WO2023040627A1 - Fully human broad-spectrum high-neutralizing-activity monoclonal antibody against sars-cov-2 and use thereof - Google Patents

Fully human broad-spectrum high-neutralizing-activity monoclonal antibody against sars-cov-2 and use thereof Download PDF

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WO2023040627A1
WO2023040627A1 PCT/CN2022/114962 CN2022114962W WO2023040627A1 WO 2023040627 A1 WO2023040627 A1 WO 2023040627A1 CN 2022114962 W CN2022114962 W CN 2022114962W WO 2023040627 A1 WO2023040627 A1 WO 2023040627A1
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monoclonal antibody
seq
ser
antibody
heavy chain
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French (fr)
Chinese (zh)
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陈薇
于长明
迟象阳
孙韩聪
张冠英
张军
李建民
范鹏飞
王步森
宰晓东
张哲�
房婷
付玲
陈郑珊
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 WO2023040627A1 publication Critical patent/WO2023040627A1/en

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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
    • C07K2317/52—Constant or Fc region; Isotype
    • 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
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention discloses an antibody and belongs to the fields of microbiology and immunology.
  • SARS-CoV-2 The pathogen of new coronary pneumonia is novel coronavirus-2 (SARS-CoV-2).
  • SARS-CoV-2 belongs to the ⁇ -coronavirus genus of Coronaviridae. Approximately 30 kb in length.
  • 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).
  • S RNA-dependent RNA polymerase
  • S Envelope protein
  • M membrane protein
  • N nucleocapsid protein
  • 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 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).
  • WHO World Health Organization
  • Alpha strain B.1.1.7
  • Beta strain B.1.351
  • Gamma strain P.1
  • Delta strain B.1.617.2
  • VOC variable of concern
  • 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.
  • 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.
  • Humanized monoclonal therapeutic antibodies to address currently circulating variants 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 sequences of amino acid sequences are shown in SEQ ID NO: 1 respectively at positions 26-33, 51-58, and 97-112; the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively shown in SEQ ID NO: 5
  • the 27-32, 50-52, 89-98 amino acid sequences are shown.
  • the monoclonal antibody is named "ZWD12" in this application.
  • amino acid sequence of the heavy chain variable region of the monoclonal 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 monoclonal 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 monoclonal antibody is shown in SEQ ID NO: 4, and the polynucleotide sequence encoding the light chain constant region of the monoclonal antibody is represented by Shown in 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 a stable engineered cell line 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 0.896 nM, and the affinity to S-ECD of Alpha strain, Beta strain, Gamma strain and Delta strain is 0.826 nM, 0.991 nM, 1.423 nM, 1.912 nM, respectively .
  • the EC 50 for the wild type of the new coronavirus was 5.860 ng/mL
  • the EC 50 for neutralizing the Alpha strain was 4.107 ng/mL
  • the EC 50 for neutralizing the Beta strain was 3.564 ng/mL.
  • the EC 50 of the Gamma strain was 3.829 ng/mL
  • the EC 50 of the neutralizing Delta strain was 7.287 ng/mL.
  • the EC 50 for the wild type of the new coronavirus is 0.103 ⁇ g/mL
  • the EC 50 for the neutralization of the Beta strain is 0.069 ⁇ g/mL
  • the EC 50 for the neutralization of the Delta strain is 0.079 ⁇ g/mL, showing that ZWD12 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 dealing with outbreaks caused by current mutant strains and possible future mutant strains.
  • FIG. 1 The binding activity diagram of antibody expression supernatant to SARS-CoV-2;
  • 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).
  • Sorting SARS-CoV-2 S-ECD-specific single memory B cells using a cell sorter (Beckman MofloXDP).
  • the sorting strategy is: CD3 - /CD19 + / IgG + /CD27 + / SARS-CoV-2 S-ECD + , as shown in Figure 1, lymphocytes are circled in Figure 1-A, and adhesions are removed by circles in Figure 1-B cells, CD3 - / CD19 + B cells are circled in Figure 1-C, IgG + /CD27 + memory B cells are circled in Figure 1-D, and SARS-CoV-2 S- cells are circled in Figure 1-E 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 an identification map of capillary electrophoresis after nested PCR amplification of H, ⁇ , ⁇ chain genes.
  • FIG. 4 shows the search results of the heavy chain variable region.
  • the V region has the highest homology of 94.79%
  • the J region has the highest homology of 87.50%
  • the D region uses reading frame 2.
  • Figure 4-B shows the search results of the light chain, the highest homology of the V region is 93.19%, and the highest homology of the J region is 92.11%.
  • 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, 72°C for 2 min, and finally extension at 72°C for 10 min.
  • the underlined part is used for fusion with upstream fragments, and the bold 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).
  • Lanes 1 and 2 in Figure 3 are the promoter-leader sequences of the heavy chain and light chain respectively, and the amplified fragment is about 750 bp, which is in line with expectations; Lane 3, 4 and 5 are the constant region-poly A tail fragments of H chain, ⁇ chain and ⁇ chain respectively, and the amplified fragments are about 2000 bp, 1350 bp and 1350 bp respectively, which are in line with expectations; lanes 6 and 7 are successfully constructed respectively The heavy chain and light chain expression cassettes of ZWD12, the amplified fragments are 3100 About bp and 2450 bp, in line with expectations; lanes 8 and 9 are the heavy chain and light chain expression cassettes of the successfully constructed ZWC6 respectively, and the amplified fragments are 3100 bp and 2450 bp respectively About bp, as expected; Lane M is Trans5K DNA Marker, molecular weight from large
  • Coating Take the recombinant SARS-CoV-2 S-ECD antigen and goat anti-human IgG (H&L) antibody (Abcam, ab97221) from a 96-well enzyme-linked plate the day before the experiment and dilute it to a concentration of 2 ⁇ g/ mL, to coat the microtiter plate, 100 ⁇ L per well, and coat overnight at 4°C.
  • H&L goat anti-human IgG
  • 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 ZWD12, 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-ZWD12-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, and the clones were selected for sequencing and identification after transformation into TOP10, and the successful light chain was constructed Expression vector pCDNA3.4-ZWD12- ⁇ .
  • 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.
  • FIG. 6 The SDS-PAGE detection results of the monoclonal antibody purified by affinity chromatography are shown in Figure 6:
  • swimming lane 1 is the reduction electrophoresis result of the monoclonal antibody ZWC6, and swimming lane 2 is the reduction electrophoresis result of the monoclonal antibody ZWD12.
  • the theoretical molecular weights of the heavy chain and the light chain are respectively 50 kDa and 25 kDa, as expected, lane M is the molecular weight marker (molecular weight from large to small: 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.
  • 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 112th amino acid sequence, the polynucleotide sequence encoding the variable region of the heavy chain is shown in SEQ ID NO: 2; the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 5, and the variable region of the light chain is shown in The amino acid sequences of the CDR1, CDR2 and CDR3 regions are respectively shown in the amino acid sequences at positions 27-32, 50-52, and 89-98 of SEQ ID NO: 5, and the polynucleotide sequence encoding the light chain variable region is represented by SEQ ID NO: 6.
  • ZWD12 specifically binds to the S-ECD protein of Alpha strain, Beta strain, Gamma strain, and Delta strain, and exhibits a dose-response relationship.
  • monoclonal antibody ZWD12 could cross-link the S-ECD protein of Alpha strain, Beta strain, Gamma strain and Delta strain.
  • the purified monoclonal antibody was serially diluted 3 times from the initial concentration (the initial concentration of ZWD12 monoclonal antibody was 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/ Then add 50 ⁇ L pseudovirus suspension of the wild type or mutant strain of the new coronavirus to each well (dilute the virus to an appropriate titer with DMEM+10% FBS), mix well, and set up a survival control (without virus and antibody) And the dead control (only virus added), incubated at 37°C in a 5% CO 2 cell incubator for 1 h.
  • the results are shown in Figure 9.
