中，在Startup下的Solution中填写HBS-EP+，Number of cycles选择3，点击下一步；在Injection Parameters界面：Ligand名称填写抗体名称，Contact time为60 s，Flow rate为10 μL/min，Stabilization period为0 s；Sample的Contact time为120 s，Flow rate为30 μL/min，Dissociation time为900 s；Regeneration中Solution为Glycine pH 1.5，Contact time为30 s，Flow rate为30 μL/min，Stabilization period为30 s；点击下一步；在Sample界面中填写分析物的信息：S-ECD分子量为134 kDa，浓度为0 nM、1.5625 nM、3.125 nM、6.25 nM、12.5 nM、25 nM、50 nM、100 nM、1.5625 nM，选择1.5625 nM作为重复，点击下一步；在System Preparations界面中设置分析温度和样品舱温度为25℃，点击下一步；在Rack Position界面中，选择Sample and Reagent Rack1，设置样品位置，按照样品位置和用量进行样品准备和放置，点击下一步；确认运行缓冲液体积达到实验所需，点击Start，对实验方法和结果进行保存，程序开始自动运行，运行时间5小时。
6) 结果分析：打开数据分析软件Biacore T200 Evaluation Software，打开运行的结果文件：点击Kinetics/Affinity，选择Surface bound；在Select Curves界面中选择0 nM和至少5个合适浓度，点击下一步；在Select Data界面中点击Kinetics；在Fit Kinetics界面中，Method选择1:1 Binding，点击fit进行数据拟合；记录结合动力学数据ka、kd、KD等。表5依据Report显示的分析数据，记录单抗ZW2G10与不同抗原的结合动力学数据ka、kd、KD。图10-15分别是ZW2G10与WT、Alpha、Beta、Gamma、Delta、Omicron株的S-ECD的亲和力常数测定图，其KD依次为0. 243 nM、0.166 nM、0.518 nM、0.615 nM、5.452 nM及0.765 nM。结果显示该中和抗体与SARS-CoV-2的野生型及目前主要变异株的S抗原均具有很好的亲和力，使其发展成新冠肺炎特效药成为可能。
表5 单抗ZW2G10与不同抗原的结合动力学数据
Figure 306921dest_path_image007
。
工业实用性
本发明提供了一种抗新型冠状病毒单克隆抗体及其应用，所述抗体易于工业化生产和药物制备，具有工业实用性。
 序列表自由内容
序列表
<110>  中国人民解放军军事科学院军事医学研究院
<120>  一种全人源抗新冠病毒广谱中和抗体ZW2G10及应用
<130>  P2022051JK
<150>  CN202210023619.9
<151>  2022-01-10
<160>  8
<170>  SIPOSequenceListing 1.0
<210>  1
<211>  128
<212>  PRT
<213>  人工序列(Artificial Sequence)
<220>
<223>  Heavy Chain Variable Region of ZW2G10
<400>  1
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1               5                   10                  15     
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr
            20                  25                  30         
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
        35                  40                  45             
Ser Gly Ile Thr Trp Asn Ser Gly Thr Ile Gly Tyr Ala Asp Ser Val
    50                  55                  60                 
Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu His
65                  70                  75                  80 
Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Leu Tyr Tyr Cys
                85                  90                  95     
Ala Lys Asp Asn Ser Asp Tyr Ser Gly Tyr Tyr Trp Glu Leu Glu Asn
            100                 105                 110        
Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
        115                 120                 125            
<210>  2
<211>  384
<212>  DNA
<213>  人工序列(Artificial Sequence)
<220>
<223>  Heavy Chain Variable Region of ZW2G10
<400>  2
gaagtgcagc tggtggagtc tgggggaggc ttggtacagc ctggcaggtc cctgagactc        60
tcctgtgcag cctctggatt cacctttgat gattatgcca tgcactgggt ccggcaagct       120
ccaggtaagg gcctggagtg ggtctcaggt attacttgga atagtggtac cataggctat       180
gcggactctg tgaagggccg attcatcatc tccagagaca acgccaagaa ctccctgcat       240
ctgcaaatga acagtctgag agttgaggac acggctttgt attactgtgc aaaagataac       300
tctgattata gtggttatta ctgggagtta gagaactacg gtatggacgt ctggggccaa       360
gggaccacgg tcaccgtctc ctca                                              384
<210>  3
<211>  330
<212>  PRT
<213>  人工序列(Artificial Sequence)
<220>
<223>  Heavy Chain Constant Region of ZW2G10
<400>  3
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1               5                   10                  15     
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
            20                  25                  30         
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
        35                  40                  45             
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
    50                  55                  60                 
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65                  70                  75                  80 
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
                85                  90                  95      
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
            100                 105                 110        
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
        115                 120                 125             
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
    130                 135                 140                
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145                 150                 155                 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
                165                 170                 175    
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
            180                 185                 190        
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
        195                 200                 205            
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
    210                 215                 220                
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225                 230                 235                 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
                245                 250                 255    
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
            260                 265                 270        
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
        275                 280                 285            
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
    290                 295                 300                
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305                 310                 315                 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
                325                 330
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<223>  Heavy Chain Constant Region of ZW2G10
<400>  4
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg        60
ggcacagcag ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg       120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca       180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc       240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc       300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga       360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct       420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg       480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac       540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag       600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc       660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag       720
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc       780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg       840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg       900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca       960
cagaagagcc tctccctgtc tccgggtaaa                                        990
<210>  5
<211>  111
<212>  PRT
<213>  人工序列(Artificial Sequence)
<220>
<223>  Light Chain Variable Region of ZW2G10
<400>  5
Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1               5                   10                  15     
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly
            20                  25                  30         
Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
        35                  40                  45             
Leu Ile Tyr Asp Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe
    50                  55                  60                 
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu
65                  70                  75                  80 
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp Ser Ser
                85                  90                  95     
Leu Ser Gly Val Val Phe Gly Gly Gly Thr Arg Leu Thr Val Leu
            100                 105                 110    
<210>  6
<211>  333
<212>  DNA
<213>  人工序列(Artificial Sequence)
<220>
<223>  Light Chain Variable Region of ZW2G10
 
