Humanized high neutralization activity anti-sars-cov-2 monoclonal antibody and application

Abstract

The application discloses a humanized high neutralization activity anti-SARS-CoV-2 monoclonal antibody, the neutralizing antibody is obtained by screening through single B cell flow sorting-antibody gene amplification pairing expression technology, has a unique CDR region, can be specifically combined with SARS-CoV-2 and can effectively neutralize the current multiple strains of international epidemic virus (Prototype strain and Omicron B.1.1.529, BF.7 and XBB.1.5 mutant strain). The application also relates to application of the antibody in preparation of a SARS-CoV-2 treatment or prevention drug and application of the antibody in preparation of a SARS-CoV-2 detection reagent. The antibody can be used for COVID-19 diagnosis, emergency prevention and / or treatment, has the characteristics of full humanization, high expression and good stability, and is suitable for industrialized preparation.

Description

Technical Field

[0001] This invention discloses a polypeptide, and more specifically, this invention discloses an antibody. Background Technology

[0002] SARS-CoV-2 has continuously evolved into various variants, including Alpha, Beta, Gamma, Delta, Lambda, and Omicron. Currently, Omicron is the dominant circulating strain, characterized by rapid transmission, high infectivity, short incubation period, and strong immune evasion capabilities. To control the incidence and severity of SARS-CoV-2, there is an urgent need to develop effective antiviral drugs, vaccines, and neutralizing antibodies.

[0003] The treatment guidelines for COVID-19 mention that convalescent plasma can be used to treat patients with rapidly progressing, severe, and critical illness. One of the main reasons for the effectiveness of plasma therapy is that convalescent patients may possess polyclonal antiviral antibodies that help kill or clear the virus. However, the main drawbacks of this method are limited availability, difficulty in quality control, and low neutralizing antibody titers. Lessons learned from SARS indicate that some non-neutralizing antibodies (nAbs) targeting non-RBD regions of the S protein may cause antibody-dependent enhancement (ADE). Therefore, developing broad-spectrum neutralizing monoclonal antibodies is essential for the prevention or treatment of COVID-19.

[0004] Studies have shown that many fragments (S1-NTD, RBD, S2) of the novel coronavirus S protein can serve as targets for broad-spectrum neutralizing antibodies. These antibodies target the S1-RBD, S1-NTD, or S2 regions, blocking the binding of SARS-CoV-2 to cell receptors, interfering with S2-mediated membrane fusion or entry into host cells, thereby inhibiting viral infection. Research has found that while NTD antibodies have strong neutralizing activity, the high glycosylation of NTDs and the generally clustered epitopes result in poor neutralization against variant strains. The S2 subunit, a key protein in viral-cell membrane fusion, is the most conserved structure in the S protein, exhibiting 63-98% sequence identity with human pathogenic coronaviruses. However, the neutralizing capacity of isolated S2 antibodies is limited, and there are no reports on in vivo protective data; whether S2 antibodies can prevent infection remains to be determined. Therefore, in comparison, RBD antibodies not only have strong neutralizing capacity but also have a broader spectrum, making them more suitable for current anti-SARS-CoV-2 drug development.

[0005] Currently, there are reports both domestically and internationally on the isolation of neutralizing antibodies against the novel coronavirus. Using methods such as single-cell sorting, antibody genome deep sequencing, and phage display, a number of humanized monoclonal antibodies targeting the RBD have been isolated, such as 1F11, 2F6, CA1, CB6, and BD-368-2. These antibodies have exhibited strong in vitro neutralizing activity (IC50 < 1 μg / mL). However, studies have found that most monoclonal antibodies used clinically cannot effectively neutralize the Omicron variant. Therefore, previously approved monoclonal antibodies such as Bamlanivimab and Etesevimab are restricted to use in individuals potentially infected with the Omicron variant. Given the increasing number of SARS-CoV-2 infections and the immune escape of variant strains, isolating more potent neutralizing antibodies as alternatives is crucial. Furthermore, various combinations of these neutralizing antibodies targeting different epitopes should be explored to develop cocktail therapies, which could more effectively prevent viral immune escape. Currently, there are no reported similar broad-spectrum antibodies or antibody compositions in this field. The purpose of this invention is to provide a group of monoclonal antibodies with high neutralizing activity against the novel coronavirus, and based on this, to provide the application of the high neutralizing activity monoclonal antibodies against the novel coronavirus in the preparation of drugs for treating novel coronavirus viral diseases. Summary of the Invention

[0006] Based on the above-mentioned objectives, this invention first provides a humanized broad-spectrum, high-neutralizing monoclonal antibody against the novel coronavirus. The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3, and the light chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody are shown below: (1) Amino acids 26-33, 51-58, and 97-115 of SEQ ID NO.1 and amino acids 27-32, 50-52, and 89-97 of SEQ ID NO.3; or (2) Amino acids 26-33, 51-57 and 96-111 of SEQ ID NO.5 and amino acids 26-34, 52-54 and 91-102 of SEQ ID NO.7; or (3) Amino acids 26-33, 51-58 and 97-115 of SEQ ID NO.9 and amino acids 26-31, 49-51 and 88-97 of SEQ ID NO.11; In a preferred embodiment, the amino acid sequences of the variable region of the heavy chain and the variable region of the light chain of the monoclonal antibody are as follows: (1) SEQ ID NO.1 and SEQ ID NO.3; In this invention, an antibody having the amino acid sequence of the variable region of the heavy chain and the variable region of the light chain is named "LY-F6HK", or (2) SEQ ID NO.5 and SEQ ID NO.7; In this invention, an antibody having the amino acid sequence of the variable region of the heavy chain and the variable region of the light chain is named “LQL-B6HL”.

