Neutralizing antibody against novel coronavirus receptor binding region or antigen binding fragment thereof and application thereof

Abstract

The invention discloses a neutralizing antibody for resisting a novel coronavirus receptor binding region or an antigen binding fragment of the neutralizing antibody and application of the neutralizing antibody, the neutralizing antibody or the antigen binding fragment of the neutralizing antibody not only has relatively strong neutralizing capacity on a currently mainly popular novel crown Omicron pedigree mutant strain, but also has cross reactivity on SARS (severe acute respiratory syndrome), and can provide effective in-vivo protective efficacy. The invention provides a new therapeutic antibody selection for resisting novel coronavirus infection, and has important application value in developing drugs and vaccines for preventing and treating novel coronavirus infection.

Description

Technical Field

[0001] The present invention relates to the field of novel coronavirus antibodies, specifically, neutralizing antibodies against novel coronavirus receptor binding regions or antigen-binding fragments thereof and applications thereof. Background Art

[0002] The pathogen of the new coronavirus infection (COVID-19) is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, referred to as the new coronavirus). SARS-CoV-2 is a single-stranded positive-sense RNA virus belonging to the genus Betacoronavirus of the family Coronaviridae. Its genome is approximately 30,000 nucleotides long and consists of six functional open reading frames (ORFs) encoding replicase (ORF1a / ORF1b) and four structural proteins (Spike protein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N)).

[0003] Currently, there are more than 11,500 new coronavirus-specific antibodies, and 5,591 antibodies (accounting for 48.56%) have been confirmed to have new coronavirus neutralizing activity. In addition, specific antibodies to severe acute respiratory syndrome coronavirus (SARS-CoV, referred to as SARS virus) can also provide certain cross-protection against new coronavirus infection. Screening out antibodies with SARS cross-protection will help develop a broader spectrum of vaccines and treatment strategies to help deal with existing and future coronavirus threats. However, in recent years, with the rapid spread of newly emerged SARS-CoV-2 variants of concern (VOCs), including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529), these variants have successively escaped most of the existing neutralizing antibodies and antibody-mediated immune responses induced by vaccination or previous infection, especially the Omicron variant. Therefore, obtaining novel coronavirus-specific antibodies against the latest variants is one of the huge challenges currently faced by technicians in this field. There is an urgent need to develop broader-spectrum and more efficient neutralizing antibodies to cope with the ever-evolving novel coronavirus, and at the same time provide technical support to guide the design of new vaccines. Summary of the invention

[0004] In order to solve the above-mentioned problems existing in the prior art, the present invention provides neutralizing antibodies or antigen-binding fragments thereof against the novel coronavirus receptor binding region and their applications.

[0005] The first object of the present invention is to provide a neutralizing antibody or an antigen-binding fragment thereof against the novel coronavirus receptor binding region.

[0006] The second object of the present invention is to provide the use of the neutralizing antibody or its antigen-binding fragment in the preparation of products for preventing, detecting or treating the new coronavirus.

[0007] The third object of the present invention is to provide a drug for preventing and / or treating the new coronavirus.

[0008] The fourth object of the present invention is to provide a biomaterial.

[0009] The fifth object of the present invention is to provide the use of the biomaterial in the preparation of antibodies or antigen-binding fragments thereof against the new coronavirus.

[0010] The sixth object of the present invention is to provide a method for preparing antibodies or antigen-binding fragments thereof against the new coronavirus.

[0011] In order to achieve the above object, the present invention is implemented by the following scheme:

[0012] Among the existing novel coronavirus-specific antibodies, the novel coronavirus RBD region-specific binding antibodies account for more than 60%, and the novel coronavirus non-RBD accounts for more than 30%. More than 90% of the novel coronavirus neutralizing antibodies bind to the novel coronavirus RBD region, so the present invention uses the novel coronavirus RBD region as the key target for the development of neutralizing antibodies.

