Neutralizing antibodies or antigen-binding fragments thereof against receptor binding domain of novel coronavirus and applications thereof

Abstract

The application discloses neutralizing antibodies or antigen-binding fragments thereof against the receptor binding domain of the novel coronavirus and application thereof, and the neutralizing antibodies or antigen-binding fragments thereof not only have strong neutralizing capacity to the currently prevailing Omicron lineage mutant strain of the novel coronavirus, but also have cross-reactivity to SARS, and can provide effective in vivo protection efficacy. The application provides a new therapeutic antibody selection for resisting the novel coronavirus infection, and has important application value in the development of drugs and vaccines for preventing and treating the novel coronavirus infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of novel coronavirus antibodies, in particular, neutralizing antibodies against the receptor binding domain of novel coronavirus or antigen-binding fragments thereof and applications thereof. BACKGROUND

[0002] The pathogen of novel coronavirus infection (COVID-19) is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, referred to as novel coronavirus), which is a single-stranded positive RNA virus belonging to the coronavirus family beta coronavirus genus, with a genome length of about 30,000 nucleotides, encoded by six functional open reading frames (ORFs) to encode replicase (ORF1a / ORF1b) and four structural proteins (spike protein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N)).

[0003] At present, more than 11,500 novel coronavirus-specific antibodies have been identified, and 5591 antibodies (accounting for 48.56%) have been confirmed to have novel coronavirus neutralizing activity. In addition, severe acute respiratory syndrome coronavirus (SARS-CoV, referred to as SARS virus) specific antibodies can also provide certain cross-protection for novel coronavirus infection, and screening antibodies with SARS cross-protection can help develop more broad-spectrum vaccines and treatment strategies to help cope with existing and future coronavirus threats. However, in recent years, with the rapid spread of newly emerging 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 the majority of 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 great challenges faced by those skilled in the art at present, and it is urgent to develop more broad-spectrum and efficient neutralizing antibodies to cope with the evolving novel coronavirus, while providing technical support for guiding the design of novel vaccines. SUMMARY

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

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

[0006] A second object of the present application is to provide the use of the neutralizing antibody or antigen-binding fragment thereof in the preparation of a product for preventing, detecting or treating the novel coronavirus.

[0007] A third object of the present application is to provide a medicine for preventing and / or treating the novel coronavirus.

[0008] A fourth object of the present application is to provide a biological material.

[0009] A fifth object of the present application is to provide the use of the biological material in the preparation of an antibody or antigen-binding fragment thereof against the novel coronavirus.

[0010] A sixth object of the present application is to provide a preparation method of an antibody or antigen-binding fragment thereof against the novel coronavirus.

[0011] In order to achieve the above objects, the present application is achieved 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 are combined with the RBD region of the novel coronavirus, so the present application takes the RBD region of the novel coronavirus as the key target for the development of neutralizing antibodies.

[0013] The full-length spike protein is 1,273 amino acids, which is translated and expressed as a trimer structure distributed on the membrane surface of the novel coronavirus, can bind to the angiotensin-converting enzyme 2 receptor (ACE2 receptor) on the surface of the host cell to mediate the entry of the novel coronavirus into the host cell, and plays a crucial role in the virus adsorption, membrane fusion, invasion and spread of the novel coronavirus infected 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 (Receptor-binding domain, RBD), which is the key region combined with the ACE2 receptor; the S2 region contains a fusion peptide, after the RBD is combined with the ACE2 receptor, the S2 subunit conformation changes, exposing the fusion peptide, mediating the fusion of the virus and the host cell, releasing the viral genome into the host cytoplasm, thereby completing the infection process. Based on the mechanism of novel coronavirus infection, the present application takes the key epitope of the spike protein of the novel coronavirus as the target, 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 receptor binding region of a novel coronavirus, comprising 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~CDR-C68-H3, wherein the amino acid sequence of CDR-C68-H1 is shown as SEQ ID NO. 2, the amino acid sequence of CDR-C68-H2 is shown as SEQ ID NO. 3, and the amino acid sequence of CDR-C68-H3 is shown as SEQ ID NO. 4; the light chain variable region (1) comprises CDR-C68-L1~CDR-C68-L3, wherein the amino acid sequence of CDR-C68-L1 is shown as 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 as 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~CDR-C74-H3, wherein the amino acid sequence of CDR-C74-H1 is shown as SEQ ID NO. 17, the amino acid sequence of CDR-C74-H2 is shown as SEQ ID NO. 18, and the amino acid sequence of CDR-C74-H3 is shown as SEQ ID NO. 19; the light chain variable region (2) comprises CDR-C74-L1~CDR-C74-L3, wherein the amino acid sequence of CDR-C74-L1 is shown as 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 as SEQ ID NO. 26.