  • the EC 50 of the monoclonal antibody ZWD12 disclosed in the present invention against the wild-type pseudovirus of the new coronavirus is 5.860 ng/mL
  • the EC 50 of neutralizing the Alpha strain pseudovirus is 4.107 ng/mL
  • neutralizing the Beta strain pseudovirus The EC 50 for neutralizing the pseudovirus of the Gamma strain was 3.829 ng/mL
  • the EC 50 for neutralizing the pseudovirus of the Delta strain was 7.287 ng/mL.
  • the results show that ZWD12 has a broad-spectrum and high-efficiency neutralizing activity against the pseudoviruses of the current main mutant strains.
  • 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 ZWD12 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 10.
  • the EC 50 of the monoclonal antibody ZWD12 disclosed in the present invention against the wild type of the new coronavirus is 0.103 ⁇ g/mL
  • the EC 50 of neutralizing the Beta strain is 0.069 ⁇ g/mL
  • the EC 50 of neutralizing the Delta strain is 0.079 ⁇ g /mL. It shows that ZWD12 has high neutralizing activity against the true viruses of the wild type, Beta and Delta variants of SARS-CoV-2.
  • 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 11-16 are the determination diagrams of the affinity constants between ZWD12 and the S-ECD and WT RBD of WT, Alpha, Beta, Gamma, and Delta strains, respectively, and the KDs are 0.896 nM, 0.826 nM, 0.991 nM, 1.423 nM, 1.912 nM and 0.356 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

A fully human monoclonal antibody ZWD12 against SARS-CoV-2. The antibody is obtained by screening using a flow sorting-single cell PCR technology and has a unique CDR, and the antigen recognition epitope of the antibody is located in an RBD region of S1 protein. The EC50 of the antibody against the wild type of SARS-CoV-2 is 0.103 μg/mL, and the EC50 neutralizing the Beta strain is 0.069 μg/mL, and the EC50 neutralizing the Delta strain is 0.079 μg/mL, showing that ZWD12 has broad-spectrum neutralizing activity against the current major mutant strains.

Description

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A fully human anti-new coronavirus broad-spectrum high neutralizing activity monoclonal antibody and its application 技术领域technical field
本发明公开了一种抗体，属于微生物学和免疫学领域。The invention discloses an antibody and belongs to the fields of microbiology and immunology.
背景技术Background technique
新冠肺炎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。The pathogen of new coronary pneumonia is novel coronavirus-2 (SARS-CoV-2). SARS-CoV-2 belongs to the β-coronavirus genus of Coronaviridae. Approximately 30 kb in length. 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). Among them, 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.
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)和Delta株 (B.1.617.2)等，这些变异株的出现对疫情防控提出了新的严峻挑战。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 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) and Delta strain (B.1.617.2), etc., which are "variants of concern (VOC)" , The emergence of these mutant strains has posed new severe challenges to the prevention and control of the epidemic.
技术问题technical problem
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。目前已有三种靶向S蛋白的单抗获得了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。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。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。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. At present, three monoclonal antibodies targeting the S protein have obtained emergency use authorization from the FDA, including Regeneron’s monoclonal antibodies REGN10933 and REGN10987 in combination, Eli Lilly’s LY-COV555 and LY-COV016, and Vir’s VIR-7831. However, with the continuous evolution and mutation of SARS-CoV-2, most of the monoclonal antibodies under development have lost their neutralizing activity against one or more mutant strains. Among them, LY-CoV555 and LY-CoV016 developed by Lilly, regeneration The REGN10933 developed by Yuan lost its neutralizing activity against the Beta strain virus. In response to virus immune escape, 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.
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。Currently, neutralizing monoclonal antibodies can be prepared by hybridoma technology, humanized transgenic mice, phage library screening and single-cell PCR technology. 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.
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，以应对当前流行的变异株以及将来可能出现的变异株。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. Humanized monoclonal therapeutic antibodies to address currently circulating variants and possible future variants.
技术解决方案technical solution
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、97-112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27-32、50-52、89-98位氨基酸序列所示。所述单克隆抗体在本申请中被命名为“ZWD12”。Based on the purpose of the above invention, 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 sequences of amino acid sequences are shown in SEQ ID NO: 1 respectively at positions 26-33, 51-58, and 97-112; the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively shown in SEQ ID NO: 5 The 27-32, 50-52, 89-98 amino acid sequences are shown. The monoclonal antibody is named "ZWD12" in this application.
在一个优选的实施方案中，所述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。In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5.
在一个更为优选的实施方案中，所述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。In a more preferred embodiment, the amino acid sequence of the heavy chain constant region of the monoclonal antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:7.
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。Second, 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.
在一个优选的实施方案中，编码所述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。In a preferred embodiment, the polynucleotide sequence encoding the heavy chain constant region of the monoclonal antibody is shown in SEQ ID NO: 4, and the polynucleotide sequence encoding the light chain constant region of the monoclonal antibody is represented by Shown in SEQ ID NO:8.
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。Thirdly, 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.
在一个优选的实施方案中，所述功能元件为线性表达框。In a preferred embodiment, the functional element is a linear expression cassette.
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。In another preferred embodiment, the functional element is a mammalian expression vector.
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。Fourth, the present invention also provides a host cell containing the above-mentioned linear expression cassette.
在一个优选的实施方案中，所述细胞为Expi 293F细胞。In a preferred embodiment, the cells are Expi 293F cells.
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。In another preferred embodiment, the cells are CHO-K1 or CHO-S cells. In the present invention, CHO-K1 or CHO-S cells can be used to construct a stable engineered cell line to realize industrial production.
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。Finally, the present invention also provides the application of the above-mentioned monoclonal antibody in the preparation of a drug for treating COVID-19.
有益效果Beneficial effect
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0.896 nM, 与Alpha株、Beta株、Gamma株、Delta株S-ECD的亲和力分别为0.826 nM、0.991 nM、1.423 nM、1.912 nM。在假病毒中和实验中，对新冠病毒野生型的EC 50是5.860 ng/mL，中和Alpha株的EC 50是4.107 ng/mL，中和Beta株的EC 50是3.564 ng/mL，中和Gamma株的EC 50是3.829 ng/mL，中和Delta株的EC 50是7.287 ng/mL。在真病毒中和实验中，对新冠病毒野生型的EC 50是0.103 μg/mL，中和Beta株的EC 50是0.069 μg/mL，中和Delta株的EC 50是0.079 μg/mL，显示ZWD12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，对于应对目前的变异株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。 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 0.896 nM, and the affinity to S-ECD of Alpha strain, Beta strain, Gamma strain and Delta strain is 0.826 nM, 0.991 nM, 1.423 nM, 1.912 nM, respectively . In the pseudovirus neutralization experiment, the EC 50 for the wild type of the new coronavirus was 5.860 ng/mL, the EC 50 for neutralizing the Alpha strain was 4.107 ng/mL, and the EC 50 for neutralizing the Beta strain was 3.564 ng/mL. The EC 50 of the Gamma strain was 3.829 ng/mL, and the EC 50 of the neutralizing Delta strain was 7.287 ng/mL. In the true virus neutralization experiment, the EC 50 for the wild type of the new coronavirus is 0.103 μg/mL, the EC 50 for the neutralization of the Beta strain is 0.069 μg/mL, and the EC 50 for the neutralization of the Delta strain is 0.079 μg/mL, showing that ZWD12 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 dealing with outbreaks caused by current mutant strains and possible future mutant strains.