[0013] The spike protein is 1,273 amino acids in length, and is translated and expressed as a trimer structure distributed on the membrane surface of the new coronavirus. It can bind to the angiotensin-converting enzyme 2 receptor (ACE2 receptor) on the surface of the host cell to mediate the entry of the new coronavirus into the host cell, and plays a vital role in the virus adsorption, membrane fusion, invasion, and transmission of the new coronavirus infecting the host cell. Each spike protein monomer is composed of two functional subunits (S1 subunit and S2 subunit). The S1 subunit contains the receptor binding domain (RBD), which is the key region for binding to the ACE2 receptor; the S2 region contains a fusion peptide. When the RBD binds to the ACE2 receptor, the conformation of the S2 subunit changes, exposing the fusion peptide, mediating the fusion of the virus and the host cell, and releasing the viral genome into the host cytoplasm, thereby completing the infection process. Combined with the mechanism of new coronavirus infection, the present invention uses the key epitopes of the spike protein of the new coronavirus as targets and obtains two specific neutralizing antibodies, namely CAV-C68 antibody and CAV-C74 antibody.

[0014] A neutralizing antibody or antigen-binding fragment thereof against a novel coronavirus receptor binding region, wherein the neutralizing antibody or antigen-binding fragment thereof comprises fragment (1) or fragment (2);

[0015] The fragment (1) comprises a heavy chain variable region (1) and a light chain variable region (1); the heavy chain variable region (1) comprises CDR-C68-H1 to CDR-C68-H3, wherein the amino acid sequence of CDR-C68-H1 is shown in SEQ ID NO.2, the amino acid sequence of CDR-C68-H2 is shown in SEQ ID NO.3, and the amino acid sequence of CDR-C68-H3 is shown in SEQ ID NO.4; the light chain variable region (1) comprises CDR-C68-L1 to CDR-C68-L3, wherein the amino acid sequence of CDR-C68-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-C68-L2 is TAS, and the amino acid sequence of CDR-C68-L3 is shown in SEQ ID NO.11;

[0016] The fragment (2) comprises a heavy chain variable region (2) and a light chain variable region (2); the heavy chain variable region (2) comprises CDR-C74-H1 to CDR-C74-H3, wherein the amino acid sequence of CDR-C74-H1 is shown in SEQ ID NO.17, the amino acid sequence of CDR-C74-H2 is shown in SEQ ID NO.18, and the amino acid sequence of CDR-C74-H3 is shown in SEQ ID NO.19; the light chain variable region (2) comprises light chain variable regions of CDR-C74-L1 to CDR-C74-L3, wherein the amino acid sequence of CDR-C74-L1 is shown in SEQ ID NO.25, the amino acid sequence of CDR-C74-L2 is KDT, and the amino acid sequence of CDR-C74-L3 is shown in SEQ ID NO.26.

[0017] Preferably, the heavy chain variable region (1) further comprises FR-C68-H1 to FR-C68-H4, wherein the amino acid sequence of FR-C68-H1 is shown in SEQ ID NO.5, the amino acid sequence of FR-C68-H2 is shown in SEQ ID NO.6, the amino acid sequence of FR-C68-H3 is shown in SEQ ID NO.7, and the amino acid sequence of FR-C68-H4 is shown in SEQ ID NO.8; the light chain variable region (1) further comprises FR-C68-L1 to FR-C68-L4, wherein the amino acid sequence of FR-C68-L1 is shown in SEQ ID NO.12, the amino acid sequence of FR-C68-L2 is shown in SEQ ID NO.13, the amino acid sequence of FR-C68-L3 is shown in SEQ ID NO.14, and the amino acid sequence of FR-C68-L4 is shown in SEQ ID NO.15.

[0018] More preferably, the amino acid sequence of the heavy chain variable region (1) is at least 95% identical to SEQ ID NO.1; the amino acid sequence of the light chain variable region (1) is at least 95% identical to SEQ ID NO.9.

[0019] More preferably, the amino acid sequence of the heavy chain variable region (1) is as shown in SEQ ID NO.1; and the amino acid sequence of the light chain variable region (1) is as shown in SEQ ID NO.9.

[0020] Preferably, the heavy chain variable region (2) further comprises FR-C74-H1 to FR-C74-H4, wherein the amino acid sequence of FR-C74-H1 is shown in SEQ ID NO.20, the amino acid sequence of FR-C74-H2 is shown in SEQ ID NO.21, the amino acid sequence of FR-C74-H3 is shown in SEQ ID NO.22, and the amino acid sequence of FR-C74-H4 is shown in SEQ ID NO.23; the light chain variable region (2) further comprises FR-C74-L1 to FR-C74-L4, wherein the amino acid sequence of FR-C74-L1 is shown in SEQ ID NO.27, the amino acid sequence of FR-C74-L2 is shown in SEQ ID NO.28, the amino acid sequence of FR-C74-L3 is shown in SEQ ID NO.29, and the amino acid sequence of FR-C74-L4 is shown in SEQ ID NO.30.

