Novel coronavirus neutralizing antibody variant and application thereof

Abstract

The invention discloses a novel coronavirus neutralizing antibody variant and application thereof, the S309-25 variant, the S309-33 variant, the S309-35 variant and the S309-39 variant provided by the invention can inhibit or neutralize the activity of SARS-CoV-2 by specifically binding to an SARS-CoV-2 spike protein RBD region, so that the SARS-CoV-2 loses the ability of binding to a receptor ACE2, and compared with a wild type antibody, the neutralizing effects are respectively improved by 42.01%, 48.38%, 39.56% and 38.52%. Particularly, the variant disclosed by the invention has an obvious inhibiting effect on an Omicron mutant strain, has relatively strong inhibiting activity and neutralizing activity on SARS-CoV-2 pseudoviruses, has the capability of inhibiting infection of various mutant strain SARS-CoV-2 pseudoviruses, is a neutralizing antibody with a broad-spectrum effect, and is good in broad-spectrum property and neutralizing activity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to a novel coronavirus neutralizing antibody variant and its application. Background Art

[0002] SARS-CoV-2 infection is caused by the binding of the receptor binding domain (RBD) of the viral spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor. Therefore, mutations in the SARS-CoV-2 spike protein primarily affect the virus's pathogenicity and transmissibility by altering the affinity of the RBD for the ACE2 receptor. As an RNA virus, SARS-CoV-2 mutates extremely rapidly.

[0003] Among them, the Omicron BA.2.86 subvariants (JN.1, KP.2, and KP.3) have become the main variants worldwide. These variants carry more receptor binding domain (RBD) mutations than the XBB and EG.5 sublineages, which are known to impair vaccine efficacy. Qian Wu et al. (https: / / doi.org / 10.1016 / j.antiviral.2025.106092) evaluated the sera of individuals who received inactivated vaccines (with or without breakthrough infection) and those who recovered from COVID-19. The results showed that the serum neutralizing activity of JN.1, KP.2, XBB.1.5, and EG.5.1 variants was significantly reduced compared with BA.2.

[0004] Faced with the challenges of these newly discovered mutant virus strains and mutant virus strains that may become prevalent in the future and evade monoclonal antibody treatment, it is particularly important to quickly screen and design detection antibodies.

[0005] In the prior art, patent application WO2022136685A1 relates to various anti-infective drug compositions based on the receptor binding domain (RBD) of a coronavirus vaccine. The invention also relates to a composite fusion antiretroviral composition comprising the S309 anti-severe acute respiratory syndrome coronavirus 2 RBD (based on VIR-7831 / sotroviamab) and an ISVD based on VHH72, as well as its use in the treatment of coronavirus infections. However, the design of more detection antibodies is still needed. Summary of the Invention

[0006] Based on the deficiencies in the prior art, the present invention provides a novel coronavirus neutralizing antibody variant and its application that can effectively inhibit the activity of multiple novel coronavirus SARS-CoV-2 mutant strains, with better neutralizing effect.

[0007] In one aspect, the present invention provides a novel coronavirus neutralizing antibody variant, which is obtained by mutating the wild-type S309 antibody, wherein the wild-type antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chain CDR3 region, and the light chain variable region comprises a light chain CDR1 region, a light chain CDR2 region, and a light chain CDR3 region;

[0008] The amino acid sequence of the heavy chain CDR1 region is GYPFTSY (amino acids 26-32 of SEQ ID NO: 15), the amino acid sequence of the light chain CDR1 region is RASQTVSSTSLA (amino acids 24-35 of SEQ ID NO: 16), and the amino acid sequence of the light chain CDR2 region is GASSRAT (amino acids 51-57 of SEQ ID NO: 16);

[0009] The neutralizing antibody variant comprises any one of the following mutations:

[0010] (1) Serine 53 in the light chain CDR2 region was mutated to tryptophan;

[0011] (2) proline 28 in the heavy chain CDR1 region was mutated to tryptophan, and serine 33 in the light chain CDR1 region was mutated to glutamic acid;

[0012] (3) Threonine 57 in the light chain CDR2 region was mutated to arginine;

[0013] (4) Proline 28 in the heavy chain CDR1 region was mutated to tryptophan, serine 53 in the light chain CDR2 region was mutated to tryptophan, and threonine 57 was mutated to arginine.