<400>  6
cagtctgtgc tgactcagcc gccctcagtc tctggggccc cagggcagag ggtcaccatc        60
tcctgcactg ggagcagctc caacatcggg gcaggttatg atgtacactg gtaccagcag       120
cttccaggaa cagcccccaa actcctcatc tatgataaca acaatcggcc ctcaggggtc       180
cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cactgggctc       240
caggctgagg atgaggctga ttattactgc cagtcgtttg acagcagcct gagtggtgtg       300
gtattcggcg gagggactcg gctgaccgtc cta                                    333
<210>  7
<211>  106
<212>  PRT
<213>  人工序列(Artificial Sequence)
<220>
<223>  Light Chain Constant Region of ZW2G10
<400>  7
Arg Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
1               5                   10                  15     
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
            20                  25                  30         
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
        35                  40                  45             
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
    50                  55                  60                  
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
65                  70                  75                  80 
Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
                85                  90                  95     
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
            100                 105    
<210>  8
<211>  318
<212>  DNA
<213>  人工序列(Artificial Sequence)
<220>
<223>  Light Chain Constant Region of ZW2G10
<400>  8
cgtcagccca aggctgcccc ctcggtcact ctgttcccac cctcgagtga ggagcttcaa        60
gccaacaagg ccacactggt gtgtctcata agtgacttct acccgggagc cgtgacagtg       120
gcctggaagg cagatagcag ccccgtcaag gcgggagtgg agaccaccac accctccaaa       180
caaagcaaca acaagtacgc ggccagcagc tacctgagcc tgacgcctga gcagtggaag       240
tcccacaaaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga gaagacagtg       300
gcccctacag aatgttca                                                     318

Claims (10)