[0021] More preferably, the amino acid sequence of the heavy chain variable region (2) is at least 95% identical to SEQ ID NO.16; the amino acid sequence of the light chain variable region (2) is at least 95% identical to SEQ ID NO.24.

[0022] More preferably, the amino acid sequence of the heavy chain variable region (2) is as shown in SEQ ID NO.16; and the amino acid sequence of the light chain variable region (2) is as shown in SEQ ID NO.24.

[0023] Preferably, the neutralizing antibody or antigen-binding fragment thereof further comprises an Fc segment. The present invention has no special limitation on the species origin of the Fc segment, including but not limited to human Fc segment, mouse Fc segment, rabbit Fc segment or biologically acceptable modifications or mutations thereof.

[0024] More preferably, the Fc segment is a human Fc segment or a biologically acceptable modification or mutation thereof.

[0025] Preferably, the fragment (1) further comprises a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.35, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.36.

[0026] Preferably, the fragment (2) further comprises a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.35, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.37.

[0027] The use of any of the neutralizing antibodies or antigen-binding fragments thereof in the preparation of products for preventing, detecting or treating the new coronavirus should also be within the scope of protection of the present invention.

[0028] A drug for preventing and / or treating the novel coronavirus, comprising any of the neutralizing antibodies or antigen-binding fragments thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0029] A biomaterial, which is any one of the following (1) to (3):

[0030] (1) a nucleic acid molecule encoding any of the neutralizing antibodies or antigen-binding fragments thereof;

[0031] (2) a recombinant expression vector comprising the nucleic acid molecule described in (1);

[0032] (3) A cell comprising the recombinant expression vector described in (2).

[0033] Preferably, the amino acid sequence of the heavy chain variable region (1) is as shown in SEQ ID NO.1, and the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region (1) in (1) is as shown in SEQ ID NO.31 or as shown in the completely complementary sequence of the sequence shown in SEQ ID NO.31.

[0034] Preferably, the amino acid sequence of the light chain variable region (1) is as shown in SEQ ID NO.9, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region (1) in (1) is as shown in SEQ ID NO.32 or as shown in the completely complementary sequence of the sequence shown in SEQ ID NO.32.

[0035] Preferably, the amino acid sequence of the heavy chain variable region (2) is as shown in SEQ ID NO.16, and the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region (2) in (1) is as shown in SEQ ID NO.33 or as shown in the completely complementary sequence of the sequence shown in SEQ ID NO.33.

[0036] Preferably, the amino acid sequence of the light chain variable region (2) is as shown in SEQ ID NO.24, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region (2) in (1) is as shown in SEQ ID NO.34 or as shown in the completely complementary sequence of the sequence shown in SEQ ID NO.34.

[0037] The present invention has no particular limitation on the source and type of the cells in (3), including but not limited to prokaryotic cells such as Escherichia coli and yeast that can conventionally carry the recombinant expression vector or animal cells such as HEK293 cells and CHO cells that can conventionally carry the recombinant expression vector, which can achieve the purpose of the present invention.

[0038] Preferably, the cells in (3) are animal cells.

[0039] More preferably, the animal cell in (3) is a HEK293 cell or a CHO cell.

[0040] The use of any of the above-mentioned biological materials in the preparation of antibodies or antigen-binding fragments thereof against the new coronavirus should also be within the scope of protection of the present invention.

[0041] A method for preparing an antibody or an antigen-binding fragment thereof against the novel coronavirus, comprising culturing the cells described in (4) of the biological material under conditions that allow expression of the monoclonal antibody or the antigen-binding fragment thereof, and recovering the monoclonal antibody or the antigen-binding fragment thereof from the cultured recombinant cell culture.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The broad-spectrum neutralizing antibodies against the novel coronavirus receptor binding region provided by the present invention not only have strong neutralizing ability against the currently prevalent novel coronavirus Omicron lineage mutant strains, but also have cross-reactivity to SARS, can provide effective in vivo protection, and provide new therapeutic antibody options for fighting novel coronavirus infection. It has important application value in the development of drugs and vaccines for the prevention and treatment of novel coronavirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The SDS-Page identification results of CAV-C68 antibody and CAV-C74 antibody, lane 1 represents CAV-C68 antibody, lane 2 represents CAV-C74 antibody, H represents heavy chain, κ / λ represents Kappa / Lambda type light chain.