[0014] Furthermore, the amino acid sequence of the heavy chain CDR2 region is STYNGN (amino acid sequence at positions 52-57 shown in SEQ ID NO: 15), the amino acid sequence of the heavy chain CDR3 region is DYTRGAWFGESLIGGFDN (amino acid sequence at positions 99-116 shown in SEQ ID NO: 15), and the amino acid sequence of the light chain CDR3 region is QQHDTSLT (amino acid sequence at positions 90-97 shown in SEQ ID NO: 16).

[0015] The amino acid sequence of the heavy chain variable region of the wild-type S309 antibody is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.

[0016] The amino acid sequence of the heavy chain constant region of the wild-type S309 antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 18.

[0017] The present invention also provides a biomaterial related to the neutralizing antibody variant, wherein the biomaterial is any one of the following:

[0018] (1) a nucleic acid molecule encoding the neutralizing antibody variant;

[0019] (2) an expression cassette containing the nucleic acid molecule described in (1);

[0020] (3) a recombinant vector containing the nucleic acid molecule described in (1), or a recombinant vector containing the expression cassette described in (2);

[0021] (4) a recombinant microorganism containing the nucleic acid molecule described in (1), or a recombinant microorganism containing the expression cassette described in (2), or a recombinant microorganism containing the recombinant vector described in (3);

[0022] (5) A cell line containing the nucleic acid molecule described in (1), or a cell line containing the expression cassette described in (2), or a cell line containing the recombinant vector described in (3).

[0023] Wherein, the recombinant microorganism described in (4) and the cell line described in (5) can express the neutralizing antibody variant.

[0024] The vectors described herein are well known to those skilled in the art, and include, but are not limited to, plasmids, phages (such as lambda phage or M13 filamentous phage), viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, or herpes viruses (such as herpes simplex virus). In one embodiment of the present invention, the vector may specifically be pTT5.

[0025] The microorganism described herein can be yeast, bacteria, algae or fungi. In one embodiment of the present invention, the microorganism can specifically be Escherichia coli DH5α.

[0026] The cells (host cells) refer to cells that can be used to introduce vectors, including but not limited to eukaryotic cells (such as yeast cells, Aspergillus cells), animal cells (such as mammalian cells, insect cells), plant cells or prokaryotic cells.

[0027] The present invention also provides a pharmaceutical composition comprising the neutralizing antibody variant and a pharmaceutically acceptable carrier.

[0028] The pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant.

[0029] The pharmaceutical composition has a neutralizing antiviral effect that inhibits or neutralizes the activity of SARS-CoV-2. The pharmaceutical composition is used to improve, prevent or treat diseases caused by SARS-CoV-2 infection and / or to inhibit SARS-CoV-2 infection.

[0030] Furthermore, the pharmaceutical composition of the present invention comprises a first antibody and a second antibody or an antigen-binding fragment thereof, wherein the first antibody is a neutralizing antibody variant of the present invention, and the second antibody is any antibody or an antigen-binding fragment thereof that neutralizes SARS-CoV-2 virus infection.

[0031] The present invention also provides the use of the neutralizing antibody variant and / or the biomaterial in the preparation of a drug for inhibiting or neutralizing the activity of SARS-CoV-2.

[0032] The inhibition or neutralization of SARS-CoV-2 activity includes specifically binding to the RBD region of the SARS-CoV-2 spike protein (S protein), thereby causing SARS-CoV-2 to lose the ability to bind to the receptor ACE2, thereby achieving an antibody blocking effect.

[0033] In the above applications, the drug that inhibits or neutralizes the activity of SARS-CoV-2 is used to improve, prevent or treat diseases caused by SARS-CoV-2 infection and / or to inhibit SARS-CoV-2 infection.

[0034] In the above application, the disease caused by the SARS-CoV-2 infection is a respiratory system infection. The respiratory system infection can be a respiratory tract infection and / or a lung infection.

[0035] The neutralizing antibody variants herein may be referred to as "neutralizing antibodies," which refer to antibodies that can neutralize, ie, prevent, inhibit, reduce, hinder or interfere with the ability of a pathogen to initiate and / or maintain infection in a host.