[0007] (3) SEQ ID NO.9 and SEQ ID NO.11; In this invention, an antibody having the amino acid sequence of the variable region of the heavy chain and the variable region of the light chain is named “LQL-D6HL”.

[0008] In a more preferred embodiment, the amino acid sequence of the constant region of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.13, and the amino acid sequence of the constant region of the light chain is shown in SEQ ID NO.15 or SEQ ID NO.17, wherein SEQ ID NO.15 is the amino acid sequence of the constant region of the Kappa-type light chain, and SEQ ID NO.17 is the amino acid sequence of the constant region of the Lamda-type light chain.

[0009] Secondly, this invention provides a polynucleotide encoding the aforementioned humanized broad-spectrum, high-potency, and highly active anti-novel coronavirus monoclonal antibody. The polynucleotide combinations encoding the variable region of the heavy chain of the monoclonal antibody and the polynucleotide combinations encoding the variable region of the light chain of the monoclonal antibody are respectively shown below: (1) SEQ ID NO.2 and SEQ ID NO.4; an antibody having a specific technical solution encoding a polynucleotide in the heavy chain variable region and a polynucleotide in the light chain variable region is named "LY-F6HK", or (2) SEQ ID NO. 6 and SEQ ID NO. 8; an antibody having a specific technical solution encoding a polynucleotide in both the heavy chain variable region and the light chain variable region is named "LQL-B6HL", or (3) SEQ ID NO.10 and SEQ ID NO.12, the antibody with a specific technical solution of the heavy chain variable region encoding a polynucleotide and the light chain variable region encoding a polynucleotide is named "LQL-D6HL".

[0010] In a preferred embodiment, the polynucleotide sequence encoding the constant region of the antibody heavy chain is shown in SEQ ID NO. 14, and the polynucleotide sequence of the constant region of the light chain is shown in SEQ ID NO. 16 or SEQ ID NO. 18, wherein SEQ ID NO. 16 is the constant region polynucleotide sequence of the Kappa type light chain, and SEQ ID NO. 18 is the constant region polynucleotide sequence of the Lamda type light chain.

[0011] Third, the present invention also provides a vector for expressing the above-mentioned humanized high- and neutralizing monoclonal antibody against the novel coronavirus. The vector contains polynucleotides encoding the variable region of the antibody heavy chain and the variable region of the antibody light chain. The vector can be a eukaryotic expression vector commonly used in genetic engineering. In a specific embodiment of the present invention, the vector is IgH (heavy chain expression vector), Igκ (κ light chain expression vector), or Igλ (λ light chain expression vector) (see Tiller et al. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning, J Immunol Methods. 2008 January 1; 329(1-2): 112–124. This prior art document is incorporated herein by reference).

[0012] Fourth, the present invention provides a host cell expressing the above-mentioned humanized, high-potency, and highly active monoclonal antibody against the novel coronavirus, wherein the host cell contains the above-mentioned vector. The host cell can be a eukaryotic host cell conventionally used in genetic engineering; in one specific embodiment of the present invention, the host cell is a 293F cell.

[0013] Fifth, the present invention provides the application of the above-mentioned humanized high-neutralizing and active anti-novel coronavirus monoclonal antibody in the preparation of drugs for the treatment and / or prevention of novel coronavirus disease. The antibody can be prepared as a clinical therapeutic drug, or as a targeted drug such as a fusion protein or antibody-drug conjugate targeting the novel coronavirus for the treatment of novel coronavirus disease or / or prevention of novel coronavirus infection.

[0014] Sixth, this invention provides the application of the above-mentioned humanized, highly neutralizing, and active anti-novel coronavirus monoclonal antibody in the preparation of novel coronavirus detection reagents. The antibody can be used for various laboratory detection applications of the novel coronavirus, such as various immunological methods for detecting the novel coronavirus based on antigen-antibody specific binding reactions, including but not limited to enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, chemiluminescent immunoassay, Western blot, etc.

[0015] Seventh, the present invention provides an antibody composition comprising a first antibody and a second antibody, wherein the amino acid sequences of the CDR1, CDR2, and CDR3 of the heavy chain variable region and the CDR1, CDR2, and CDR3 of the light chain variable region of the first antibody are as shown in amino acids 26-33, 51-58, and 97-115 of SEQ ID NO.1 and amino acids 27-32, 50-52, and 89-97 of SEQ ID NO.3, respectively; and the amino acid sequences of the CDR1, CDR2, and CDR3 of the heavy chain variable region and the CDR1, CDR2, and CDR3 of the light chain variable region of the second antibody are as shown in amino acids 26-33, 51-57, and 96-111 of SEQ ID NO.5 and amino acids 26-34, 52-54, and 91-102 of SEQ ID NO.7, respectively; or, the CDR1, CDR2, and CDR3 of the heavy chain variable region of the second antibody... The amino acid sequences of the CDR1, CDR2, and CDR3 of the light chain variable region are shown as amino acids 26-33, 51-58, and 97-115 of SEQ ID NO. 9 and amino acids 26-31, 49-51, and 88-97 of SEQ ID NO. 11, respectively. In this invention, the "first" antibody and the "second" antibody do not have a priority order; they are only used to distinguish between two different antibodies. In a specific definition of this invention, the first antibody refers to "LY-F6HK," which has different antigenic recognition epitopes from the other two antibodies "LQL-B6HL" and "LQL-D6HL" provided in this invention. "LQL-B6HL" and "LQL-D6HL," however, have the same antigenic recognition epitopes, and the two antibodies compete for epitopes. Therefore, in this invention, the composition includes an antibody “LY-F6HK” and at least one of two antibodies, “LQL-B6HL” or “LQL-D6HL”, to exert sufficient binding to the antigenic epitopes of the novel coronavirus and block the virus from invading the body's cells.