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

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

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

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

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

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

[0023] Preferably, the neutralizing antibody or antigen-binding fragment thereof further comprises an Fc fragment, and the present application does not have special limitations on the species origin of the Fc fragment, including but not limited to human Fc fragment, murine Fc fragment, rabbit Fc fragment or biologically acceptable modifications or mutations thereof.

[0024] More preferably, the Fc fragment is a human Fc fragment 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 as set forth in SEQ ID NO. 35, and the amino acid sequence of the light chain constant region is as set forth 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 as set forth in SEQ ID NO. 35, and the amino acid sequence of the light chain constant region is as set forth in SEQ ID NO. 37.

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

[0028] A medicine 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 biological material, which is any 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 of (1);

[0032] (3) a cell comprising the recombinant expression vector of (2).

[0033] Preferably, the amino acid sequence of the heavy chain variable region (1) is as set forth 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 set forth in SEQ ID NO. 31 or as set forth in the complete complementary sequence of SEQ ID NO. 31.

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

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

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

[0037] The source and type of the cell in (3) are not particularly limited in the present application, and include, but are not limited to, Escherichia coli, yeast, and other conventional prokaryotic cells or HEK293 cells, CHO cells, and other conventional animal cells that can carry the recombinant expression vector.

[0038] Preferably, the cell in (3) is an animal cell.

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

[0040] The use of any of the biomaterials in the preparation of an antibody or antigen-binding fragment thereof against the novel coronavirus should also be within the protection scope of the present application.

[0041] A method for preparing an antibody or antigen-binding fragment thereof against the novel coronavirus, wherein the cell in (4) of the biomaterial is cultured under conditions allowing the expression of the monoclonal antibody or antigen-binding fragment thereof, and the monoclonal antibody or antigen-binding fragment thereof is recovered from the cultured recombinant cell culture.

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

[0043] The broad-spectrum neutralizing antibody against the receptor binding region of the novel coronavirus provided by the application not only has strong neutralizing ability to the currently prevailing Omicron lineage mutant strain of the novel coronavirus, but also has cross-reactivity to SARS, can provide effective in vivo protection efficacy, provides a new therapeutic antibody selection for anti-novel coronavirus infection, and has important application value in the development of drugs and vaccines for preventing and treating novel coronavirus infection. BRIEF DESCRIPTION OF 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, and Kappa / Lambda represents Kappa / Lambda type light chain.

[0045] Figure 2 The binding specificity identification results of CAV-C68 antibody and CAV-C74 antibody; A is the binding of each antibody to the RBD protein of the novel coronavirus; B is the binding of each antibody to the spike protein of the novel coronavirus.

[0046] Figure 3 The inhibition rate statistical results of CAV-C68 antibody and CAV-C74 antibody on 15 kinds of pseudoviruses.

[0047] Figure 4 The IC statistical results of CAV-C68 antibody and CAV-C74 antibody on 15 kinds of pseudoviruses. 50 The heat map of the statistical results.

[0048] Figure 5 The neutralization activity identification results of CAV-C68 antibody and CAV-C74 antibody on 4 kinds of novel coronavirus live viruses; A to D are the inhibition rate statistical results of novel coronavirus WT strain, Delta strain, XBB.1 strain and EG.5 strain, respectively; E is the IC 50 statistical results of 4 kinds of novel coronavirus live viruses.

[0049] Figure 6 The determination results of the viral load in K18-hACE2 mice infected with the novel coronavirus EG.5 strain under the treatment of CAV-C68 antibody and CAV-C74 antibody. DETAILED DESCRIPTION

[0050] The application will be further described in conjunction with the accompanying drawings and specific embodiments, which are used to explain the application and are not intended to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used are commercially available unless otherwise specified. The operations involving live SARS-CoV-2 virus in the following examples were carried out in a biosafety level 3 laboratory (BSL-3), and the relevant experimental procedures were strictly reviewed and approved by the Guangdong Health Commission 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 receptor binding domain of the novel coronavirus

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

[0053] Using a human single-cell expansion platform, blood samples from recovered COVID-19 patients were collected to isolate peripheral blood mononuclear cells (PBMCs), which were sorted by 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, i.e. CD19+CD27 high CD38 high plasmablast B cells, were obtained.

[0054] 2. Acquisition of antibody sequences

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

[0056] The IMGT v-quest antibody alignment tool (https: / / www.imgt.org) was used to define the domains, and the domains of the CAV-C68 antibody and the CAV-C74 antibody 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 SYADSVKGRFTISRDNSKNTLYLRMNNLRAEDTAVYFCAKEDGTMVTYPDY WGQGTLVTVSS (SEQ ID NO.1); the underlined parts are the complementarity 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 SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQ SYSTPPWT FGQGTKVEIK (SEQ ID NO.9); the underlined parts are the complementarity 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 (SEQID 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 ISWNSGGI GYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC AKDIGFCSGESCYDDYYYYYGMDVWGQGTTVTVSS (SEQ ID NO. 16); wherein the underlined portions are CDRs CDR-C74-H1 (SEQ ID NO. 17), CDR-C74-H2 (SEQ ID NO. 18), and CDR-C74-H3 (SEQ ID NO. 19), respectively, and the remaining portions are FRs 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), respectively.