[0036] Preferably, the neutralizing antibodies of the present invention are capable of neutralizing pseudoviruses of multiple mutant strains of SARS-CoV-2, exemplary SARS-CoV-2 mutant strains include but are not limited to Delta, Omicron or JN.1 variants.

[0037] Beneficial effects of the present invention:

[0038] The S309-25 (HCDR1-P28W & LCDR1-S33E), S309-33 (LCDR2-S53W), S309-35 (LCDR2-T57R) and S309-39 (HCDR1-P28W & LCDR2-S53W-T57R) variants provided by the present invention can inhibit or neutralize the activity of SARS-CoV-2 by specifically binding to the RBD region of the SARS-CoV-2 spike protein, causing SARS-CoV-2 to lose the ability to bind to the receptor ACE2. Compared with the wild-type antibody, the neutralization effect is increased by 42.01%, 48.38%, 39.56% and 38.52%, respectively.

[0039] At the same time, the variants of the present invention have a significant inhibitory effect on the main prevalent strain of SARS-CoV-2 (Omicron mutant), have strong inhibitory and neutralizing activity against SARS-CoV-2 pseudoviruses, and have the ability to inhibit infection by multiple mutant SARS-CoV-2 pseudoviruses. They are neutralizing antibodies with broad spectrum effects and good neutralizing activity. The present invention has very broad prospects and important significance in the fields of pharmaceutical applications and so on. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 SDS-PAGE purity verification of the new coronavirus WT, Delta, Omicron, and JN.1RBD.

[0041] Figure 2 The purity of 6 antibodies was verified by SDS-PAGE.

[0042] Figure 3 ELISA was used to verify the binding ability of antibodies to WT, Delta, Omicron, and JN.1RBD; among them, AH are LY-CoV016, AZD8895, LY-CoV555, REGN10987, S309, CR3022, TB1, and TB2 antibodies, respectively.

[0043] Figure 4 This is the crystal structure of the 7TLY antigen-antibody complex.

[0044] Figure 5 Molecular docking of the Pose 3 antigen-antibody complex structure.

[0045] Figure 6 This is a virtual screening diagram from the crystal structure of the 7TLY antigen-antibody complex.

[0046] Figure 7 This is a virtual screening diagram from the Pose 3 antigen-antibody complex structure.

[0047] Figure 8 Agarose gel electrophoresis diagram for S309-11-14 vector construction.

[0048] Figure 9 Agarose gel electrophoresis diagram for S309-15-17 vector construction.

[0049] Figure 10 Agarose gel electrophoresis diagram for the S309-31-35 vector construction.

[0050] Figure 11 This is the agarose gel electrophoresis diagram of EcoRI and BamHI double enzyme digestion of pTT5.

[0051] Figure 12 Figure 3 is the SDS-PAGE purity test graph of S309 variants; A is the S309 variant selected based on the free energy ranking from 7TLY virtual screening; B is the S309 variant selected based on the free energy ranking from Pose 3 virtual screening; C is the S309 variant with combined site mutations.

[0052] Figure 13 The affinity of a single point mutation detected by ELISA (S309-11-16) is shown in Figure 1. A represents the affinity of a single point mutation detected by ELISA for Omicron; and B represents the affinity of a single point mutation detected by ELISA for JN.1.

[0053] Figure 14 The affinity of the single point mutation (S309-31-35) was detected by ELISA; wherein, A is the affinity of the single point mutation to Omicron detected by ELISA; B is the affinity of the single point mutation to JN.1 detected by ELISA.

[0054] Figure 15 Affinity of combined mutations detected by ELISA; wherein, A is affinity of combined mutations detected by ELISA to Omicron; B is affinity of combined mutations detected by ELISA to JN.1.

[0055] Figure 16 Affinity of combined mutations detected by ELISA; wherein, A is affinity of combined mutations detected by ELISA to Omicron; B is affinity of combined mutations detected by ELISA to JN.1.

[0056] Figure 17 ELISA was used to detect the affinity of the combined mutations to WT (A) and Delta (B).

[0057] Figure 18 The affinity of the variants for Omicron was tested by SPR.

[0058] Figure 19SPR detects the affinity of variants to JN.1; among them, AD represents the affinity of S309 antibody, HCDR1-P28W, HCDR1-P28W&LCDR1-S33E, and HCDR1-P28W&LCDR2-S53W-T57R variants to JN.1, respectively.