[0045] Figure 2 These are the binding specificity identification results of CAV-C68 antibodies and CAV-C74 antibodies; A is the binding status of each antibody with the SARS-CoV-2 RBD protein; B is the binding status of each antibody with the SARS-CoV-2 spike protein.

[0046] Figure 3 The figures are the statistical results of the inhibition rates of CAV-C68 antibody and CAV-C74 antibody against 15 pseudoviruses.

[0047] Figure 4 IC of CAV-C68 antibody and CAV-C74 antibody against 15 pseudoviruses 50 Heat map of statistical results.

[0048] Figure 5 The results of the neutralization activity identification of CAV-C68 antibody and CAV-C74 antibody against four live novel coronaviruses; A to D are the statistical results of the inhibition rate of the novel coronavirus WT strain, Delta strain, XBB.1 strain and EG.5 strain respectively; E is the IC 50 Statistical results.

[0049] Figure 6 These are the results of measuring the viral load in K18-hACE2 mice infected with the EG.5 strain of the new coronavirus under treatment with CAV-C68 and CAV-C74 antibodies. DETAILED DESCRIPTION

[0050] The present invention is further elaborated in detail below in conjunction with the drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials available from commercial sources. The operations involving SARS-CoV-2 live viruses in the following examples were all carried out in a biosafety level 3 laboratory (BSL-3), and the relevant experimental procedures were strictly reviewed and approved by the Health Commission of Guangdong Province and the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University (Ethics Number: 20230413).

[0051] Example 1 Isolation, cloning, expression and purification of antibodies against the novel coronavirus receptor binding region

[0052] 1. Isolation and purification of peripheral blood mononuclear cells (PBMC)

[0053] Using the human single-cell expansion platform, blood samples from recovered COVID-19 patients were collected to separate peripheral blood mononuclear cells (PBMCs), which were then sorted using flow cytometry. The antibodies used were PB anti-human CD19 (Biolegend, 302224), APC anti-human CD27 flow cytometry antibody (Biolegend, 302810), and PE anti-human CD38 flow cytometry antibody (Biolegend, 303516). Single plasmablast B cells that were positive for all three antibodies, namely CD19+CD27, were obtained. high CD38 high of plasmablastic B cells.

[0054] 2. Obtaining antibody sequences

[0055] The antibody variable region genes were obtained by RT-PCR and single-cell nested PCR, and the variable region fragments of the heavy and light chains of two anti-new coronavirus antibodies (CAV-C68 antibody and CAV-C74 antibody) were obtained.

[0056] The domains were defined using the IMGT v-quest antibody comparison tool (https: / / www.imgt.org). The domains of the CAV-C68 and CAV-C74 antibodies were specifically divided as follows:

[0057] The amino acid sequence of the heavy chain variable region of the CAV-C68 antibody (IGHV3-30 / IGHD4-23 / IGHJ4) is: QVQLVESGGGVVQPGRSLRLSCTAS GFTFSSYG MHWVRQAPGKGLEWVAV ISSDGSYK SYADSVKGRFTISRDNSKNTLYLRMNNLRAEDTAVYFC AKEDGTMV TYPDY WGQGTLVTVSS (SEQ ID NO.1); wherein the underlined parts are the complementary determining regions CDR-C68-H1 (SEQ ID NO.2), CDR-C68-H2 (SEQ ID NO.3) and CDR-C68-H3 (SEQ ID NO.4), and the remaining parts are the framework regions FR-C68-H1 (SEQ ID NO.5), FR-C68-H2 (SEQ ID NO.6), FR-C68-H3 (SEQ ID NO.7) and FR-C68-H4 (SEQ ID NO.8).