[0060] The amino acid sequence of the heavy chain variable region of the CAV-C74 antibody (IGHV3-73 / IGLJ2) is: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMDWVRQAPGKGLEWVSAISSGSDTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAADGYSSSSYMDVWGQGTTVTVSS (SEQ ID NO. 1). ALPKQY AYWYQQKPGRAPVLVIY KDT ERPSGIPERFSGSSSGTIVTLTVSGVQAEDEADYYC QS ADSSGSYGNVV GGGGTKLTVL (SEQ ID NO. 24); wherein the underlined portions are CDRs CDR-C74-L1 (SEQ ID NO. 25), CDR-C74-L2, and CDR-C74-L3 (SEQ ID NO. 26), respectively, and the remaining portions are FRs 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), respectively.

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

[0062] (1) Construction of recombinant expression vector

[0063] The coding sequence (SEQ ID NO. 31) of the heavy chain variable region of the CAV-C68 antibody (SEQ ID NO. 1) was ligated between the EcoRI site and the NheI site of a human IgG1 antibody heavy chain expression vector (pFUSE-CHIg-hG1, Invivogen) expressing the heavy chain constant region (SEQ ID NO. 35), thereby obtaining a heavy chain expression vector of the CAV-C68 antibody, denoted 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 ligated between the Age I site and the Xho I site of a human IgG1 antibody kappa type light chain expression vector (pFUSE2-CLIg-rhK, Invivogen) expressing the light chain constant region (SEQ ID NO. 36), thereby obtaining a heavy chain expression vector of the CAV-C68 antibody, designated 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 ligated between the EcoRI site and the Nhe I site of a human IgG1 antibody heavy chain expression vector (pFUSE-CHIg-hG1, Invivogen) expressing the heavy chain constant region (SEQ ID NO. 35), thereby obtaining a heavy chain expression vector of the CAV-C74 antibody, designated 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 ligated between the Age I site and the Xho I site of a human IgG1 antibody lambda type light chain expression vector (pFUSE2-CLIg-hl2, Invivogen) expressing the light chain constant region (SEQ ID NO. 37), thereby obtaining a heavy chain expression vector of the CAV-C74 antibody, designated as pFUSE-C74-L vector.

[0067] (2) Eukaryotic expression of the antibody

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

[0069] (3) Affinity purification of the antibody

[0070] The cell supernatant was centrifuged to remove cell impurities. The purified Protein A was washed with 1 × PBS, and the cell supernatant of step one was mixed with the beads after centrifugation, placed in a slow horizontal shaker, and incubated overnight at 4 °C. The next day, centrifugation was performed at 3000 rpm for 15 min at 4 °C, and 50 mL of 1 × PBS was added to wash the unbound proteins and other impurities, which was repeated twice. After centrifugation to remove the supernatant, 0.1 M Glycine-HCL was added to elute the target protein. Tris-HCl was added to quickly neutralize the Glycine-HCL, stabilize the buffer PH, and ensure protein stability. A 30 kDa ultrafiltration tube was used to replace the protein buffer with 1 × PBS, which was repeated three times to obtain the preliminary purified CAV-C68 antibody and CAV-C74 antibody. The SEC chromatography column Superose™ 6 Increase 10 / 300 GL was used for further separation and purification.

[0071] Denaturing reducing gel (Reducing SDS-Page) identification, as shown in Figure 1 the heavy chain and light chain of CAV-C68 antibody and CAV-C74 antibody both meet the expected protein size, indicating that the preparation of the two antibodies is successful.

[0072] Example 2 Specificity identification of anti-SARS-CoV-2 receptor binding region

[0073] 1. ELISA method

[0074] This example identifies the specificity of CAV-C68 antibody and CAV-C74 antibody obtained in Example 1 by ELISA, and the specific method is as follows:

[0075] In the 96-well enzyme-labeled plate, 2 μg / mL SARS-CoV-2 RBD protein (Sino Biological, 40592-V08B) or spike protein (Sino Biological, 40589-V08B1-B) was coated, and 10 μg / mL CAV-C68 antibody or CAV-C74 antibody was added in a three-fold gradient dilution, and S309 antibody (a SARS cross-reactive new crown RBD specific antibody in the prior art “Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody” (DOI: 10.1038 / s41586-020-2349-y) “S309”) was used as a positive control, and PBS was used as a negative control; then HRP-labeled goat anti-human IgG secondary antibody was added, and the microplate spectrophotometer (BioTek) was used to measure the absorbance at 405 nm.