[0059] Figure 20 ELISA detection of antigen detection of HRP-labeled variants at different binding times; A is the antigen detection result after incubation for 5 minutes; B is the antigen detection result after incubation for 30 minutes.

[0060] Figure 21 Flow cytometry was used to detect the blocking effect of S309 and its variants on Omicron. DETAILED DESCRIPTION

[0061] Example 1: Antigen plasmid construction and expression

[0062] The four genes of the new coronavirus WT, Delta, Omicron and JN.1RBD (the corresponding amino acid sequences are shown in SEQ ID No. 1-4, respectively) were synthesized by Qingke Biotechnology and constructed on the pTT5 expression vector with a 10×His tag.

[0063] The day before transfection, 2.0 × 10 6 293 cells were inoculated at a density of 10 cells / mL, and the cell density on the second day of culture could reach 4.0×10 6 About cells / mL;

[0064] After cell counting on the second day of culture, the cell viability was >95% and the viable cell density was >4.0×10 6 cells / mL, can be used directly; if the cell density is lower than 4.0×10 6 cells / mL, the cells were collected by centrifugation (1000 rpm, 5 min) and the cells were plated at 4.0×10 6 Resuspend in 293CD01 culture medium at a density of cells / mL;

[0065] Prepare two 15 mL sterile centrifuge tubes, add 5 mL of 293CD01 culture medium and 100 μg of antigen expression vector to one of them, and gently pipette to mix;

[0066] Take another centrifuge tube, add 5 mL of 293 CD01 culture medium and 300 μL of PEI transfection solution (1 mg / mL), and mix gently by pipetting;

[0067] Transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid and mix gently by pipetting;

[0068] The plasmid-vector complex was prepared by standing at room temperature for 10-15 minutes;

[0069] adding the plasmid-vector complex to the cell culture medium and culturing;

[0070] After culturing for 24 h, adding 10 mL of feed medium 293F01 can further increase the viable cell density and protein expression;

[0071] After 72 h of culture, adding 1 mL of feed medium 293F03 can maintain cell viability and increase protein expression;

[0072] After culturing for 5-7 days, if the cell viability is lower than 60%, the culture is terminated.

[0073] After the supernatant was purified by nickel column, the purity was verified by SDS-PAGE. Figure 1 The antigen bands shown were consistent in size and had high purity.

[0074] Example 2: Antibody plasmid construction and expression

[0075] At Qingke Bio, we synthesized the heavy and light chain expression vectors of eight antibodies, including AZD8895 (PDBID: 7L7D), CR3022 (PDBID: 7JN5), LY-CoV016 (PDBID: 7C01), Ly-CoV555 (PDBID: 7KMG), REGN10987 (PDBID: 9LYP), S309 (PDB ID: 6WPT), TB1, and TB2 (the heavy chain sequences are shown in SEQ ID NOs: 5, 7, 9, 11, 13, 15, 19, and 21, respectively; the light chain sequences are shown in SEQ ID NOs: 6, 8, 10, 12, 14, 16, 20, and 22, respectively; the light chain is fused to human kappa (SEQ ID NO: 17), and the heavy chain is fused to human IgG1 (SEQ ID NO: 18)).

[0076] Antibody transfection was performed with reference to Example 1 (50 μg of light chain expression vector and 50 μg of heavy chain expression vector).

[0077] The supernatant was purified by Protein A column, and SDS-PAGE showed that the purity was high ( Figure 2 ).

[0078] Example 3: Selection of antibodies

[0079] Add 100 μL / well of 0.25 μg / mL WT, Delta, Omicron or JN.1RBD-His to a 96-well plate and coat overnight at 4 degrees Celsius. Discard the supernatant, add 200 μL / well of 0.05% PBST, wash 3 times, add 200 μL / well of 5% MPBST, and incubate at 37 degrees Celsius for 2 hours. Wash 3 times, add 100 μL / well of gradient diluted antibodies, and incubate at 37 degrees Celsius for 1 hour. Wash 3 times, add 100 μL / well of 5000-fold diluted horseradish peroxidase-labeled goat anti-human IgG (H+L) (Shanghai Biyuntian Biotechnology Co., Ltd., A0201), and incubate at 37 degrees Celsius for 1 hour. Wash 3 times, add 100 μL / well of TMB color development solution, and develop at room temperature for 5-30 minutes. Add 50 μL / well of stop solution, and detect OD450 ( Figure 3 ), the results showed that LY-CoV016, AZD8895, and LY-CoV555 only bound to WT and Delta, lost the ability to bind to Omicron and JN.1, while S309 and TB1 could still bind to Omicron and JN.1.