[0058] The amino acid sequence of the light chain variable region of the CAV-C68 antibody (IGKV1-39 / IGKJ1) is: DIQMTQSPSSLSASVGDRVTITCRAS QSINNY LQWFQQKPGKAPKLLIY TAS SL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPWTFGQGTKVEIK (SEQ ID NO.9); wherein the underlined parts are the complementary determining regions CDR-C68-L1 (SEQ ID NO.10), CDR-C68-L2 and CDR-C68-L3 (SEQ ID NO.11), and the remaining parts are the framework regions FR-C68-L1 (SEQ ID NO.12), FR-C68-L2 (SEQ ID NO.13), FR-C68-L3 (SEQ ID NO.14) and FR-C68-L4 (SEQ ID NO.15).

[0059] The amino acid sequence of the heavy chain variable region of the CAV-C74 antibody (IGHV3-9 / IGHD2-15 / IGHJ6) is: EVQLVESGGGLVQPGRSLRLSCAAS GFTFGDYA MHWVRQAPGKGLEWVSG IS WNSGGI GYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC AKDIGFCS GESCYDDYYYYYGMDVWGQGTTVTVSS (SEQ ID NO.16); wherein the underlined parts are the complementary determining regions CDR-C74-H1 (SEQ ID NO.17), CDR-C74-H2 (SEQ ID NO.18) and CDR-C74-H3 (SEQ ID NO.19), and the remaining parts are the framework regions FR-C74-H1 (SEQ ID NO.20), FR-C74-H2 (SEQ ID NO.21), FR-C74-H3 (SEQ ID NO.22) and FR-C74-H4 (SEQ ID NO.23).

[0060] The amino acid sequence of the light chain variable region of the CAV-C74 antibody (IGLV3-25 / IGLJ2) is: SYELTQPPSVSVSPGQTATITCSGD ALPKY AYWYQQKPGRAPVLVIY KDT ER PSGIPERFSGSSSGTIVTLTVSGVQAEDEADYYC QS ADSSGSYGNVV FGGGTKL TVL (SEQ ID NO.24); wherein the underlined parts are the complementary determining regions CDR-C74-L1 (SEQ ID NO.25), CDR-C74-L2 and CDR-C74-L3 (SEQ ID NO.26), and the remaining parts are the framework regions FR-C74-L1 (SEQ ID NO.27), FR-C74-L2 (SEQ ID NO.28), FR-C74-L3 (SEQ ID NO.29) and FR-C74-L4 (SEQ ID NO.30).

[0061] 3. Recombinant expression, purification and identification of monoclonal antibodies

[0062] (1) Construction of recombinant expression vector

[0063] The coding sequence (SEQ ID NO.31) of the heavy chain variable region (SEQ ID NO.1) of the CAV-C68 antibody was connected to the EcoRI site and NheI site of the human IgG1 antibody heavy chain expression vector (pFUSE-CHIg-hG1, Invivogen) expressing the heavy chain constant region (SEQ ID NO.35) to obtain the heavy chain expression vector of the CAV-C68 antibody, which was recorded as pFUSE-C68-H vector.

[0064] The coding sequence (SEQ ID NO.32) of the light chain variable region (SEQ ID NO.9) of the CAV-C68 antibody was connected to the AgeI site and XhoI site of the human IgG1 antibody κ-type light chain expression vector (pFUSE2-CLIg-rhK, Invivogen) expressing the light chain constant region (SEQ ID NO.36) to obtain the heavy chain expression vector of the CAV-C68 antibody, which was recorded as pFUSE-C68-L vector.

[0065] The coding sequence (SEQ ID NO.33) of the heavy chain variable region (SEQ ID NO.16) of the CAV-C74 antibody was connected to the EcoRI site and NheI site of the human IgG1 antibody heavy chain expression vector (pFUSE-CHIg-hG1, Invivogen) expressing the heavy chain constant region (SEQ ID NO.35) to obtain the heavy chain expression vector of the CAV-C74 antibody, which was recorded as pFUSE-C74-H vector.

[0066] The coding sequence (SEQ ID NO.34) of the light chain variable region (SEQ ID NO.24) of the CAV-C74 antibody was connected to the AgeI site and XhoI site of the human IgG1 antibody λ-type light chain expression vector (pFUSE2-CLIg-hl2, Invivogen) expressing the light chain constant region (SEQ ID NO.37) to obtain the heavy chain expression vector of the CAV-C74 antibody, which was recorded as pFUSE-C74-L vector.