附图说明Description of drawings
图1. 流式细胞仪单细胞分选图；Figure 1. Sorting diagram of single cells by flow cytometry;
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；Figure 2. Capillary electrophoresis identification map of H, κ, λ chain genes after nested PCR amplification;
图3. 单克隆抗体重链和轻链线性表达框的核酸电泳鉴定图谱； Figure 3. Nucleic acid electrophoresis identification pattern of monoclonal antibody heavy chain and light chain linear expression cassette;
图4. 单克隆抗体可变区序列的检索结果输出图；Figure 4. The output image of the retrieval results of the variable region sequences of monoclonal antibodies;
图5. 抗体表达上清与SARS-CoV-2的结合活性图；Figure 5. The binding activity diagram of antibody expression supernatant to SARS-CoV-2;
图6. 亲和层析纯化后的单抗SDS-PAGE检测图谱；Figure 6. SDS-PAGE detection pattern of monoclonal antibody purified by affinity chromatography;
图7. ELISA检测单抗ZWD12与S蛋白、S1蛋白、RBD蛋白的结合活性随浓度变化的曲线图； Figure 7. ELISA detection curve of the binding activity of monoclonal antibody ZWD12 to S protein, S1 protein, and RBD protein as a function of concentration;
图8. ELISA检测ZWD12的交叉结合活性；Figure 8. ELISA detection of cross-binding activity of ZWD12;
图9. ZWD12对新冠假病毒的广谱中和活性；Figure 9. The broad-spectrum neutralizing activity of ZWD12 against the new coronavirus;
图10. ZWD12在细胞模型上对真病毒的EC 50测定曲线图； Figure 10. ZWD12 on the cell model to the EC50 assay curve of true virus;
图11. ZWD12与WT株 S-ECD蛋白的结合动力学曲线图；Figure 11. The binding kinetics curve of ZWD12 and WT strain S-ECD protein;
图12. ZWD12与Alpha株S-ECD蛋白的结合动力学曲线图；Figure 12. The binding kinetics curve between ZWD12 and Alpha strain S-ECD protein;
图13. ZWD12与Beta 株S-ECD蛋白及的结合动力学曲线图；Figure 13. The binding kinetics curve between ZWD12 and Beta strain S-ECD protein;
图14. ZWD12与Gamma株S-ECD蛋白的结合动力学曲线图；Figure 14. The binding kinetics curve of ZWD12 and Gamma strain S-ECD protein;
图15. ZWD12与Delta株S-ECD蛋白的结合动力学曲线图；Figure 15. The binding kinetics curve of ZWD12 and Delta strain S-ECD protein;
图16. ZWD12与WT株RBD蛋白的结合动力学曲线图。Figure 16. The binding kinetics curve between ZWD12 and WT strain RBD protein.
本发明的实施方式Embodiments of the present invention
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。The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are exemplary only, and do not constitute any limitation to the protection scope defined by the claims of the present invention.
实施例1 人源抗SARS-CoV-2单克隆抗体的筛选和制备Example 1 Screening and Preparation of Human Anti-SARS-CoV-2 Monoclonal Antibody
1. 血液样品的采集1. Collection of Blood Samples
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。After obtaining informed consent, 20 mL of blood samples were collected from the recipients immunized with the recombinant novel coronavirus vaccine 14 days after the second immunization for subsequent experiments.
2. 流式分选记忆B细胞2. Flow cytometric sorting of memory B cells
将采集的血样利用Ficoll密度梯度离心法分离PBMC，过程如下：The collected blood samples were separated from PBMC by Ficoll density gradient centrifugation, and the process was as follows:
1）取新鲜抗凝全血，EDTA抗凝。1) Take fresh anticoagulated whole blood and anticoagulate with EDTA.
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。2) Add the same volume of separation liquid as the blood sample into the centrifuge tube, spread the blood sample above the liquid surface of the separation liquid, and keep the interface between the two liquid surfaces clear.
3）配平，室温，水平转子800 g，加减速度3，离心30 min。3) Trim, room temperature, horizontal rotor 800 g, acceleration and deceleration speed 3, centrifuge for 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。4) After centrifugation, 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, and 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).
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。5) Carefully pipette the buffy coat into a new 50 mL centrifuge tube, dilute it 3 times with PBS, and invert to mix. Centrifuge at room temperature with a horizontal rotor at 600 g for 10 min, and discard the supernatant. Repeat the wash 2 times.
6）用PBS将淋巴细胞重悬备用。6) Resuspend the lymphocytes in PBS for later use.
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。7) Count the cells used for sorting, according to the recommended dosage in the table below, first add all antibodies and antigens except Anti-His tag and Anti-FLAG tag antibodies, incubate at 4°C for 1 h, then wash with PBS+2% FBS Twice, add Anti-His tag and Anti-FLAG For tag antibody, supplement the reaction system with PBS+2% FBS, and incubate at 4°C for 1 h.
表1. 流式分选荧光抗体/抗原Table 1. Fluorescent Antibodies/Antigens for Flow Cytometry
Figure 961804dest_path_image001
Figure 961804dest_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。8) Wash repeatedly 2-3 times with PBS containing 2% FBS, resuspend in 1 mL FPBS, remove cell clusters with a 40 μm cell sieve, and store in the dark at 4°C for sorting.
9）使用细胞分选仪（Beckman MofloXDP）分选SARS-CoV-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 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 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。 9) Sorting SARS-CoV-2 S-ECD-specific single memory B cells using a cell sorter (Beckman MofloXDP). The sorting strategy is: CD3 - /CD19 + / IgG + /CD27 + / SARS-CoV-2 S-ECD + , as shown in Figure 1, lymphocytes are circled in Figure 1-A, and adhesions are removed by circles in Figure 1-B cells, CD3 - / CD19 + B cells are circled in Figure 1-C, IgG + /CD27 + memory B cells are circled in Figure 1-D, and SARS-CoV-2 S- cells are circled in Figure 1-E ECD + memory B cells. A single memory B cell was directly sorted into a 96-well plate, and 20 μL 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.
结果：分选获得456个SARS-CoV-2 S-ECD +的记忆B细胞。 Results: 456 SARS-CoV-2 S-ECD + memory B cells were obtained by sorting.
3. 利用单细胞-PCR技术扩增全人源单抗可变区基因3. Using single cell-PCR technology to amplify the variable region gene of fully human monoclonal antibody
1）反转录PCR1) Reverse transcription PCR
参考说明书（QIAGEN，210212），程序简单介绍如下：Refer to the manual (QIAGEN, 210212), the procedure is briefly introduced as follows:
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：456 single cells were sorted by flow cytometry. Add all of the following heavy chain (heavy chain, H), Kappa light chain (kappa chain, κ), and Lamda light chain (Lamda chain, λ) subtype-specific primers (see Table 2 for primer sequences). Primers:
H：5′ L-VH 1、5′ L-VH 3、5′ L-VH 4/6，5′L-VH 5、HuIgG-const-anti、3′ Cm CH1H: 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 543–566κ: 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′ Cλλ: 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Table 2 Reverse transcription PCR primers
Figure 941261dest_path_image002
Figure 941261dest_path_image002
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。The PCR reaction system contains: 6 μL of 5× buffer, 1.2 μL of dNTP, 1.2 μL of reverse transcriptase (Qiagen, 210212), primers as above, single-cell template, and make up to 30 μL with water.
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。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.
2）巢式PCR2) Nested 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。Take 1 μL of the reverse transcription product as a template, and perform nested PCR reaction to amplify the variable regions of H, κ, and λ, and the primers for amplifying the variable regions of the heavy chain, κ light chain, and λ light chain are as follows Table 3 shows.
表3. 巢式PCR引物Table 3. Nested PCR Primers
Figure 720998dest_path_image003
Figure 720998dest_path_image003
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。The PCR reaction system contains: 5 μL of 10× buffer, 4 μL of 2.5 mM dNTP, 0.5 μL of DNA polymerase (Quanshijin Biotechnology Co., Ltd., AP141), primers as above, 1 μL of reverse transcription product as template, water supplement Make up to 50 μL. 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.
3）毛细管电泳3) Capillary electrophoresis
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。QIAGEN DNA Fast for nested PCR amplification products Analysis Cartridge (Qiagen, 929008) was used for capillary electrophoresis, and a clone that successfully amplified both the heavy chain and light chain genes in a single cell was considered a successful pairing clone. Fig. 2 is an identification map of capillary electrophoresis after nested PCR amplification of H, κ, λ chain genes.