[0080] Example 4: Obtaining the crystal structure of the antigen-antibody complex

[0081] We obtained the crystal structures of S309 and Omicron 7TLY from the PDB database ( Figure 4 ).

[0082] However, due to the lack of crystal structures of S309 and JN.1, we first extracted the antibody variable region structure and RBD structure from the crystal structure 6WS6 of S309 and the crystal structure 8X4H of SARS-CoV-2JN.1Spike JN.1, respectively. ZDOCK was then used to rigidly dock the crystal structure 6WS6 of S309 and the crystal structure 8X4H of JN.1, and 54,000 conformations were obtained. 171 conformations were obtained by filtering by setting the known interaction site information of the antigen and antibody. Cluster analysis of these Poses showed that there were 21 clusters, and then RDOCK analysis was performed, and finally Pose 3 ranked first was obtained (Table 1). After comparison, it has a more similar structure compared with the known crystal structure 6WPT of S309 and Wild Type and the crystal structure 7TLY of S309 and Omicron ( Figure 5 ).

[0083] Table 1 Molecular docking of S309 and JN.1 RBD

[0084]

[0085] Example 5: Virtual Screening

[0086] The crystal structure 7TLY of S309 and Omicron and the complex structure Pose 3 of S309 and JN.1 were imported. The proteins were assigned CHARMm force field. Omicron or JN.1 was used as ligand. The amino acid residues within 5 angstroms around the ligand were selected and named as mutation groups. The amino acids in the mutation groups were mutated into 20 amino acids respectively. The free energy after mutation was calculated ( Figure 6 、 Figure 7 ).

[0087] Example 6: Variant plasmid construction and expression

[0088] HCDR1-P28W (S309-11), HCDR1-S31F (S309-12), HCDR3-S109E (S309-13), LCDR1-T32E (S309-15) and LCDR1-S33E (S309-16) were selected based on the free energy ranking from 7TLY virtual screening, as well as the free energy ranking from Pose 3 Free energy ranking of virtual screening HCDR1-P28G (S309-31), LCDR1-S31H (S309-32), LCDR2-S53W (S309-33), LCDR2-R55W (S309-34) and LCDR2-T57R (S309-35) were selected for mutation (the above HCDR1-P28W indicates that the 28th amino acid in the heavy chain variable region of the S309 antibody (i.e., in the CDR1 region) is mutated from proline (P) to tryptophan (W); LCDR1-S33E indicates that the 33rd amino acid in the light chain variable region of the S309 antibody (i.e., in the CDR1 region) is mutated from serine (S) to glutamic acid (E). The same applies to the others. The amino acid sequence of the heavy chain variable region of the S309 antibody is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16. NO: 16), and designed mutation primers by Snapgene to amplify the first half and the second half of the variant ( Figure 8 、 Figure 9 、 Figure 10 ), and double-digested pTT5 ( Figure 11 ) homologous recombination, transformed into DH5α, and the correct single clone was selected for plasmid extraction.

[0089] Antibody expression and purification were performed with reference to Example 2, and SDS-PAGE showed that the purity was high ( Figure 12 ).

[0090] Example 7: ELISA detection of affinity of single point mutations

[0091] Add 100 μL / well of 0.25 μg / mL Omicron or JN.1RBD-His to a 96-well plate and coat overnight at 4°C. Discard the supernatant, add 200 μL / well of 0.05% PBST, wash three times, add 200 μL / well of 5% MPBST, and incubate at 37°C for 2 hours. Wash three times, add 100 μL / well of serially diluted antibodies, and incubate at 37°C for 1 hour. Wash three times, add 100 μL / well of 5000-fold diluted horseradish peroxidase-conjugated goat anti-human IgG (H+L) (Shanghai Beyotime Biotechnology Co., Ltd., A0201), and incubate at 37°C for 1 hour. Wash three times, add 100 μL / well of TMB colorimetric solution, and develop at room temperature for 5-30 minutes. Add 50 μL / well of stop solution, and measure OD450 on an analyzer.