  1. 一种抗SARS-CoV-2的全人源单克隆抗体，其特征在于，所述单克隆抗体的重链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:1第26-33、51-58、97-117位氨基酸序列所示；轻链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:5第26-34、52-54、91-101位氨基酸序列所示。
  2. 根据权利要求1所述的抗SARS-CoV-2的全人源单克隆抗体，其特征在于，所述抗SARS-CoV-2的全人源单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，轻链可变区的氨基酸序列如SEQ ID NO: 5所示。
  3. 根据权利要求2所述的抗体，其特征在于，所述抗SARS-CoV-2的全人源单克隆抗体的重链恒定区的氨基酸序列如SEQ ID NO:3所示，轻链恒定区的氨基酸序列如SEQ ID NO:7所示。
  4. 一种编码权利要求1-3任一所述抗SARS-CoV-2的全人源单克隆抗体的重链和轻链的多核苷酸，其特征在于，编码所述抗SARS-CoV-2的全人源单克隆抗体的重链可变区的多核苷酸序列由SEQ ID NO:2所示，编码所述抗SARS-CoV-2的全人源单克隆抗体的轻链可变区的多核苷酸序列由SEQ ID NO:6所示。
  5. 根据权利要求4所述的多核苷酸，其特征在于，编码所述抗SARS-CoV-2的全人源单克隆抗体的重链恒定区的多核苷酸序列由SEQ ID NO:4所示，编码所述抗SARS-CoV-2的全人源单克隆抗体的轻链恒定区的多核苷酸序列由SEQ ID NO:8所示。
  6. 一种表达权利要求5所述编码抗SARS-CoV-2的全人源单克隆抗体重链和轻链的多核苷酸的功能元件，其特征在于，所述功能元件为线性表达框或哺乳动物表达载体。
  7. 一种含有权利要求6所述功能元件的宿主细胞。
  8. 根据权利要求7所述的宿主细胞，其特征在于，所述细胞为Expi 293F细胞。
  9. 根据权利要求7所述的宿主细胞，其特征在于，所述细胞为CHO-K1或CHO-S细胞。
  10. 权利要求1-3任一所述的抗SARS-CoV-2的全人源单克隆抗体在制备COVID-19治疗或预防药物中的应用。
PCT/CN2022/121155 2022-01-10 2022-09-24 一种全人源抗新冠病毒广谱中和抗体zw2g10及应用 WO2023130770A1 (zh)

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Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114031685B (zh) * 2022-01-10 2022-03-25 中国人民解放军军事科学院军事医学研究院 一种全人源抗新冠病毒中和抗体zw2g10及应用
CN114292327B (zh) * 2022-03-10 2022-07-12 中国科学院微生物研究所 一种广谱新型冠状病毒的人源抗体及其应用
CN114456264B (zh) * 2022-03-24 2023-02-03 中国科学院微生物研究所 一种新型冠状病毒稀有广谱表位的人源抗体及其应用
CN114573691B (zh) * 2022-03-28 2022-10-04 广州医科大学附属市八医院 一株人源中和抗体或其抗原结合片段及其应用
CN115043935B (zh) * 2022-03-31 2023-06-27 深圳市人民医院 靶向新冠病毒的纳米抗体及其制备方法和应用
GB202204813D0 (en) * 2022-04-01 2022-05-18 Bradcode Ltd Human monoclonal antibodies and methods of use thereof
CN114907484B (zh) * 2022-04-20 2022-12-09 中国科学院微生物研究所 一种抗新冠突变株包括奥密克戎四种亚型的双特异人源抗体
WO2024006472A1 (en) * 2022-06-30 2024-01-04 Vir Biotechnology, Inc. Antibodies that bind to multiple sarbecoviruses