[0067] (2) Eukaryotic expression of antibodies

[0068] HEK293T cells were cultured according to conventional methods. When the cell density reached 80%, pFUSE-C68-H and pFUSE-C68-L were transiently transfected into HEK293T cells for expression of CAV-C68 antibody, and pFUSE-C74-H and pFUSE-C74-L were transfected into HEK293T cells for expression of CAV-C74 antibody. After culturing in a carbon dioxide incubator at 37°C for 4 days, the cell supernatant was collected.

[0069] (3) Antibody affinity purification

[0070] Remove cell impurities from the cell supernatant by centrifugation. Wash and purify Protein A with 1×PBS. After centrifugation, mix the cell supernatant from step one with the beads, place in a slow horizontal shaker, and incubate overnight at 4°C. The next day, centrifuge at 3000rpm for 15min at 4°C, add 50mL 1×PBS to wash unbound proteins and other impurities, and repeat twice. After centrifugation to remove the supernatant, add 0.1M Glycine-HCL to elute the target protein. Add Tris-HCl to quickly neutralize Glycine-HCL, stabilize the buffer pH, and ensure protein stability. Use a 30kDa ultrafiltration tube to replace the protein buffer with 1×PBS, repeat three times, and obtain preliminary purified CAV-C68 antibodies and CAV-C74 antibodies. Use SEC chromatography column Superose TM 6Increase 10 / 300GL further separation and purification.

[0071] Identified by reducing SDS-Page, Figure 1 As shown, the heavy and light chains of CAV-C68 and CAV-C74 antibodies were consistent with the expected protein sizes, indicating that the two antibodies were successfully prepared.

[0072] Example 2 Specific identification of the anti-novel coronavirus receptor binding region

[0073] 1. ELISA method

[0074] In this example, the specificity of the CAV-C68 antibody and the CAV-C74 antibody obtained in Example 1 was identified by ELISA. The specific method is as follows:

[0075] 2 μg / mL SARS-CoV-2 RBD protein (Sino Biological, 40592-V08B) or spike protein (Sino Biological, 40589-V08B1-B) was coated in a 96-well ELISA plate, and 10 μg / mL of three-fold gradient dilutions of CAV-C68 antibody or CAV-C74 antibody, as well as S309 antibody (the "S309" in the prior art "Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody" (DOI: 10.1038 / s41586-020-2349-y) is a new coronavirus RBD-specific antibody that cross-reacts with SARS) were added as positive controls, and PBS was used as a negative control; then HRP-labeled goat anti-human IgG secondary antibody was added, and the absorbance was measured at 405 nm using a microplate spectrophotometer (BioTek).

[0076] 2. Identification results

[0077] like Figure 2 As shown in A and B in the figure, the CAV-C68 antibody and CAV-C74 antibody prepared in Example 1 can strongly bind to the RBD protein and spike protein of the new coronavirus, and are specific antibodies for the new coronavirus.

[0078] Example 3 Identification of Neutralizing Activity of Anti-novel Coronavirus Receptor Binding Region

[0079] 1. Pseudovirus neutralization activity

[0080] Referring to the method in the prior art “Rapid isolation and immune profiling of SARS-CoV-2specific memory B cell in convalescent COVID-19 patients via LIBRA-seq” (DOI: 10.1038 / s41392-021-00610-7), 14 SARS-CoV-2 pseudoviruses (WT pseudovirus, Alpha pseudovirus, Beta pseudovirus, Gamma pseudovirus, Delta pseudovirus, BA.1 pseudovirus, BA.2 pseudovirus, BA.4 / 5 pseudovirus, BQ.1.1 pseudovirus, XBB.1.5 pseudovirus, XBB.2.3 pseudovirus, XBB.1.9.2 pseudovirus, EG.5 pseudovirus and JN.1 pseudovirus) and SARS-CoV pseudovirus were prepared by a lentiviral three-plasmid pseudovirus packaging system.

[0081] The neutralizing activity of the CAV-C68 and CAV-C74 antibodies prepared in Example 1 was detected using the above 15 pseudoviruses. The specific steps are as follows:

[0082] Three-fold serial dilutions of 10 μg / mL CAV-C68 antibody or CAV-C74 antibody were mixed with 650 TCID 50 After pre-incubation with pseudovirus mixture, HEK293T stable cells (TaKaRa, 631289) overexpressing ACE2 receptor were added, which was recorded as the antibody experimental group; stable cells with only pseudovirus added were recorded as the positive virus group; stable cells without pseudovirus and antibody added were recorded as the negative cell group; after further culture for 48 hours, the fluorescence intensity of each group was detected by the firefly luciferase assay system (Promega), and the inhibition rate was calculated as follows: inhibition rate (%) = (1-(fluorescence intensity of antibody experimental group-fluorescence intensity of negative cell group) / (fluorescence intensity of positive virus group-fluorescence intensity of negative cell group) × 100%, and IC 50 .