[0092] from Figure 13 It can be seen that HCDR1-P28W has a significant improvement in affinity for both Omicron and JN.1, while LCDR1-S33E has an increased affinity for Omicron, but a slight decrease in affinity for JN.1.

[0093] from Figure 14 It can be seen that LCDR2-S53W and LCDR2-T57R have significant improvements on Omicron and JN.1, while HCDR1-P28G and LCDR2-R55W have slightly decreased Omicron and JN.1. LCDR1-S31H has slightly improved Omicron and JN.1.

[0094] Example 8: ELISA detection of affinity of combined mutations

[0095] HCDR1-P28W, LCDR1-S33E, LCDR1-S31H, LCDR2-S53W and LCDR2-T57R were randomly expressed and their affinity to Omicron was determined. Figure 15 As can be seen from the results, the two-point combination mutation (S309-25) of HCDR1-P28W and LCDR1-S33E is significantly higher than that of the wild-type antibody and the single-point mutation variants of HCDR1-P28W and LCDR1-S33E.

[0096] In the affinity test of JN.1, affected by the LCDR1-S33E mutation, the combined mutation of HCDR1-P28W and LCDR1-S33E (S309-25) was lower in affinity than the single point mutation of HCDR1-P28W, but overall, it was improved compared to the wild type.

[0097] from Figure 16It can be seen that in the affinity test of Omicron and JN.1, it can be found that the three-point combination mutation of HCDR1-P28W, LCDR2-S53W and LCDR2-T57R (S309-39) showed stronger affinity than the two-point combination mutation of HCDR1-P28W and LCDR2-S33E.

[0098] from Figure 17 As can be seen in the affinity test of WT and Delta, it can be found that the two-point combination mutation of HCDR1-P28W and LCDR2-S33E (S309-25) and the three-point combination mutation of HCDR1-P28W, LCDR2-S53W and LCDR2-T57R (S309-39) have similar affinities to the S309 wild type, and these mutations do not affect the binding of the antibody to WT and Delta.

[0099] Example 9: Biacore determination of the affinity of variants for Omicron and JN.1

[0100] from Figure 18 and Figure 19 As can be seen in Tables 2 and 3, the HCDR1-P28W variant increased the binding rate for Omicron and JN.1 by 2.3-fold and 1.5-fold, respectively, and the affinity by 2.3-fold and 1.9-fold. The LCDR1-T32E variant increased the binding rate for Omicron by 5.9-fold. The two-point combination mutation of HCDR1-P28W and LCDR1-S33E increased the affinity for Omicron and JN.1 by 3.4-fold and 3.7-fold, respectively. The three-point combination mutation of HCDR1-P28W, LCDR2-S53W, and LCDR2-T57R increased the binding rate for Omicron by 2.5-fold and the binding rate for JN.1 by 4.3-fold.

[0101] Table 2 Biacore determination of the affinity of S309 and its variants for Omicron

[0102]

[0103] Table 3 Biacore determination of the affinity of S309 and different variants for JN.1

[0104]

[0105] Example 10: ELISA detection of antigens of HRP-labeled variants at different binding times

[0106] We labeled S309, S309-11 (HCDR1-P28W), S309-15 (LCDR1-T32E), S309-25 (HCDR1-P28W & LCDR1-S33E), and S309-39 (HCDR1-P28W & LCDR2-S53W-T57R) using an HRP labeling kit (Biodragona, BF06095S).

[0107] 100 μL / well of 0.1 μg / mL Omicron RBD-His (B.1.1.529) was coated in a 96-well plate and blocked with 5% MPBST. Then, different concentrations of S309, S309-11, S309-15, S309-25, and S309-39-HRP labeled antibodies were added and incubated for 5 minutes and 30 minutes, respectively. The plates were washed and developed immediately. It was found that the S309-25-HRP labeled antibody showed a strong signal at a concentration of 100 nM after incubation for 5 minutes ( Figure 20 ), while the S309-39-HRP labeled antibody was incubated for 5 minutes and showed stronger signals at concentrations of 33.3 and 100nM. This is because the two variants have faster binding speeds and have good application value in the rapid detection of new coronaviruses.