4）序列分析4) Sequence Analysis
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4所示，图4-A显示重链可变区的检索结果，V区同源性最高为94.79%，J区同源性最高为87.50%，D区使用读框2。图4-B显示轻链的检索结果，V区同源性最高为93.19%，J区同源性最高为92.11%。The positive clones identified by PCR were subjected to DNA sequence determination and analysis, and the variable region search was performed on the IMGT website (http://www.imgt.org/IMGT_vquest/analysis). It was a typical antibody sequence, which was in line with expectations. The search results are shown in Figure 4. Figure 4-A shows the search results of the heavy chain variable region. The V region has the highest homology of 94.79%, the J region has the highest homology of 87.50%, and the D region uses reading frame 2. Figure 4-B shows the search results of the light chain, the highest homology of the V region is 93.19%, and the highest homology of the J region is 92.11%.
4. 线性表达框表达抗体4. Expression of Antibodies in a Linear Expression Cassette
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，Kappa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，Lamda型轻链恒定区序列由SEQ ID NO:9所示，DNA编码序列由SEQ ID NO:10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。Compared with traditional expression vector construction methods, the construction of linear expression cassettes is faster. 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. Region (obtained from single cell amplification), antibody constant region (Shenggong Biosynthesis, heavy chain constant region sequence by SEQ Shown in ID NO:3, the DNA coding sequence is represented by SEQ Shown in ID NO:4, the Kappa type light chain constant region sequence is represented by SEQ Shown in ID NO:7, the DNA coding sequence is represented by SEQ Shown in ID NO:8, the constant region sequence of the Lambda type light chain is represented by SEQ Shown in ID NO:9, the DNA coding sequence is represented by SEQ As shown in ID NO:10, the poly A tail (Genbank accession number: X03896.1) was ligated, and the DNA in linear form was transfected into cells for antibody expression.
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：The specific process is to connect and construct each PCR fragment through in vitro overlap extension PCR technology:
1）扩增启动子-前导序列1) Amplify the promoter-leader sequence
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：Using pMD-CMVH and pMD-CMVL as templates, the promoter-leader sequence fragments of the heavy chain and light chain were amplified, respectively. The PCR reaction system for amplifying the heavy chain promoter-leader sequence fragment includes: template plasmid pMD-CMVH 10 ng, 10× buffer 5 μL, 2.5 mM dNTP 4 μL, DNA polymerase 0.5 μL, 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:
5'-ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTAATTTTAAAAGGTGTC-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'-ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTAATTTTAAAAGGTGTC-3'), the encoded amino acid sequence is MNFGLSLIFLVLILKGV. The PCR reaction system for amplifying the light chain promoter-leader sequence fragment includes: template plasmid pMD-CMVL 10 ng, 10× buffer 5 μL, 2.5 mM dNTP 4 μL, DNA polymerase 0.5 μL, 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:
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。5'-ATGGATTCACAGGCCCAGGTTCTTATGTTACTGCTGCTATGGGTATC TGGTACCTGTGGG, 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 μL with water.
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。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.
2）扩增抗体恒定区-多聚A尾片段2) Amplify the antibody constant region-poly A tail fragment
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。The H chain constant region-poly A tail fragment PCR system contains: template plasmid pMD-TKH 10 ng, 10× buffer 5 μL, 2.5 mM dNTP 4 μL, DNA polymerase 0.5 μL, primer 5'-CH (5' -ACCAAGGGCCCATCGGTCTTCCCC-3'), primer 3'-TK-POLY(A) (5'- AAGTGTAGCGGTCACGCTGCGCGTAACC -3'), water to make up to 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。The κ chain constant region-poly A tail fragment PCR system contains: template plasmid pMD-TKκ 10 ng, 10× buffer 5 μL, 2.5 mM dNTP 4 μL, DNA polymerase 0.5 μL, primer 5'-Cκ (5' - ACTGTGGCTGCACCATCTGTCTTC -3'), primer 3'-TK-POLY(A) (5'- AAGTGTAGCGGTCACGCTGCGCGTAACC -3'), water to make up to 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。The λ chain constant region-poly A tail fragment PCR system contains: template plasmid pMD-TKλ 10 ng, 10× buffer 5 μL, 2.5 mM dNTP 4 μL, DNA polymerase 0.5 μL, primer 5'-Cλ (CTACGTCAGCCCAAGGCTGCCCCC) , Primer 3'-TK-POLY(A) (5'- AAGTGTAGCGGTCACGCTGCGCGTAACC -3'), water to make up to 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。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 2 min, and finally extension at 72°C for 10 min.
3）扩增抗体可变区3) Amplify the antibody variable region
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。Take 1 μL of the nested PCR product as a template and use TransStart Taq DNA polymerase and according to the product instructions, respectively use the corresponding mixed primers to amplify the H chain, κ chain, and λ chain of the antibody. The corresponding primers are shown in Table 4 below.
表4.  PCR引物Table 4. PCR Primers
Figure 178524dest_path_image004
Figure 178524dest_path_image004
Figure 992896dest_path_image005
Figure 992896dest_path_image005
※  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。※ The underlined part is used for fusion with upstream fragments, and the bold bold part is used for fusion with downstream fragments.
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。The PCR reaction system contains: 5 μL of 10× buffer, 4 μL of 2.5 mM dNTP, 0.5 μL of DNA polymerase (Quanshijin Biotechnology Co., Ltd., AP141), primers as above, 1 μL of nested PCR product as template, water supplement Make up to 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。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.
4）分别扩增重链和轻链的线性表达框4) Amplify the linear expression cassettes of heavy chain and light chain respectively
PCR反应体系中包括：The PCR reaction system includes:
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。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 μL, 12.5 mM dNTP 4 μL, DNA polymerase (Quanshijin Biotechnology Co., Ltd., AP151-13 ) 0.5 μL, primer 5'-CMV-UP (5'- GATATACGCGTTGACATTGATTATTGAC -3') and 3'-TK-POLY(A) (5'- AAGTGTAGCGGTCACGCTGCGCGTAACC -3', make up to 50 μL with water.
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。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.
5）PCR产物回收纯化和定量5) PCR product recovery, purification and quantification
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。The PCR reaction product was directly recovered with the recovery kit of OMEGA Company. DNA quantification: PCR recovery products were quantified using Nano (GE Healthcare).
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。 6) Cell seeding: 293T cells were seeded in 96-well cell culture plates at 2×10 5 /mL, and cultured overnight at 37°C in a cell incubator containing 5% CO 2 .
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。 7) Cell co-transfection: The next day, add 20 μL of serum-free Opti-MEM medium to each well of the 96-well plate, and add 0.1 μg of the PCR products of the successfully constructed heavy chain and light chain linear expression cassettes, mix well and add 0.4 μL of transfection reagent Turbofect (Thermo Scientific, R0531) was incubated together for 15-20 min and then added dropwise to the overnight cultured 293T cell culture wells. After culturing at 37°C for 48 h in a cell incubator containing 5% CO 2 , the cell culture supernatant was collected for use.