[0016] In a preferred embodiment, the amino acid sequences of the heavy chain variable region and the light chain variable region of the first antibody are shown in SEQ ID NO.1 and SEQ ID NO.3, respectively, and the amino acid sequences of the heavy chain variable region and the light chain variable region of the second antibody are shown in SEQ ID NO.5 and SEQ ID NO.7, respectively; or, as shown in SEQ ID NO.9 and SEQ ID NO.11.

[0017] Finally, this invention provides the application of the above-described antibody compositions in the preparation of drugs for the treatment and / or prevention of COVID-19. The three antibodies provided by this invention not only possess high neutralizing activity but also broad-spectrum neutralization, exhibiting strong neutralizing ability against multiple variants of the novel coronavirus currently circulating worldwide. Therefore, the application of antibody compositions containing at least two or more of the antibodies of this invention, such as a combination of LQL-D6HL and LY-F6HK or LQL-B6HL and LY-F6HK, can more effectively enhance the broad-spectrum neutralizing ability against multiple variants of the novel coronavirus. Thus, antibody compositions containing this invention can be used in cocktail therapy for the treatment of COVID-19 and / or for the prevention of COVID-19 infection.

[0018] The humanized, highly neutralizing monoclonal antibody against the novel coronavirus provided by this invention was obtained through screening using single B cell flow cytometry sorting-antibody gene amplification pairing expression technology. It possesses a unique CDR region, specifically binds to SARS-CoV-2, and can effectively neutralize multiple currently circulating international viruses (Prototype and Omicron variants B.1.1.529, BF.7, XBB.1.5), exhibiting significant broad-spectrum neutralizing ability against multiple circulating novel coronavirus strains worldwide. Therefore, the antibody provided by this invention, and compositions containing both antibodies, can be used to prepare emergency prevention and / or treatment drugs for COVID-19, featuring full humanization, high expression, and good stability, making it suitable for industrialization. Furthermore, the monoclonal antibody provided by this invention can also be used to prepare SARS-CoV-2 virus detection reagents for detecting viral antigens and for identifying effective neutralizing antigenic epitopes. Attached Figure Description

[0019] Figure 1. Results of biotinylated RBD protein titer assay, where A. RBD biotinylation efficiency was detected by magnetic bead assay; B. Biotinylation efficiency was detected by ELISA. Figure 2 Schematic diagram of RBD-specific B cells sorted by flow cytometry; Figure 3ELISA results of antibody binding ability against RBD; where A. Binding ability curves of 3 monoclonal antibodies and Prototype-RBD; B. Binding ability curves of 3 monoclonal antibodies and Omicron B.1.1.529-RBD; Figure 4. Affinity test results of LY-F6HK detected using BLI technology; where A. Interaction curve between monoclonal antibody and Prototype-RBD; B. Interaction curve between monoclonal antibody and Omicron B.1.1.529-RBD; Figure 5. Affinity detection results of LQL-B6HL using BLI technology; where A. Interaction curve between monoclonal antibody and Prototype-RBD; B. Interaction curve between monoclonal antibody and Omicron B.1.1.529-RBD; Figure 6. Affinity detection results of LQL-D6HL using BLI technology; where A is the interaction curve between the monoclonal antibody and Prototype-RBD; and B is the interaction curve between the monoclonal antibody and Omicron B.1.1.529-RBD. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0021] Example 1: Synthesis, expression, biotinylation, and staining of novel coronavirus RBD probes (1) Based on the data published by Genbank (NC_045512), the full-length RBD gene sequence carrying the 6×His-Avi (His-His-His-His-His-His-Glu-Lys-Asn-Glu-Gln-Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn-Trp-Phe-Asp-Ile-Thr-Asn-Trp-Leu-Trp-Tyr-ILe-Lys-Lys-Lys) tag was synthesized.

[0022] (2) After being digested with EcoRI and EcoRV, the clones were re-ligated into the eukaryotic expression vector pDRVI1.0 (constructed and preserved by the inventors). After selection, the clones were sequenced and found to be correct.

[0023] (3) Transfect the two probe plasmids into 293F cells for expression. After 5-6 days, centrifuge the culture medium to collect the cell supernatant and purify the antigen protein through a nickel column.

[0024] (4) Biotinylate the probe protein using the BirA 500 Biotinylate Ligase Kit (BirA500, Avidity). Dissolve 1 mg of molecular probe protein in 0.7 mL of PBS buffer, add 0.1 mL of 10× buffer A and 0.1 mL of 10× buffer B, then add 4 μL of BirA 500 enzyme, mix well, and incubate at 30°C for 30 minutes; transfer the mixture to a 10 KD concentration tube, add 10 mL of PBS, centrifuge at 4000 g for 15 min until the remaining volume is 0.5 mL, repeat this operation 5 times; collect the concentrated protein and determine the concentration, then store it at -80°C. To detect the biotinylation activity of the molecular probe, 60 μL of streptomycin-labeled agarose + 10 μg protein + 500 μL PBS were placed in a shaker at room temperature for 30 min, briefly centrifuged, and washed three times with 1 mL PBS. The last wash was performed by aspirating as much liquid as possible, leaving approximately 30 μL of agarose. Simultaneously, 30 μL of 2×SDS gel loading buffer (100 mM pH 6.8 Tris-HCl, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM β-mercaptoethanol) was prepared. The agarose and loading buffer were mixed 1:1, and 20 μL was incubated at 100℃ for 5-10 min, followed by SDS-PAGE electrophoresis. After electrophoresis, an appropriate amount of Coomassie brilliant blue was added for staining. After destaining in a shaker, the intensity of the electrophoretic bands was observed to determine the biotinylation efficiency. A band intensity of at least 50% was sufficient for fluorescent labeling.