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111333722A (zh) * 2020-03-03 2020-06-26 江苏省疾病预防控制中心(江苏省公共卫生研究院) SARS-CoV-2抑制剂及其应用
CN111592595A (zh) * 2020-04-27 2020-08-28 南京医科大学 抗新型冠状病毒SARS-Cov-2的中和性抗体及其应用
CN111690059A (zh) * 2020-06-19 2020-09-22 武汉生物制品研究所有限责任公司 一种抗SARS-CoV-2的单克隆抗体1D7
CN111718411A (zh) * 2020-06-19 2020-09-29 武汉生物制品研究所有限责任公司 一种抗SARS-CoV-2的单克隆抗体1F2
CN111732655A (zh) * 2020-07-01 2020-10-02 中国人民解放军军事科学院军事医学研究院 靶向RBD的高中和活性抗SARS-CoV-2全人源单克隆抗体及应用
CN111732654A (zh) * 2020-06-19 2020-10-02 武汉生物制品研究所有限责任公司 一种抗SARS-CoV-2的单克隆抗体1E10
CN112175073A (zh) * 2020-09-30 2021-01-05 上海市公共卫生临床中心 冠状病毒的中和抗体或其抗原结合片段
CN113735970A (zh) * 2021-09-20 2021-12-03 中国人民解放军军事科学院军事医学研究院 一种抗新冠病毒全人源广谱中和抗体及应用
WO2022003530A1 (en) * 2020-06-30 2022-01-06 Fondazione Toscana Life Sciences Neutralizing antibodies to sars coronavirus-2
CN114031685A (zh) * 2022-01-10 2022-02-11 中国人民解放军军事科学院军事医学研究院 一种全人源抗新冠病毒广谱中和抗体zw2g10及应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI296930B (en) * 2005-05-11 2008-05-21 Dev Center Biotechnology Recombinant enterovirus 71 neutralizing antibodies and use thereof
CN105753983A (zh) * 2005-12-13 2016-07-13 阿斯利康(瑞典)有限公司 胰岛素样生长因子特异性结合蛋白及其用途
CN113388029B (zh) * 2020-03-12 2022-07-05 中国科学院武汉病毒研究所 针对新型冠状病毒的中和性人源单克隆抗体及其应用
AU2021271388A1 (en) * 2020-05-11 2022-12-15 Chang Gung Memorial Hospital, Linkou Novel monoclonal antibodies against SARS-CoV-2 and uses thereof
CN113735969B (zh) * 2021-09-20 2023-05-12 中国人民解放军军事科学院军事医学研究院 一种全人源抗新冠病毒广谱高中和活性单克隆抗体及应用

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111333722A (zh) * 2020-03-03 2020-06-26 江苏省疾病预防控制中心(江苏省公共卫生研究院) SARS-CoV-2抑制剂及其应用
CN111592595A (zh) * 2020-04-27 2020-08-28 南京医科大学 抗新型冠状病毒SARS-Cov-2的中和性抗体及其应用
CN111690059A (zh) * 2020-06-19 2020-09-22 武汉生物制品研究所有限责任公司 一种抗SARS-CoV-2的单克隆抗体1D7
CN111718411A (zh) * 2020-06-19 2020-09-29 武汉生物制品研究所有限责任公司 一种抗SARS-CoV-2的单克隆抗体1F2
CN111732654A (zh) * 2020-06-19 2020-10-02 武汉生物制品研究所有限责任公司 一种抗SARS-CoV-2的单克隆抗体1E10
WO2022003530A1 (en) * 2020-06-30 2022-01-06 Fondazione Toscana Life Sciences Neutralizing antibodies to sars coronavirus-2
CN111732655A (zh) * 2020-07-01 2020-10-02 中国人民解放军军事科学院军事医学研究院 靶向RBD的高中和活性抗SARS-CoV-2全人源单克隆抗体及应用
CN112175073A (zh) * 2020-09-30 2021-01-05 上海市公共卫生临床中心 冠状病毒的中和抗体或其抗原结合片段
CN113735970A (zh) * 2021-09-20 2021-12-03 中国人民解放军军事科学院军事医学研究院 一种抗新冠病毒全人源广谱中和抗体及应用
CN114031685A (zh) * 2022-01-10 2022-02-11 中国人民解放军军事科学院军事医学研究院 一种全人源抗新冠病毒广谱中和抗体zw2g10及应用

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