线性表达框的PCR融合扩增核酸电泳检测结果见图3，图3中泳道1，2分别是重链、轻链的启动子-前导序列，扩增片段750 bp左右，符合预期；泳道3，4，5分别是H链、κ链、λ链的恒定区-多聚A尾片段，扩增片段分别为2000 bp、1350 bp、1350 bp左右，符合预期；泳道6，7分别是构建成功的ZWD12的重链、轻链表达框，扩增片段分别为3100 bp、2450 bp左右，符合预期；泳道8，9分别是构建成功的ZWC6的重链、轻链表达框，扩增片段分别为3100 bp、2450 bp左右，符合预期；泳道M是Trans5K DNA Marker，分子量从大到小分别为5000、3000、2000、1500、1000、750、500、250、100 bp。The detection results of PCR fusion amplified nucleic acid electrophoresis of the linear expression frame are shown in Figure 3. Lanes 1 and 2 in Figure 3 are the promoter-leader sequences of the heavy chain and light chain respectively, and the amplified fragment is about 750 bp, which is in line with expectations; Lane 3, 4 and 5 are the constant region-poly A tail fragments of H chain, κ chain and λ chain respectively, and the amplified fragments are about 2000 bp, 1350 bp and 1350 bp respectively, which are in line with expectations; lanes 6 and 7 are successfully constructed respectively The heavy chain and light chain expression cassettes of ZWD12, the amplified fragments are 3100 About bp and 2450 bp, in line with expectations; lanes 8 and 9 are the heavy chain and light chain expression cassettes of the successfully constructed ZWC6 respectively, and the amplified fragments are 3100 bp and 2450 bp respectively About bp, as expected; Lane M is Trans5K DNA Marker, molecular weight from large to small are 5000, 3000, 2000, 1500, 1000, 750, 500, 250, 100 bp.
5. ELISA筛选具有结合活性的抗体5. ELISA screening of antibodies with binding activity
1) 包被：实验前一天96孔酶联板，取重组的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。1) Coating: Take the recombinant SARS-CoV-2 S-ECD antigen and goat anti-human IgG (H&L) antibody (Abcam, ab97221) from a 96-well enzyme-linked plate the day before the experiment and dilute it to a concentration of 2 μg/ mL, to coat the microtiter plate, 100 μL per well, and coat overnight at 4°C.
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。2) Blocking: Wash 3 times with a plate washer (BIO-TEK, 405_LS) on the day of the experiment, add 100 µL of blocking solution to each well, and incubate at 37°C for 1 hour.
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。3) Sample incubation: wash the plate 3 times, add 50 µL transfected cell culture supernatant and 50 µL diluent, and incubate at 37°C for 1 hour.
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。4) Secondary antibody incubation: wash the plate 3 times, dilute the HPR-labeled goat anti-human IgG secondary antibody (Abcam, ab97225) at 1:10000 with diluent, add 100 µL per well to the corresponding well of the ELISA plate, and keep at 37°C Incubate for 1 hour.
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。5) Color development: Wash the plate 3 times, add 100 µL of TMB single-component chromogenic solution to each well, develop color for 6 min, and keep in the dark at room temperature, then add 50 µL of stop solution to each well to terminate the reaction.
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。。 6) Use a microplate reader to detect the OD value at 450-630nm, and use the wells without the sample to be tested as negative controls, and the wells with OD 450-630 > 2.1 times the negative control as positive. .
结果：将42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21株单抗与SARS-CoV-2 S-ECD能够特异性结合,如图5所示。Results: 42 monoclonal antibodies were expressed and their binding activity to SARS-CoV-2 S-ECD was identified. The results showed that 21 monoclonal antibodies could specifically bind to SARS-CoV-2 S-ECD, as shown in Figure 5.
6.表达载体的构建和酶切鉴定6. Construction of expression vector and identification of enzyme digestion
对ZWD12构建轻、重链重组表达质粒，进行单抗的表达制备。The light and heavy chain recombinant expression plasmids were constructed for ZWD12, and the monoclonal antibody was expressed and prepared.
1) pCDNA3.4-ZWD12-H表达质粒构建：1) pCDNA3.4-ZWD12-H expression plasmid construction:
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-ZWD12-H。Using the linear expression cassette as a template, 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-ZWD12-H.
2) pCDNA3.4-ZWD12-κ表达质粒构建：2) Construction of pCDNA3.4-ZWD12-κ expression plasmid:
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-ZWD12-κ。Using the light chain expression cassette as a template, 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, and the clones were selected for sequencing and identification after transformation into TOP10, and the successful light chain was constructed Expression vector pCDNA3.4-ZWD12-κ.
3) 单抗的瞬时表达和亲和层析纯化3) Transient expression and affinity chromatography purification of monoclonal antibody
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6 ：泳道1为单抗ZWC6的还原电泳结果，泳道2为单抗ZWD12的还原电泳结果，重链和轻链的理论分子量分别为50 kDa和25 kDa，符合预期，泳道M为分子量标记（分子量从大到小依次为：250，130，100，70，55，55，35，25，15，10 kDa）。 Using the Expi293 expression system, mix 15 μg of the heavy chain and 15 μg 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. Use at least 3 column volumes of 20 mM PBS to wash the column. After the baseline is stable, wash with 20% ethanol column. The SDS-PAGE detection results of the monoclonal antibody purified by affinity chromatography are shown in Figure 6: Swimming lane 1 is the reduction electrophoresis result of the monoclonal antibody ZWC6, and swimming lane 2 is the reduction electrophoresis result of the monoclonal antibody ZWD12. The theoretical molecular weights of the heavy chain and the light chain are respectively 50 kDa and 25 kDa, as expected, lane M is the molecular weight marker (molecular weight from large to small: 250, 130, 100, 70, 55, 55, 35, 25, 15, 10 kDa).
实施例2. 抗体ZWD12识别表位分析Example 2. Antibody ZWD12 recognition epitope analysis
1) 包被：实验前一天96孔酶联板，取重组的SARS-CoV-2 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。1) Coating: Take the recombinant SARS-CoV-2 S-ECD antigen, S1 antigen, RBD antigen and S2 antigen on the 96-well enzyme-linked plate one day before the experiment and dilute to a concentration of 2 μg/mL with coating solution, and coat the enzyme Plate, 100 μL per well, coated overnight at 4°C.
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。2) Blocking: Wash 3 times with a plate washer (BIO-TEK, 405_LS) on the day of the experiment, add 100 µL of blocking solution to each well, and incubate at 37°C for 1 hour.
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。3) Sample incubation: wash the plate 3 times, add 100 μL diluent to each well except the first well, dilute the antibody to 1 μg/mL in the first well, 4-fold serial dilution, 100 μL/well, set three for each antibody Duplicate the wells and incubate at 37°C for 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。4) Secondary antibody incubation: wash the plate 3 times, dilute the HPR-labeled goat anti-human IgG secondary antibody (Abcam, ab97225) at 1:10000 with diluent, add 100 µL per well to the corresponding well of the ELISA plate, and keep at 37°C Incubate for 1 hour.
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。5) Color development: Wash the plate 3 times, add 100 µL of TMB single-component chromogenic solution to each well, develop color for 6 min, and keep in the dark at room temperature, then add 50 µL of stop solution to each well to terminate the reaction.
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。 6) Use a microplate reader to detect the OD value at 450-630nm, and use the wells without the sample to be tested as negative controls, and the wells with OD 450-630 > 2.1 times the negative control as positive.
结果：检测ZWD12与不同抗原表位的结合活性，具体见图7， ZWD12与SARS-CoV-2 WT的S-ECD、S1和RBD蛋白均特异结合，呈现剂量反应关系。结果表明，单抗ZWD12识别的表位位于S1蛋白的RBD区。Results: The binding activity of ZWD12 to different epitopes was detected, as shown in Figure 7. ZWD12 specifically binds to the S-ECD, S1 and RBD proteins of SARS-CoV-2 WT, showing a dose-response relationship. The results showed that the epitope recognized by monoclonal antibody ZWD12 was located in the RBD region of S1 protein.
单抗ZWD12的序列分析结果如下：The sequence analysis results of monoclonal antibody ZWD12 are as follows:
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、97-112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27-32、50-52、89-98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。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 112th amino acid sequence, the polynucleotide sequence encoding the variable region of the heavy chain is shown in SEQ ID NO: 2; the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 5, and the variable region of the light chain is shown in The amino acid sequences of the CDR1, CDR2 and CDR3 regions are respectively shown in the amino acid sequences at positions 27-32, 50-52, and 89-98 of SEQ ID NO: 5, and the polynucleotide sequence encoding the light chain variable region is represented by SEQ ID NO: 6.