[0025] (5) The biotinylation activity of the two probes was detected by enzyme-linked immunosorbent assay (ELISA). Dilute the novel coronavirus antigen to 2 μg / mL with PBS and add 100 μL to each well of a 96-well ELISA plate. Incubate overnight at 4°C. The next day, wash three times with PBST (PBS containing 0.05% Tween 20). Add 370 μL of blocking buffer (PBS containing 2% milk and 5% FBS) to each well and block at room temperature for 1 hour. Wash three times with PBST (PBS containing 0.05% Tween 20). Add 100 μL of blocking buffer to each well. Add 25 μL of diluted biotinylated probe protein (the stock concentration of biotinylated probe protein is 50 μg / mL, diluted 5 times in blocking buffer before use) to each well in the first row. Mix well and add 25 μL from each well in the first row to the second row. Mix well and add 25 μL to each well in the third row, and so on until the last row, discarding 25 μL (forming a five-fold dilution gradient between each row). Incubate at 37°C for 1 hour. Wash the plate 5 times with PBST (PBS containing 0.05% Tween 20) for 1 hour; dilute horseradish peroxidase-labeled streptavidin (Sigma KPL HRP-SA) 1000 times in blocking buffer containing 0.05% Tween 20, add 100 μL to each well, incubate at 37°C for 1 hour, wash the plate 5 times with PBST; add 100 μL of substrate to each well, incubate at room temperature for 20 minutes, then add 50 μL of sulfuric acid stop solution to each well to immediately stop the reaction, and record the reading on a microplate reader. Figure 1 shows the titer of biotinylated RBD protein. Lane A represents the biotinylation efficiency of RBD as determined by the magnetic bead method. Lanes 1-2 show the successful biotinylation of RBD protein by streptavidin magnetic beads after applying 10 μg of biotin. Lane 3 shows 10 μg of biotinylated RBD, and lane 4 is the molecular weight marker. Figure 1A shows that the biotinylation efficiency of RBD exceeds 50%. Figure 1B shows the biotinylation efficiency as determined by ELISA, with an end-titer probe concentration of 0.0032 μg / mL. The results indicate that the biotinylation of RBD was successful.

[0026] (6) Biotinylated probe protein fluorescently labeled RBD-Avi probe protein fluorescently labeled with PE (phycoerythrin) for use in single-cell flow cytometry sorting.

[0027] Example 2: Screening of humanized monoclonal antibodies against SARS-CoV-2 2.1 Preparation of cell lysis buffer: 20 μL of cell lysis buffer per well, containing 0.5 μL RNase-degrading enzyme, 5 μL 5× First Strand buffer, 1.25 μL 0.1M DTT, 0.0625 μL Igepal, and 13.25 μL water. Cover with sealing film and store at 4°C until use.

[0028] 2.2 Sample preparation: (1) Resuscitation of PBMCs from recovered patients after novel coronavirus infection: After removing the cryopreserved cell tubes from liquid nitrogen, quickly place them in a 37°C water bath. When the cells have thawed to the point of having an ice core, remove them from the water and open them in a biosafety cabinet. Slowly add R10 + Benzonase medium (5 mL R10 + Benzonase medium per cell line). Centrifuge at 1500 rpm for 10 minutes, discard the supernatant, resuspend the cells in the residual liquid, add 10 mL R10, mix well, and take 50 μL for cell counting. Centrifuge at 1500 rpm for 10 minutes. Adjust cell concentration: Discard the supernatant, resuspend the cells in the residual liquid, adjust the concentration using R10 medium, and add 2.5 × 10⁶ cells / well to a 96-well U-shaped plate. 6 One per hole; (2) Prepare a 2 mM EDTA / PBS solution, hereinafter referred to as E-PBS; (3) Place the 96-well cell plate in a centrifuge and centrifuge at 4 ℃ and 2000 rpm for 3 minutes. Discard the supernatant (operate in a biosafety cabinet and place the paper towel in the autoclave). (4) Add 50 μL of Vivid working solution to each well (Prepare Vivid (UV) working solution: dilute with PBS at a ratio of 1:1000 and mix well), mix well, and incubate on ice in the dark for 20 minutes. (5) Add 150 μL of E-PBS to each well and centrifuge at 4 ℃ and 2000 rpm for 3 minutes; (6) Discard the supernatant, add 50 μL of extracellular antibody mixture (2 μL Anti-CD3-Pacific Blue, 2 μL Anti-CD8-Pacific Blue, 1.5 μL Anti-CD14-Pacific Blue, 1 μL Anti-CD19-BV510, 2 μL Anti-CD20-ECD, 2.5 μL Anti-CD27-APC-cy7, 5 μL Anti-IgG-FITC, 2.5 μL Anti-IgM-PercpCy5.5, 2.5 μL Anti-PD-1-PECy7, 1 μL Anti-CXCR5-APC-R700, 5 μL Anti-CXCR3-PECy5, 1 μL Anti-CD45RA-BV650, 1 μL Anti-CD4-BV605, 5 μL Anti-RBD-PE, make up the difference with E-PBS if necessary), mix well, and incubate on ice in the dark for 60 minutes. (7) Add 150 μL of E-PBS to each well, centrifuge at 4 ℃ and 2000 rpm for 3 minutes, and discard the supernatant; (8) Add 200 μL of E-PBS to each well, centrifuge at 4 ℃ and 2000 rpm for 3 minutes, and discard the supernatant; (9) Add 200 μL of E-PBS to each well, mix well, filter the cell suspension of the same sample into the same branching tube, and store at 4°C in the dark for sorting.