[0108] Example 11: Flow cytometry analysis of the blocking effect of variants on Omicron

[0109] Take 100 μL of 1×10 6 To Vero E6 cells (150 cells / mL), add antibodies at a final concentration of 12 or 48 μg / mL and 0.5 μg / mL Omicron RBD-His mixture, incubate at 4°C for 1 hour, wash twice with 200 μL of 2% FBS, add 1:200 diluted FITC-Goat anti-His Antibody, incubate at 4°C for 1 hour, wash twice with 200 μL of 2% FBS, resuspend, and detect on flow cytometer.

[0110] We tested the binding of different concentrations of Omicron and JN.1RBD to Vero E6, and selected concentrations of 0.5 and 1 μg / mL Omicron RBD to test the antibody blocking effect.

[0111] Table 4 Blocking effect of S309 and its variants on Omicron RBD

[0112]

[0113] The results showed that at a concentration of 0.5 μg / mL, 48 μg / mL of S309-25 (HCDR1-P28W & LCDR1-S33E), S309-33 (LCDR2-S53W), S309-35 (LCDR2-T57R) and S309-39 (HCDR1-P28W & LCDR2-S53W-T57R) showed better blocking effect ( Figure 21 , Table 4).

Claims

1. A novel coronavirus neutralizing antibody variant, characterized in that Obtained by mutation of the wild-type S309 antibody, the wild-type antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises a heavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chain CDR3 region, and the light chain variable region comprises a light chain CDR1 region, a light chain CDR2 region, and a light chain CDR3 region; Among them, the amino acid sequence of the heavy chain CDR1 region is GYPFTSY, the amino acid sequence of the light chain CDR1 region is RASQTVSSTSLA, and the amino acid sequence of the light chain CDR2 region is GASSRAT; The neutralizing antibody variant comprises any one of the following mutations: (1) Serine 53 in the light chain CDR2 region was mutated to tryptophan; (2) proline 28 in the heavy chain CDR1 region was mutated to tryptophan, and serine 33 in the light chain CDR1 region was mutated to glutamic acid; (3) Threonine 57 in the light chain CDR2 region was mutated to arginine; (4) Proline 28 in the heavy chain CDR1 region was mutated to tryptophan, serine 53 in the light chain CDR2 region was mutated to tryptophan, and threonine 57 was mutated to arginine.

2. The neutralizing antibody variant according to claim 1, characterized in that The amino acid sequence of the heavy chain CDR2 region is STYNGN, the amino acid sequence of the heavy chain CDR3 region is DYTRGAWFGESLIGGFDN, and the amino acid sequence of the light chain CDR3 region is QQHDTSLT.

3. The neutralizing antibody variant according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the wild-type S309 antibody is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

4. A biomaterial related to the neutralizing antibody variant according to any one of claims 1 to 3, characterized in that: The biological material is any one of the following: (1) A nucleic acid molecule encoding the neutralizing antibody variant according to any one of claims 1 to 3; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant vector containing the nucleic acid molecule described in (1), or a recombinant vector containing the expression cassette described in (2); (4) a recombinant microorganism containing the nucleic acid molecule described in (1), or a recombinant microorganism containing the expression cassette described in (2), or a recombinant microorganism containing the recombinant vector described in (3); (5) A cell line containing the nucleic acid molecule described in (1), or a cell line containing the expression cassette described in (2), or a cell line containing the recombinant vector described in (3).

5. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the neutralizing antibody variant according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

6. Use of the neutralizing antibody variant according to any one of claims 1 to 3 and / or the biomaterial according to claim 4 in the preparation of a drug for inhibiting or neutralizing the activity of SARS-CoV-2.

7. The use according to claim 6, characterized in that The drug for inhibiting or neutralizing the activity of SARS-CoV-2 is used to improve, prevent or treat diseases caused by SARS-CoV-2 infection and / or to inhibit SARS-CoV-2 infection.

8. The use according to claim 7, characterized in that The disease caused by the SARS-CoV-2 infection is a respiratory system infection.

9. The use according to claim 6, characterized in that The SARS-CoV-2 is SARS-CoV-2 itself, or a Delta, Omicron or JN.1 variant of SARS-CoV-2.

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