实施例3：抗体ZWD12的交叉结合活性鉴定Example 3: Identification of cross-binding activity of antibody ZWD12
对ZWD12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8所示。ZWD12与Alpha株、Beta株、Gamma株、Delta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ZWD12能够交叉结合Alpha株、Beta株、Gamma株、Delta株的S-ECD蛋白。The cross-binding activity between ZWD12 and the S protein of SARS-CoV-2 variants of concern (Variants of concern) was identified using the same method as above, and the results are shown in Figure 8. ZWD12 specifically binds to the S-ECD protein of Alpha strain, Beta strain, Gamma strain, and Delta strain, and exhibits a dose-response relationship. The results showed that monoclonal antibody ZWD12 could cross-link the S-ECD protein of Alpha strain, Beta strain, Gamma strain and Delta strain.
实施例4. 抗体ZWD12的假病毒中和活性鉴定Example 4. Pseudovirus neutralization activity identification of antibody ZWD12
1) 将纯化单抗用培养基 DMEM+10% FBS 自初始浓度（ZWD12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。 1) The purified monoclonal antibody was serially diluted 3 times from the initial concentration (the initial concentration of ZWD12 monoclonal antibody was 3.7 μg/ml) with medium DMEM+10% FBS, added to a 96-well culture plate, and set up 3 replicate wells with a volume of 50 μL/ Then add 50 μL pseudovirus suspension of the wild type or mutant strain of the new coronavirus to each well (dilute the virus to an appropriate titer with DMEM+10% FBS), mix well, and set up a survival control (without virus and antibody) And the dead control (only virus added), incubated at 37°C in a 5% CO 2 cell incubator for 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。 2) Digest HEK293T cells with 0.25% trypsin, dilute them with medium (DMEM+10% FBS) to a concentration of 2.5×10 5 cells/mL, inoculate them into 96-well cell culture plates, and inoculate at a volume of 100 μL/well , placed in a 37°C 5% CO 2 cell incubator and cultured overnight.
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。3) After 48 h, discard 100 μL of cell culture supernatant, add 100 μL of chromogenic substrate, and incubate in the dark for 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。 4) Transfer 150 μL to a 96-well white microplate, and use the Tecan Spark multifunctional microplate detector to read the Luciferase signal value; use (Luc sample well-Luc death control) / (Luc survival control-Luc death control) Calculate the cell viability, use GraphPad Prism 8 to fit the curve, and calculate the EC 50 value of the antibody.
结果见图9，本发明公开的单抗ZWD12对新冠病毒野生型假病毒的EC 50是5.860 ng/mL，中和Alpha株假病毒的EC 50是4.107 ng/mL，中和Beta株假病毒的EC 50是3.564 ng/mL，中和Gamma株假病毒的EC 50是3.829 ng/mL，中和Delta株假病毒的EC 50是7.287 ng/mL。结果显示ZWD12对目前的主要变异株的假病毒具有广谱高效中和活性。 The results are shown in Figure 9. The EC 50 of the monoclonal antibody ZWD12 disclosed in the present invention against the wild-type pseudovirus of the new coronavirus is 5.860 ng/mL, the EC 50 of neutralizing the Alpha strain pseudovirus is 4.107 ng/mL, and neutralizing the Beta strain pseudovirus. The EC 50 for neutralizing the pseudovirus of the Gamma strain was 3.829 ng/mL, and the EC 50 for neutralizing the pseudovirus of the Delta strain was 7.287 ng/mL. The results show that ZWD12 has a broad-spectrum and high-efficiency neutralizing activity against the pseudoviruses of the current main mutant strains.
实施例5. 抗体ZWD12的真病毒中和活性鉴定Example 5. Identification of euvirus neutralizing activity of antibody ZWD12
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。 1) Digest Vero E6 cells with 0.25% trypsin, dilute them with medium (DMEM+10% FBS) to a concentration of 3×10 5 cells/mL, and inoculate them into 96-well cell culture plates with an inoculation volume of 100 μL/ Wells were incubated overnight in a 37°C 5% CO 2 cell incubator.
2) 实验当天，将纯化单抗用培养基 DMEM+2% FBS 自初始浓度（ZWD12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。 2) On the day of the experiment, the purified monoclonal antibody was added to a 96-well culture plate with a volume of 120 μL/well from the initial concentration (the initial concentration of ZWD12 monoclonal antibody was 100 μg/ml, 3-fold serial dilution) in the culture medium DMEM+2% FBS; Add 120 μL 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.
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。 3) Discard the cell culture supernatant in the 96-well plate, and add 200 μL of co-incubated virus-antibody mixed suspension to each well; set up a survival control (no virus and antibody) and death control (only virus), and set Incubate for 72 h at 37°C in a 5% CO 2 incubator.
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。4) Discard the cell culture supernatant after 72 h, add 50 μL of crystal violet staining solution to stain at room temperature for 30 min, discard the staining solution, add 200 μL/well of pure water, and repeat washing 6 times.
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。 5) Discard the washing solution, pat dry the water in the plate wells with absorbent paper, add 100 μL of decolorization solution to fully dissolve, take the OD 620 as a reference, and measure the OD 570 value with a microplate reader; use (OD sample well-OD dead control) /(OD survival control-OD death control) to calculate the cell viability, use GraphPad Prism 8 to fit the curve, and calculate the EC 50 value of the antibody.
结果见图10，本发明公开的单抗ZWD12的对新冠病毒野生型的EC 50是0.103 μg/mL，中和Beta株的EC 50是0.069 μg/mL，中和Delta株的EC 50是0.079 μg/mL。说明ZWD12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。 The results are shown in Figure 10. The EC 50 of the monoclonal antibody ZWD12 disclosed in the present invention against the wild type of the new coronavirus is 0.103 μg/mL, the EC 50 of neutralizing the Beta strain is 0.069 μg/mL, and the EC 50 of neutralizing the Delta strain is 0.079 μg /mL. It shows that ZWD12 has high neutralizing activity against the true viruses of the wild type, Beta and Delta variants of SARS-CoV-2.
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Example 6. Surface Plasmon Resonance (SPR) Determination of the Affinity of Monoclonal Antigen to S Antigen
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。1) Prepare buffer solution: Measure 50mL 10×HBS-EP+ buffer solution and 450mL deionized water, mix well and put into a 500mL buffer bottle.
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。2) 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, unscrew the cap of the desalting column, 1500 Centrifuge at g for 1 min to remove the original liquid in the column, add 300 μL of HBS-EP+ buffer, centrifuge at 1500 g for 1 min, repeat 4 times, put the desalting column in a new collection tube, add 100 μL of protein solution to the column, 1500 g After centrifugation for 2 min, the filtered liquid was collected and the protein concentration was determined using NanoVue.
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。3) Turn on the Biocore T200 machine, insert the inlet tube A into the HBS-EP+ buffer bottle, put the ProteinA chip, and run the Prime program.
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。4) Sample preparation: Dilute the ligand (antibody) to 0.5 μg/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.
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，RBD分子量为26.5 kDa，浓度为0 nM、0.78125 nM、1.5625 nM、3.125 nM、6.25 nM、12.5 nM、25 nM、50 nM、100 nM、0.78125 nM，选择0.78125 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。5) Use the multi-cycle detection method for sample detection: open Kinetics/Affinity in Run/Wizard, select 2-1 or 4-3 for Flow path, select Protein A for Chip type, select Ligand capture, and click Next; in the Setup interface , fill in HBS-EP+ in Solution under Startup, select 3 for Number of cycles, and click Next; in the Injection Parameters interface: Fill in the antibody name for Ligand name, Contact time is 60 s, Flow rate is 10 μL/min, Stabilization The period is 0 s; the Contact time of the Sample is 120 s, the Flow rate is 30 μL/min, and the Dissociation Time is 900 s; Solution in Regeneration is Glycine pH 1.5, Contact time is 30 s, Flow rate is 30 μL/min, Stabilization The period is 30 s; click Next; fill in the analyte information in the Sample interface: S-ECD molecular weight is 134 kDa, RBD molecular weight is 26.5 kDa at 0 nM, 0.78125 nM, 1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 0.78125 nM, select 0.78125 nM as the repeat, click Next; in System In the Preparations interface, set the analysis temperature and sample compartment temperature to 25°C, click Next; in the Rack In the Position interface, select Sample and Reagent Rack1, set the sample position, prepare and place the sample according to the sample position and amount, and click Next; confirm that the volume of the running buffer meets the requirements of the experiment, click Start, and save the experimental method and results. The program starts to run automatically, and the running time is 5 hours.