[0029] (10) Single-staining tube control: Set up one unstained tube control (add 1 drop of compensating microspheres, 200 μL E-PBS) and 15 individually stained tubes (each tube added 1 drop of compensating microspheres, 2 μL Anti-CD3-PacificBlue, 1 μL Anti-CD19-BV510, 2 μL Anti-CD20-ECD, 2.5 μL Anti-CD27-APC-cy7, 5 μL Anti-IgG-FITC, 2.5 μL Anti-IgM-PercpCy5.5, 2.5 μL Anti-PD-1-PECy7, 1 μL Anti-CXCR5-APC-R700, 5 μL Anti-CXCR3-PECy5, 1 μL Anti-CD45RA-BV650, 1 μL Anti-CD4-BV605, 5 μL Anti-RBD-PE, 50 μL UV working solution), mix well, and incubate at room temperature in the dark for 20 minutes. After 1 minute, add 150 μL of E-PBS to each well, centrifuge at 2000 rpm for 3 minutes at 4 ℃, discard the supernatant, and resuspend in 200 μL of E-PBS.

[0030] 2.3 Single B cell flow cytometry sorting: CD3 selection - CD8 - CD14 - CD19 + CD20 + CD27 + IgG + IgM - RBD + The cells were sorted, and a total of 157 cells were sorted. First, single lymphocyte populations were identified, followed by CD3+ cell populations. - CD8 - CD14 - Live cells were used to exclude T cells and macrophages, and then CD19 cells were circled. + CD20 + B cells, then CD27 + Memory B cells, then circle IgG. + IgM - B cells were selected, and memory B cells that bound to the probe RBD were circled. One of these B cells was sorted into each well of a 96-well plate containing the following lysis buffer (Table 1). After sorting, the 96-well plates were immediately sealed with sealing film, solidified on dry ice, and then transferred to a -80°C freezer overnight. PCR was performed the next day. Results: The results of flow cytometry sorting are shown in Figure 2.

[0031] Table 1. B cell lysis buffer system

[0032] 2.4 The fully human antibody variable region gene was amplified using RT-PCR technology. The RT-PCR reaction system shown in Table 2 was prepared, with 6 μL added to each well for RT-PCR reaction.

[0033] Table 2. RT-PCR reaction system

[0034] RT-PCR reaction program settings: 42℃ for 10 min, 25℃ for 10 min, 50℃ for 50 min, 94℃ for 5 min, and store at 4℃.

[0035] (1) Two rounds of PCR reaction were performed, and the reaction system was as follows: First-round PCR reaction system (Table 3) Supplementary primer sequences (Table 4) Table 3. First-round PCR reaction system

[0036] Table 4 Primer sequences used for the first round of PCR amplification

[0037] The target fragments amplified by the primers used in the first round of PCR amplification are shown in Table 5.

[0038] Table 5. Amplification target fragments using primers for the first round of PCR amplification.

[0039] The procedure for the first round of PCR reaction is shown in Table 6.

[0040] Table 6. First Round PCR Procedure

[0041] After the first round of PCR reaction was completed, the second round of PCR reaction was carried out. The system for the second round of PCR reaction is shown in Table 7.

[0042] Table 7. Second round PCR reaction system

[0043] The primer sequences for the second round of PCR are shown in Table 8.

[0044] Table 8. Primer sequences for the second round of PCR

[0045] The target fragments amplified by the primers used in the second round of PCR amplification are shown in Table 9.

[0046] Table 9. Amplification target fragments using primers for the second round of PCR amplification

[0047] The procedure for the second round of PCR reaction is shown in Table 10.

[0048] Table 10. Second Round PCR Procedure

[0049] (2) Electrophoresis, sequencing, and family analysis The products from the second round of PCR were detected by electrophoresis, and the heavy and light chain products were directly sequenced. The sequencing results were analyzed using the antibody family gene database (http: / / www.imgt.org / IMGT_vquest / vquest), and antibody variable region sequences with restriction enzyme sites (Heavy chain: 5'-Age I, 3'-Sal I; Kappa chain: 5'-Age I, 3'-BsiWI; Lambda chain: 5'-Age I, 3'-Xho I) were designed and synthesized to obtain paired clones of heavy and light chains.

[0050] 2.5 Construction of monoclonal antibody expression vector and plasmid transformation The synthesized gene was digested with the corresponding enzymes and recovered by gel electrophoresis. The variable region gene was then ligated with the corresponding vectors IgH (heavy chain expression vector), Igκ (κ light chain expression vector), and Igλ (λ light chain expression vector) using T4 DNA ligase (see Tiller et al. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning, J Immunol Methods. 2008 January 1; 329(1-2): 112–124. This prior art document is incorporated herein by reference). The mixture was incubated overnight at 16°C in a ligation apparatus and transformed. 10 μL of the ligation product was added to 50 μL of DH5α competent cells, shaken well, incubated on ice for 30 min, and then heat-shocked at 42°C for 45 s. After placing the centrifuge tubes on ice for 2 minutes, add 1 mL of non-resistant LB medium, incubate at 37°C with shaking at 200 rpm for 1 hour, centrifuge at 4000 rpm for 4 minutes, and spread the remaining bacterial culture onto resistant LB plates. Incubate upside down at 37°C for 14-16 hours. Pick single colonies and inoculate them into resistant LB liquid medium, incubate at 37°C with shaking at 200 rpm for 14-16 hours, and then perform plasmid extraction (Omega Plasmid Midi Kit).

[0051] 2.6 Antibody Expression and Purification The concentration of 293F cells was adjusted to 1.2 × 10⁻⁶. 6Cells were cultured at 133 antibodies / mL for 2 hours. Solution A: 500 μg of antibody heavy chain DNA and 500 μg of antibody light chain DNA were added to 25 mL of opti-MEM. Solution B: 5 mL of PEI transfection reagent was added to 25 mL of opti-MEM and incubated for 5 minutes. Solutions A and B were mixed and incubated for 20 minutes. The mixture was then added dropwise to 1 L of 293F cells while shaking. The cells were incubated at 37°C with shaking for 5 days using 8% CO2. The antibody was purified using a Protein A affinity column (GE Health). The antibody concentration was measured using a NanoDrop2000 micro-volume spectrophotometer (Thermo). The cells were incubated at 4°C until detection. This step resulted in the expression of 133 antibodies in the light / heavy chain pairing (antibody amount > 10 μg, antibody concentration > 10 μg / mL).