[0083] like Figure 3 As shown in the figure, the inhibition rate of CAV-C68 antibody and CAV-C74 antibody against 14 SARS-CoV-2 pseudoviruses and SARS-CoV pseudoviruses was dose-dependent, and both had obvious neutralizing effects. Figure 4 As shown, the IC values ​​of CAV-C68 antibody and CAV-C74 antibody against 15 pseudoviruses 50 They fluctuated in the ranges of 0.01-3.938 nM and 0.076-0.878 nM, respectively, indicating that CAV-C68 antibody and CAV-C74 antibody had a high level of neutralizing activity and cross-reacted with SARS virus.

[0084] 2. Live virus neutralization activity

[0085] The neutralizing ability of CAV-C68 antibody and CAV-C74 antibody prepared in Example 1 against live SARS-CoV-2 virus was determined by viral plaque reduction neutralization test (FRNT).

[0086] After incubation with an equal volume of 200 FFU SARS-CoV-2 live virus (WT strain, Delta strain, XBB.1 strain or EG.5 strain) of 45 μL 50 μg / mL three-fold serial dilution of antibody, the plate was transferred to a 96-well plate pre-laid with Vero E6 cells and cultured for 24 hours. The plate was fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. The plate was then stained with SARS-CoV-2 nucleocapsid protein rabbit antibody (Sino Biological, 245 40143-T62) and peroxidase affinity purified goat anti-rabbit IgG (H+L) (Jackson, 111-246 035-144) in turn. Virus spots were observed and read and automatically counted with a CTL ImmunoSpot S6 Ultra analyzer to calculate the inhibition rate.

[0087] like Figure 5 As shown in A to D in Figure 1, the inhibition rates of CAV-C68 and CAV-C74 antibodies against four types of live novel coronaviruses were dose-dependent and had broad-spectrum neutralizing activity; Figure 5 As shown in E, the IC values ​​of CAV-C68 antibody and CAV-C74 antibody against four live novel coronaviruses 50 They were 2.626-16.59 μg / mL and 2.094-7.353 μg / mL respectively, with a high level of neutralization activity.

[0088] Example 4 In vivo protective effect against novel coronavirus receptor binding region

[0089] 1. SARS-CoV-2 attack

[0090] In this example, 6-week-old K18-hACE2 transgenic mice were used as experimental subjects, and a challenge experiment was conducted in an ABSL-3 laboratory. The specific method is as follows:

[0091] One day before the challenge, hACE2 mice were intraperitoneally injected with 200 μg / mouse of CAV-C68 antibody or CAV-C74 antibody prepared in Example 1, and treated with an equal amount of DPBS as a negative control (control); on the day of the challenge, SARS-CoV-2 live virus (EG.5 strain) was used at 5×10 4 PFU / mice infected intranasally with hACE2.

[0092] 2. Determination of infection status

[0093] Three days after the challenge, lung tissues of the mice were collected and the virus titer was determined (n=3 per group).