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ZWD12与不同抗原的结合动力学数据ka、kd、KD。图11-16分别是ZWD12与WT、Alpha、Beta、Gamma、Delta株的S-ECD和WT RBD的亲和力常数测定图，其KD依次为0.896 nM、0.826 nM、0.991 nM、1.423 nM、1.912 nM及0.356 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。6) Result analysis: Open the data analysis software Biacore T200 Evaluation Software, open the running result file: click Kinetics/Affinity, select Surface bound; select 0 nM and at least 5 appropriate concentrations in the Select Curves interface, and click Next; click Kinetics in the Select Data interface; in the Fit Kinetics interface, select 1:1 Binding for Method, and click fit for data fitting; record Combine kinetic data ka, kd, KD, etc. Table 5 records the binding kinetic data ka, kd, and KD of monoclonal antibody ZWD12 and different antigens according to the analysis data displayed in the Report. Figures 11-16 are the determination diagrams of the affinity constants between ZWD12 and the S-ECD and WT RBD of WT, Alpha, Beta, Gamma, and Delta strains, respectively, and the KDs are 0.896 nM, 0.826 nM, 0.991 nM, 1.423 nM, 1.912 nM and 0.356 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.
表5 单抗ZWD12与不同抗原的结合动力学数据Table 5 The binding kinetic data of monoclonal antibody ZWD12 to different antigens
Figure 877676dest_path_image006
Figure 877676dest_path_image006
工业实用性Industrial Applicability
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。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.
序列表自由内容Sequence Listing Free Content
序列表sequence listing
 the
<110>  中国人民解放军军事科学院军事医学研究院<110> Academy of Military Medical Sciences, Chinese People's Liberation Army
 the
<120>  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<120> A fully human anti-new coronavirus broad-spectrum high neutralizing activity monoclonal antibody and its application
 the
<130>  P2022048JK<130> P2022048JK
 the
<150>  CN202111102531.8<150> CN202111102531.8
<151>  2022-08-23<151> 2022-08-23
 the
<160>  10<160> 10
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<170>  SIPOSequenceListing 1.0<170> SIP Sequence Listing 1.0
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<210>  1<210> 1
<211>  123<211> 123
<212>  PRT<212> PRT
<213>  人工序列(Artificial Sequence)<213> Artificial Sequence
 the
<220><220>
<223>  重链可变区氨基酸序列<223> Heavy Chain Variable Region Amino Acid Sequence
 the
 the
<400>  1<400> 1
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1               5                   10                  15      1 5 10 15
Thr Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Ser Phe Ser Asn Tyr Thr Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Ser Phe Ser Asn Tyr
            20                  25                  30                  20 25                                
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
        35                  40                  45              `` 35 40 45
Ala Asn Ile Lys Gln Asp Gly Ser Glu Thr His Tyr Val Asp Ser Val Ala Asn Ile Lys Gln Asp Gly Ser Glu Thr His Tyr Val Asp Ser Val
    50                  55                  60                  the 50 55                  
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65                  70                  75                  80  65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Phe Cys Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Phe Cys
                85                  90                  95              85               95
Val Lys Asp Arg Thr Asp Trp Glu Leu Ile Arg Gly Tyr Phe Gly His Val Lys Asp Arg Thr Asp Trp Glu Leu Ile Arg Gly Tyr Phe Gly His
            100                 105                 110                 100 105 110
Trp Gly Gln Gly Thr Gln Ile Thr Val Ser Ser Trp Gly Gln Gly Thr Gln Ile Thr Val Ser Ser
        115                 120             `` 115 120
 the
<210>  2<210> 2
<211>  369<211> 369
<212>  DNA<212> dna
<213>  人工序列(Artificial Sequence)<213> Artificial Sequence
 the
<220><220>
<223>  重链可变区DNA序列<223> heavy chain variable region DNA sequence
 the
 the
<400>  2<400> 2
gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggac cctgagactc        60gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggac cctgagactc 60
tcctgtgtag cgtccggatt cagttttagt aattattgga tgagctgggt tcgccaggct       120tcctgtgtag cgtccggatt cagttttagt aattattgga tgagctgggt tcgccaggct 120
ccagggaagg ggttggagtg ggtggccaat ataaagcaag atggaagtga gacacactat       180ccagggaagg ggttggagtg ggtggccaat ataaagcaag atggaagtga gacacactat 180
gtggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat       240gtggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240
ctgcaaatga acagcctgag agccgacgac acggctgtgt atttctgtgt gaaagatagg       300ctgcaaatga acagcctgag agccgacgac acggctgtgt atttctgtgt gaaagatagg 300
actgactggg agctaatacg gggctacttt ggtcactggg gccagggaac ccagatcacc       360actgactgggg agctaatacg gggctacttt ggtcactggg gccagggaac ccagatcacc 360
gtctcctca                                                               369gtctcctca 369
 the
<210>  3<210> 3
<211>  330<211> 330
<212>  PRT<212> PRT
<213>  人工序列(Artificial Sequence)<213> Artificial Sequence
 the
<220><220>
<223>  重链恒定区氨基酸序列<223> Amino acid sequence of heavy chain constant region
 the
 the
<400>  3<400> 3
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1               5                   10                  15      1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
            20                  25                  30                  20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
        35                  40                  45              `` 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
    50                  55                  60                  the 50 55 60 ``
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65                  70                  75                  80  65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
                85                  90                  95              85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
            100                 105                 110                 100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
        115                 120                 125             `` 115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
    130                 135                 140                 the 130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145                 150                 155                 160 145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
                165                 170                 175             165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
            180                 185                 190                 180 185                            
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
        195                 200                 205             `` 195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
    210                 215                 220                 the 210 215                  
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225                 230                 235                 240 225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
                245                 250                 255             245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
            260                 265                 270                 260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
        275                 280                 285             `` 275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
    290                 295                 300                 the 290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305                 310                 315                 320 305 310                               320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
                325                 330         325 330
 the
<210>  4<210> 4
<211>  990<211> 990
<212>  DNA<212> dna
<213>  人工序列(Artificial Sequence)<213> Artificial Sequence
 the
<220><220>
<223>  重链恒定区DNA序列<223> heavy chain constant region DNA sequence
 the
 the
<400>  4<400> 4
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg        60gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg       120ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca       180tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct agatcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc       240ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc       300tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga       360aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctggggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct       420ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg       480gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac       540tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag       600agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc       660gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag       720aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc       780ctgaccaaga accagtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg       840gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg       900ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca       960cagcaggggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 960
cagaagagcc tctccctgtc tccgggtaaa                                        990cagaagagcc tctccctgtc tccgggtaaa 990
 the
<210>  5<210> 5
<211>  109<211> 109
<212>  PRT<212> PRT
<213>  人工序列(Artificial Sequence)<213> Artificial Sequence
 the
<220><220>
<223>  轻链可变区氨基酸序列<223> Amino Acid Sequence of Light Chain Variable Region
 the
 the
<400>  5<400> 5