[0052] Example 3 Identification of humanized monoclonal antibodies against SARS-CoV-2 3.1 Detection of antigen-antibody binding ability The binding capacity of SARS-CoV-2 monoclonal antigens was determined by ELISA. RBD proteins from the Prototype and Omicron B.1.1.529 variants were diluted to 0.5 μg / mL with PBS, and 100 μL was added to each well of a 96-well ELISA plate (Corning Costar), incubated overnight at 4°C. The plates were washed 5 times with PBS-T solution (0.05% Tween-20); 300 μL of blocking buffer (PBS, 2% BSA + 5% skim milk powder) was added to each well, and the plates were blocked at 37°C for 2 hours. The plates were washed 5 times with PBS-T. Antibodies were serially diluted 5-fold with blocking buffer at a starting concentration of 10 μg / mL, and 100 μL of each sample was added to each ELISA plate, incubated at 37°C for 1 hour. The plates were washed 5 times with PBS-T. Add 100 μL of horseradish enzyme-labeled goat anti-human IgG (H+L) (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) diluted 1:5000 with blocking buffer to each well, and incubate at 37°C for 1 hour. Wash the plate 5 times with PBS-T. Add 100 μL of TMB chromogenic solution (Beijing Jinhao Pharmaceutical Co., Ltd.), and incubate at room temperature in the dark for 15 minutes. Add 50 μL of stop solution (Beijing Jinhao Pharmaceutical Co., Ltd.) to each well to stop the reaction, and read the absorbance (OD) values ​​at dual wavelengths of 450nm-630nm using a microplate reader.

[0053] From 133 tested antibodies, we screened out three monoclonal antibodies with strong binding ability against the SARS-CoV-2 prototype, OmicronB.1.1.529, BF.7, and XBB.1.5 variants. These three antibodies are designated LY-F6HK, LQL-B6HL, and LQL-D6HL, respectively. The binding ability of the three antibodies against different variants is shown in Table 11 and Figure 3. In Figure 3, VRC01 is an anti-HIV neutralizing antibody, serving as a negative control.

[0054] Table 11. End-titer values ​​of neutralizing antibodies

[0055] 3.2 Antigen-antibody affinity kinetics determination The affinity of LY-F6HK, LQL-D6HL for RBD proteins of Prototype and Omicron B.1.1.529 mutant strains was detected using the Octet Red 96 system (Fortebio, USA) and a streptavidin sensor via BLI technology. LY-F6HK, LQL-B6HL and LQL-D6HL were diluted to a concentration of 2 μg / mL with buffer (PBS solution containing 0.1% BSA + 0.02% Tween 20) and then immobilized on a protein sensor (Sartorius AG, Germany) for 120 seconds. After a 120-second washing step with PBST, the biosensor probes were immersed in wells containing serially diluted RBDs (25 nM, 12.5 nM, 6.25 nM, 3.13 nM, and 1.56 nM) and bound for 120 seconds, followed by a 300-second dissociation step. KD values ​​were calculated using a 1:1 binding model in data analysis software 9.0 (see Table 12 below), and the results are shown in Figure 4. This indicates that these three antibodies have extremely strong affinity for RBDs.

[0056] Table 12. Affinity values ​​of antigen-antibody

[0057] 3.3 Detection of neutralizing activity of antibodies against SARS-CoV-2 pseudovirus (1) Packaging of pseudoviruses: The full-length S protein gene of artificially synthesized SARS-CoV-2 (GenBank: MN908947) was inserted into the pcDNA3.1 expression plasmid to construct pcDNA-SARS-CoV2-S. The mutation sites of the pseudoviruses with mutations are shown in Table 13 and were introduced into the S gene.

[0058] Table 13. Pseudovirus mutation sites

[0059] 3×10 6 3 million 293T cells were seeded in T75 cell culture flasks and cultured at 37°C with 5% CO2 for 20-24 hours. Transfection was performed using Fugene 6 Transfection Reagent (Promega, Cat#E2691): 30 μg of plasmid pcDNA-SARS-CoV-2-S was transfected into 293T cells, and 1.05 × 10⁻⁶ cells were added simultaneously. 6 293T cells were infected with G*ΔG-VSV virus with TCID50, and the culture medium was changed after 8 hours. 24 hours after transfection, the culture supernatant was collected and filtered to obtain pseudoviruses containing SARS-CoV-2 S protein, which were then frozen and stored at -80°C.

[0060] (2) Neutralization experiment of antibodies against pseudoviruses: Add 100 μL of serially diluted antibody buffer to each well of a 96-well plate. Then, dilute the pseudovirus to 1.3 × 10⁻⁶ using DMEM complete medium. 4 Add 50 μL of TCID50 / mL to each well in columns 3-11, making the pseudovirus concentration 650 TCID50 / well. Incubate the 96-well plate in a cell culture incubator (37°C, 5% CO2) for 1 hour. After half an hour of incubation, remove the prepared Vero cells, discard the culture medium, and wash the cells with PBS buffer; discard the PBS, add trypsin-EDTA for digestion, centrifuge, and resuspend the cells in complete culture medium. Count the cells. Dilute the cell suspension to 2 × 10⁻⁶. 5 Cells / mL. Incubate for 1 hour, then add 100 μL of cells to each well of a 96-well plate, making the cell density 2 × 10⁶ cells per well. 4Gently shake the 96-well plate back and forth and side to side to disperse the cells evenly in the wells. Place the 96-well plate in a cell culture incubator and incubate at 37°C and 5% CO2 for 48 hours. Remove the 96-well plate from the cell culture incubator and use a multichannel pipette to aspirate 150 μL of supernatant from each sample well. Then add 100 μL of luciferase assay reagent and react at room temperature in the dark for 2 minutes. After the reaction, mix well by shaking on a plate shaker and read the luminescence value using a multi-functional plate reader. Calculate the neutralization inhibition rate: Calculate the IC50 of the antibody based on the neutralization inhibition rate results.