[0094] 3. Test results

[0095] like Figure 6 As shown in the figure, compared with the control group, the viral load in hACE2 mice treated with CAV-C68 and CAV-C74 antibodies was significantly reduced, almost below the detection limit. This shows that both CAV-C68 and CAV-C74 antibodies have in vivo protective effects and can effectively resist the infection of the new coronavirus.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A neutralizing antibody or antigen-binding fragment thereof against the novel coronavirus receptor binding region, characterized in that: The neutralizing antibody or antigen-binding fragment thereof comprises fragment (1) or fragment (2); The fragment (1) comprises a heavy chain variable region (1) and a light chain variable region (1); the heavy chain variable region (1) comprises CDR-C68-H1 to CDR-C68-H3, wherein the amino acid sequence of CDR-C68-H1 is shown in SEQ ID NO.2, the amino acid sequence of CDR-C68-H2 is shown in SEQ ID NO.3, and the amino acid sequence of CDR-C68-H3 is shown in SEQ ID NO.4; the light chain variable region (1) comprises CDR-C68-L1 to CDR-C68-L3, wherein the amino acid sequence of CDR-C68-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-C68-L2 is TAS, and the amino acid sequence of CDR-C68-L3 is shown in SEQ ID NO.11; The fragment (2) comprises a heavy chain variable region (2) and a light chain variable region (2); the heavy chain variable region (2) comprises CDR-C74-H1 to CDR-C74-H3, wherein the amino acid sequence of CDR-C74-H1 is shown in SEQ ID NO.17, the amino acid sequence of CDR-C74-H2 is shown in SEQ ID NO.18, and the amino acid sequence of CDR-C74-H3 is shown in SEQ ID NO.19; the light chain variable region (2) comprises light chain variable regions of CDR-C74-L1 to CDR-C74-L3, wherein the amino acid sequence of CDR-C74-L1 is shown in SEQ ID NO.25, the amino acid sequence of CDR-C74-L2 is KDT, and the amino acid sequence of CDR-C74-L3 is shown in SEQ ID NO.

26.

2. The neutralizing antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain variable region (1) further comprises FR-C68-H1 to FR-C68-H4, wherein the amino acid sequence of FR-C68-H1 is shown in SEQ ID NO.5, the amino acid sequence of FR-C68-H2 is shown in SEQ ID NO.6, the amino acid sequence of FR-C68-H3 is shown in SEQ ID NO.7, and the amino acid sequence of FR-C68-H4 is shown in SEQ ID NO.8; The light chain variable region (1) further comprises FR-C68-L1 to FR-C68-L4, wherein the amino acid sequence of FR-C68-L1 is shown in SEQ ID NO.12, the amino acid sequence of FR-C68-L2 is shown in SEQ ID NO.13, the amino acid sequence of FR-C68-L3 is shown in SEQ ID NO.14, and the amino acid sequence of FR-C68-L4 is shown in SEQ ID NO.

15.

3. The neutralizing antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The amino acid sequence of the heavy chain variable region (1) is at least 95% identical to SEQ ID NO.1; the amino acid sequence of the light chain variable region (1) is at least 95% identical to SEQ ID NO.

9.

4. The neutralizing antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain variable region (2) further comprises FR-C74-H1 to FR-C74-H4, wherein the amino acid sequence of FR-C74-H1 is shown in SEQ ID NO.20, the amino acid sequence of FR-C74-H2 is shown in SEQ ID NO.21, the amino acid sequence of FR-C74-H3 is shown in SEQ ID NO.22, and the amino acid sequence of FR-C74-H4 is shown in SEQ ID NO.23; the light chain variable region (2) further comprises FR-C74-L1 to FR-C74-L4, wherein the amino acid sequence of FR-C74-L1 is shown in SEQ ID NO.27, the amino acid sequence of FR-C74-L2 is shown in SEQ ID NO.28, the amino acid sequence of FR-C74-L3 is shown in SEQ ID NO.29, and the amino acid sequence of FR-C74-L4 is shown in SEQ ID NO.

30.

5. The neutralizing antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The amino acid sequence of the heavy chain variable region (2) is at least 95% identical to SEQ ID NO.16; the amino acid sequence of the light chain variable region (2) is at least 95% identical to SEQ ID NO.

24.

6. Use of the neutralizing antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 in the preparation of products for preventing, detecting or treating the new coronavirus.

7. A drug for preventing and / or treating novel coronavirus, characterized in that: It comprises the neutralizing antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier and / or excipient.

8. A biomaterial, characterized in that It is any one of the following (1) to (3): (1) A nucleic acid molecule encoding the neutralizing antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; (2) a recombinant expression vector comprising the nucleic acid molecule described in (1); (3) A cell comprising the recombinant expression vector described in (2).

9. Use of the biomaterial according to claim 8 in the preparation of antibodies or antigen-binding fragments thereof against the new coronavirus.

10. A method for preparing an antibody or an antigen-binding fragment thereof against the new coronavirus, characterized in that: The cell of claim 8 is cultured under conditions that allow expression of the monoclonal antibody or the antigen-binding fragment thereof, and the monoclonal antibody or the antigen-binding fragment thereof is obtained from the cell culture.

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