Asp Ile Val Met Thr Gln Thr Pro Ser Ser Leu Ser Leu Ser Pro Gly Asp Ile Val Met Thr Gln Thr Pro Ser Ser Leu Ser Leu Ser Pro Gly
1               5                   10                  15      1 5 10 15
Asp Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Asn Ile Ile Asn Tyr Asp Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Asn Ile Ile Asn Tyr
            20                  25                  30                  20 25 30
Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile
        35                  40                  45              `` 35 40 45 the
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
    50                  55                  60                  the 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65                  70                  75                  80  65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ile Ile Trp Pro Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ile Ile Trp Pro Pro
                85                  90                  95              85 90 95
Tyr Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Tyr Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg
            100                 105                         100 105
 the
<210>  6<210> 6
<211>  327<211> 327
<212>  DNA<212> dna
<213>  人工序列(Artificial Sequence)<213> Artificial Sequence
 the
<220><220>
<223>  轻链可变区DNA序列<223> Light chain variable region DNA sequence
 the
 the
<400>  6<400> 6
gatattgtga tgacccagac tccatcctcc ctgtctttgt ctccagggga cagagccacc        60gatattgtga tgacccagac tccatcctcc ctgtctttgt ctccagggga cagagccacc 60
ctctcctgca gggccagtga gaatattatc aattacttag cctggtacca acagagacct       120ctctcctgca gggccagtga gaatattatc aattacttag cctggtacca acagagacct 120
ggccagtctc ccaggctcct catctatgat gcgtccaaca gggccactgg catcccggcc       180ggccagtctc ccaggctcct catctatgat gcgtccaaca gggccactgg catcccggcc 180
aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct       240aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240
gaagattttg cagtttatta ctgtcagcag cgtatcatct ggcctccgta cacttttggc       300gaagattttg cagtttatta ctgtcagcag cgtatcatct ggcctccgta cacttttggc 300
caggggacca aagtggatat caaacgt                                           327caggggacca aagtggatat caaacgt 327
 the
<210>  7<210> 7
<211>  106<211> 106
<212>  PRT<212> PRT
<213>  人工序列(Artificial Sequence)<213> Artificial Sequence
 the
<220><220>
<223>  Kappa轻链恒定区氨基酸序列<223> Amino acid sequence of Kappa light chain constant region
 the
 the
<400>  7<400> 7
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1               5                   10                  15      1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr
            20                  25                  30                  20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
        35                  40                  45              `` 35 40                                
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
    50                  55                  60                  the 50 55 60
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65                  70                  75                  80  65 70                   80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
                85                  90                  95              85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
            100                 105             100 105
 the
<210>  8<210> 8
<211>  318<211> 318
<212>  DNA<212> dna
<213>  人工序列(Artificial Sequence)<213> Artificial Sequence
 the
<220><220>
<223>  Kappa型轻链恒定区DNA序列<223> Kappa light chain constant region DNA sequence
 the
 the
<400>  8<400> 8
actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga        60actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 60
actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg       120actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 120
aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc       180aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 180
aaggacagca cctacagcct cagcagtacc ctgacgctga gcaaagcaga ctacgagaaa       240aaggacagca cctacagcct cagcagtacc ctgacgctga gcaaagcaga ctacgagaaa 240
cacaaagtct acgcctgcga agtcacccat cagggcctga gttcgcccgt cacaaagagc       300cacaaagtct acgcctgcga agtcacccat cagggcctga gttcgcccgt cacaaagagc 300
ttcaacaggg gagagtgt                                                     318ttcaacaggg gagagtgt 318
 the
<210>  9<210> 9
<211>  106<211> 106
<212>  PRT<212> PRT
<213>  人工序列(Artificial Sequence)<213> Artificial Sequence
 the
<220><220>
<223>  Lamda型轻链恒定区氨基酸序列<223> Lambda light chain constant region amino acid sequence
 the
 the
<400>  9<400> 9
Arg Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Arg Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
1               5                   10                  15      1 5 10 15  
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
            20                  25                  30                  20 25 30
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
        35                  40                  45              `` 35 40                      
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
    50                  55                  60                  the 50 55 60
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
65                  70                  75                  80  65 70                 80
Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
                85                  90                  95              85 90 95
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
            100                 105             100 105
 the
<210>  10<210> 10
<211>  318<211> 318
<212>  DNA<212> dna
<213>  人工序列(Artificial Sequence)<213> Artificial Sequence
 the
<220><220>
<223>  Lamda型轻链恒定区DNA序列<223> Lambda type light chain constant region DNA sequence
 the
 the
<400>  10<400> 10
cgtcagccca aggctgcccc ctcggtcact ctgttcccac cctcgagtga ggagcttcaa        60cgtcagccca aggctgcccc ctcggtcact ctgttcccac cctcgagtga ggagcttcaa 60
gccaacaagg ccacactggt gtgtctcata agtgacttct acccgggagc cgtgacagtg       120gccaacaagg ccaacactggt gtgtctcata agtgacttct acccgggagc cgtgacagtg 120
gcctggaagg cagatagcag ccccgtcaag gcgggagtgg agaccaccac accctccaaa       180gcctggaagg cagatagcag ccccgtcaag gcgggagtgg agaccaccac accctccaaa 180
caaagcaaca acaagtacgc ggccagcagc tacctgagcc tgacgcctga gcagtggaag       240caaagcaaca acaagtacgc ggccagcagc tacctgagcc tgacgcctga gcagtggaag 240
tcccacaaaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga gaagacagtg       300tcccacaaaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga gaagacagtg 300
gcccctacag aatgttca                                                     318gcccctacag aatgttca 318

Claims (1)

  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、97-112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27-32、50-52、89-98位氨基酸序列所示。An anti-SARS-CoV-2 fully human monoclonal antibody, characterized in that the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the monoclonal antibody are respectively as shown in SEQ ID NO: 1 No. 26 -33, 51-58, and 97-112 amino acid sequences; the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the light chain variable region are shown in SEQ ID NO: 5 No. 27-32, 50-52, 89-98, respectively The amino acid sequence is shown.
    2. 根据权利要求1所述的单克隆抗体，其特征在于，所述单克隆抗体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。2. The monoclonal antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is as SEQ ID NO: Shown in 5.
    3. 根据权利要求2所述的抗体，其特征在于，所述单克隆抗体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。3. The antibody according to claim 2, wherein the amino acid sequence of the heavy chain constant region of the monoclonal antibody is as shown in SEQ ID NO:3, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:7 Show.
    4. 一种编码权利要求1-3任一所述单克隆抗体重链和轻链的多核苷酸，其特征在于，编码所述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。4. A polynucleotide encoding the heavy chain and light chain of the arbitrary described monoclonal antibody of claim 1-3, it is characterized in that, the polynucleotide sequence of the heavy chain variable region of encoding described monoclonal antibody consists of SEQ As shown in ID NO:2, the polynucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:6.
    5. 根据权利要求4所述的多核苷酸，其特征在于，编码所述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。5. The polynucleotide according to claim 4, wherein the polynucleotide sequence encoding the heavy chain constant region of the monoclonal antibody is shown in SEQ ID NO: 4, encoding the light of the monoclonal antibody The polynucleotide sequence of the chain constant region is shown by SEQ ID NO:8.
    6. 一种表达权利要求5所述编码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。6. A functional element expressing the polynucleotide encoding the heavy chain and light chain of the monoclonal antibody according to claim 5, wherein the functional element is a linear expression cassette or a mammalian expression vector.
    7. 一种含有权利要求6所述功能元件的宿主细胞。7. A host cell containing the functional element of claim 6.
    8. 根据权利要求要求7所述的宿主细胞，其特征在于，所述细胞为Expi 293F细胞。8. The host cell according to claim 7, wherein the cell is an Expi 293F cell.
    9. 根据权利要求要求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。9. The host cell according to claim 7, wherein the cell is a CHO-K1 or CHO-S cell.
    10. 权利要求1-3任一所述的单克隆抗体在制备COVID-19治疗药物中的应用。10. The application of the monoclonal antibody described in any one of claims 1-3 in the preparation of COVID-19 therapeutic drug.
PCT/CN2022/114962 2021-09-20 2022-08-25 Fully human broad-spectrum high-neutralizing-activity monoclonal antibody against sars-cov-2 and use thereof WO2023040627A1 (en)

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