[0061]

[0062] The neutralizing ability of the three antibodies, LY-F6HK, LQL-B6HL and LQL-D6HL, against different variant pseudoviruses is shown in Table 14.

[0063] Table 14. Neutralizing ability of antibodies against different variant pseudoviruses (IC50, μg / mL)

[0064] 3.4 Detection of neutralizing activity of antibodies against live SARS-CoV-2 virus One day in advance, Vero-E6 cells were cultured at a rate of 2.5 × 10⁻⁶. 4 Cells were seeded at a rate of [number] cells / well in 48-well plates and cultured in MEM medium containing 10% FBS and penicillin antibody. Antibody preparation: The antibody was diluted in MEM medium containing 5% FBS and penicillin antibody. Based on a final plate volume of 500 μL / well and 3 replicates per concentration, 750 μL (250 μL / well) of antibody was prepared for each concentration, prepared at twice the effective concentration. Serial dilutions were performed starting from the highest concentration, at multiples (e.g., 3-fold). Positive and negative controls were also included. The prepared antibody was added to the 48-well plates and transferred to a P3 laboratory. The novel coronavirus stock solution was diluted in MEM medium containing 5% FBS, and the virus solution (250 μL / well) was added to the prepared antibody at a 1:1 volume ratio. The plates were incubated at 37°C for 3 hours. The Vero-E6 cells in logarithmic growth phase were aspirated, and the incubated antibody-virus mixture (500 μL / well) was added. The plates were incubated at 37°C for 7 days. Subsequent testing: Observe cytopathic effects, estimate the concentration of neutralizing antibodies with a 50% neutralization percentage, and calculate it as IC50. 50The results are shown in Table 15. All three monoclonal antibodies effectively neutralized the live viruses of the Prototype, Omicron B.1.1.529, and BA.5 variants. LY-F6HK was found to have the strongest neutralizing ability against the Prototype and Omicron BA.5 strains, with IC50 values ​​of 0.020 μg / mL and 0.081 μg / mL, respectively.

[0065] Table 15. Neutralizing ability of antibodies against live virus (IC50, μg / mL)

[0066] The amino acid and nucleotide sequences of the three antibodies were obtained by sequencing the clones encoding the above-mentioned antibody strains as follows: The nucleotide sequence of the heavy chain variable region of antibody LY-F6HK is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1. Specifically, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown as amino acids 26-33, 51-58, and 97-115 of SEQ ID NO.1, respectively. The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.3. Specifically, the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown as amino acids 27-32, 50-52, and 89-97 of SEQ ID NO.3, respectively.

[0067] The nucleotide sequence of the heavy chain variable region of antibody LQL-B6HL is shown in SEQ ID NO.6, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.5. Specifically, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown as amino acids 26-33, 51-57, and 96-111 in SEQ ID NO.5, respectively. The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7. Specifically, the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown as amino acids 26-34, 52-54, and 91-102 in SEQ ID NO.7, respectively.

[0068] The nucleotide sequence of the heavy chain variable region of antibody LQL-D6HL is shown in SEQ ID NO.10, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.9. Specifically, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown at positions 26-33, 51-58, and 97-115 of SEQ ID NO.9, respectively. The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.11. Specifically, the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown at positions 26-31, 49-51, and 88-97 of SEQ ID NO.11, respectively.

[0069] The polynucleotide sequence of the heavy chain constant region of the above-mentioned antibody is shown in SEQ ID NO. 14, and the polynucleotide sequence of the light chain constant region is shown in SEQ ID NO. 16 (kappa chain) or SEQ ID NO. 18 (lambda chain). The amino acid sequence of the antibody heavy chain constant region is shown in SEQ ID NO. 13, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO. 15 or SEQ ID NO. 17. In a specific embodiment of the present invention, the light chain constant region of antibody LY-F6HK adopts a Kappa chain; the light chain constant regions of antibodies LQL-B6HL and LQL-D6HL both adopt a Lambda chain.

[0070] Example 4: Determination of anti-SARS-CoV-2 humanized monoclonal antibody combination 4.1 Epitope Pairing Detection of Monoclonal Antibody Combinations Epitopes of a combination of two monoclonal antibodies were detected using the Octet Red 96 system (Fortebio, USA) and a streptavidin sensor via the Octetin-tandem method.

[0071] (1) Dilute the biotinylated RBD to 1 µg / mL as a fixative; (2) Dilute the human monoclonal antibody with buffer (PBS solution containing 0.1% BSA + 0.02% Tween 20) to a concentration of 30 µg / mL; (3) Select the SA biosensor probe and pre-wet it with buffer solution; (4) Set the molecular interaction program as follows: baseline 30 s, loading 120 s, baseline 30 s, 1st antibody 180 s, baseline 30 s, 2nd antibody 180 s, regeneration 30 s; (5) The 96-well plate containing the sample is then tested on the instrument; (6) After the detection is completed, ForteBio Data Analysis 9.0 software is used for analysis to obtain the antibody reaction signal.

[0072] Pairwise combinations of the monoclonal antibodies LY-F6HK, LQL-B6HL, and LQL-D6HL were performed. The results showed that the reaction signals of LY-F6HK, LQL-B6HL, and LQL-D6HL were all greater than 70%, indicating that the binding epitope of LY-F6HK is different from that of LQL-B6HL and LQL-D6HL, and there is no competition between the antibodies. However, the reaction signal of LQL-B6HL and LQL-D6HL was 7%, indicating that there is epitope competition between the two antibodies (Table 16).

[0073] Table 16. Detection values ​​of monoclonal antibody competitive epitopes

[0074] 4.2 Detection of neutralizing activity of combined antibodies against SARS-CoV-2 pseudovirus Three monoclonal antibodies (LY-F6HK, LQL-B6HL, and LQL-D6HL) were combined in pairs in equal proportions. The neutralizing ability of these combined antibodies against SARS-CoV-2 Prototype, Omicron B.1.1.529, Omicron BF.7, and Omicron XBB.1.5 variants was tested using a pseudovirus neutralization method (details in Section 3.3). The results are shown in Table 17. The combined antibodies LY-F6HK + LQL-D6HL and LQL-D6HL + LQL-B6HL showed strong neutralizing ability against Prototype and Omicron variants. Furthermore, epitope detection revealed no competing epitopes between the LY-F6HK and LQL-D6HL antibodies, further indicating that the antibody combination of LY-F6HK and LQL-D6HL could serve as a candidate neutralizing antibody for the prevention and treatment of SARS-CoV-2.

[0075] Table 17. Neutralizing ability of antibodies against different variant pseudoviruses (IC50, μg / mL) .

Claims

1. A humanized, highly neutralizing, and active monoclonal antibody against the novel coronavirus, characterized in that, The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region and CDR1, CDR2, and CDR3 in the light chain variable region of the monoclonal antibody are shown below: (1) Amino acids 26-33, 51-58, and 97-115 of SEQ ID NO.1 and amino acids 27-32, 50-52, and 89-97 of SEQ ID NO.3; or (2) Amino acids 26-33, 51-57 and 96-111 of SEQ ID NO.5 and amino acids 26-34, 52-54 and 91-102 of SEQ ID NO.7; or (3) Amino acids 26-33, 51-58 and 97-115 of SEQ ID NO.9 and amino acids 26-31, 49-51 and 88-97 of SEQ ID NO.

11.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown below: (1) SEQ ID NO.1 and SEQ ID NO.3; or (2) SEQ ID NO.5 and SEQ ID NO.7; or (3) SEQ ID NO.9 and SEQ ID NO.

11.

3. The monoclonal antibody according to claim 2, characterized in that, The amino acid sequence of the heavy chain constant region of the monoclonal antibody is shown in SEQ ID NO.13, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.15 or SEQ ID NO.

17.

4. A polynucleotide encoding the humanized, highly neutralizing, and active monoclonal antibody against the novel coronavirus as described in claim 2 or 3, characterized in that, The polynucleotides encoding the heavy chain variable region and the polynucleotides encoding the light chain variable region of the antibody are shown below: (1) SEQ ID NO.2 and SEQ ID NO.4; or (2) SEQ ID NO.6 and SEQ ID NO.8; or (3) SEQ ID NO.10 and SEQ ID NO.

12.

5. The polynucleotide according to claim 4, characterized in that, The polynucleotide sequence encoding the heavy chain constant region of the antibody is shown in SEQ ID NO.14, and the polynucleotide sequence encoding the light chain constant region of the antibody is shown in SEQ ID NO.16 or SEQ ID NO.

18.

6. A vector expressing the humanized, highly neutralizing, anti-novel coronavirus monoclonal antibody as described in claim 2 or 3, characterized in that, The vector contains the polynucleotide encoding the heavy chain variable region of the antibody as described in claim 4 and the polynucleotide encoding the light chain variable region of the antibody.

7. A host cell expressing the humanized, highly neutralizing, and active anti-novel coronavirus monoclonal antibody as described in claim 2 or 3, characterized in that, The host cell contains the vector as described in claim 6.

8. The use of the humanized, highly neutralizing, and active anti-novel coronavirus monoclonal antibody according to any one of claims 1-3 in the preparation of medicaments for the treatment and / or prevention of novel coronavirus disease.

9. The use of the humanized, highly neutralizing, and active anti-novel coronavirus monoclonal antibody according to any one of claims 1-3 in the preparation of a novel coronavirus detection reagent.

10. An antibody composition comprising a first antibody and a second antibody, characterized in that, The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region and CDR1, CDR2, and CDR3 in the light chain variable region of the first antibody are as shown in amino acids 26-33, 51-58, and 97-115 of SEQ ID NO.1 and amino acids 27-32, 50-52, and 89-97 of SEQ ID NO.3, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region and CDR1, CDR2, and CDR3 in the light chain variable region of the second antibody are as shown in amino acids 26-33, 51-57, and 96-111 of SEQ ID NO.5 and amino acids 26-34, 52-54, and 91-102 of SEQ ID NO.7, respectively. Alternatively, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region and CDR1, CDR2, and CDR3 in the light chain variable region of the second antibody are as shown in SEQ ID NO.

1. The amino acids at positions 26-33, 51-58, and 97-115 of SEQ ID NO. 9 and positions 26-31, 49-51, and 88-97 of SEQ ID NO. 11 are shown.

11. The antibody composition according to claim 10, characterized in that, The amino acid sequences of the heavy chain variable region and the light chain variable region of the first antibody are shown in SEQ ID NO.1 and SEQ ID NO.3, respectively; and the amino acid sequences of the heavy chain variable region and the light chain variable region of the second antibody are shown in SEQ ID NO.5 and SEQ ID NO.7, respectively; or, as shown in SEQ ID NO.9 and SEQ ID NO.

11.

12. The use of the antibody composition of claim 10 or 11 in the preparation of medicaments for the treatment and / or prevention of novel coronavirus disease.

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