[0076] 2. Identification results

[0077] like Figure 2 As shown in A and B in Figure 1, 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 cells 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 using a lentiviral three-plasmid pseudovirus packaging system.

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

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

[0083] As shown in Figure 3 , the inhibition rates of CAV-C68 antibody and CAV-C74 antibody to 14 kinds of SARS-CoV-2 pseudovirus and SARS-CoV pseudovirus were dose-dependent, and both had obvious neutralization effect. As shown in Figure 4 , the IC 50 of CAV-C68 antibody and CAV-C74 antibody to 15 kinds of pseudovirus respectively fluctuated in the range of 0.01-3.938 nM and 0.076-0.878 nM, indicating that CAV-C68 antibody and CAV-C74 antibody had high level of neutralization activity, and had cross reaction to SARS virus.

[0084] 2. Neutralization activity of live virus

[0085] The neutralization ability of CAV-C68 antibody and CAV-C74 antibody prepared in Example 1 to live coronavirus was determined by virus plaque reduction neutralization test (FRNT).

[0086] After 45 μL of 50 μg / mL antibody diluted in three steps was incubated with an equal volume of 200 FFU of SARS-CoV-2 live virus (WT strain, Delta strain, XBB.1 strain or EG.5 strain), it was moved to a 96-well plate pre-coated with Vero E6 cells, cultured for 24 hours, fixed with 4 % paraformaldehyde, permeabilized with 0.2 % Triton X-100, sequentially 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), observed the virus plaque, and read and automatically counted by CTL ImmunoSpot S6 Ultra analyzer, and the inhibition rate was calculated.

[0087] As shown in Figure 5 A-D, the inhibition rates of CAV-C68 antibody and CAV-C74 antibody to 4 kinds of live coronavirus were dose-dependent, and had broad-spectrum neutralization activity; as shown in Figure 5 E, the IC 50 of CAV-C68 antibody and CAV-C74 antibody to 4 kinds of live coronavirus were 2.626-16.59 μg / mL and 2.094-7.353 μg / mL respectively, and had high level of neutralization activity.

[0088] Example 4 In vivo protective effect of anti-SARS-CoV-2 receptor binding region

[0089] 1. SARS-CoV-2 challenge

[0090] This example used 6-week-old K18-hACE2 transgenic mice as experimental objects to perform challenge experiments in an ABSL-3 laboratory, and the specific method was as follows:

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

[0092] 2. Infection determination

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

[0094] 3. Detection results

[0095] As shown in Figure 6 Compared with the control group, the viral load in hACE2 mice treated with CAV-C68 antibody and CAV-C74 antibody was significantly reduced, almost below the detection limit. It is shown that CAV-C68 antibody and CAV-C74 antibody both have in vivo protective effect and can effectively resist the infection of the new coronavirus.

[0096] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. For ordinary skilled persons in the art, on the basis of the above description and ideas, other different forms of changes or variations can also be made, and here it is not necessary and impossible to exhaust all the embodiments. Any modification, equivalent replacement and improvement made within the spirit and principles of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A neutralizing antibody or antigen-binding fragment thereof against the receptor binding domain of SARS-CoV-2, 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~CDR-C68-H3, wherein the amino acid sequence of CDR-C68-H1 is shown as SEQ ID NO. 2, the amino acid sequence of CDR-C68-H2 is shown as SEQ ID NO. 3, and the amino acid sequence of CDR-C68-H3 is shown as SEQ ID NO. 4; the light chain variable region (1) comprises CDR-C68-L1~CDR-C68-L3, wherein the amino acid sequence of CDR-C68-L1 is shown as 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 as 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~CDR-C74-H3, wherein the amino acid sequence of CDR-C74-H1 is shown as SEQ ID NO. 17, the amino acid sequence of CDR-C74-H2 is shown as SEQ ID NO. 18, and the amino acid sequence of CDR-C74-H3 is shown as SEQ ID NO. 19; the light chain variable region (2) comprises CDR-C74-L1~CDR-C74-L3, wherein the amino acid sequence of CDR-C74-L1 is shown as 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 as 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~FR-C68-H4, wherein the amino acid sequence of FR-C68-H1 is shown as SEQ ID NO. 5, the amino acid sequence of FR-C68-H2 is shown as SEQ ID NO. 6, the amino acid sequence of FR-C68-H3 is shown as SEQ ID NO. 7, and the amino acid sequence of FR-C68-H4 is shown as SEQ ID NO.

8. The light chain variable region (1) further comprises FR-C68-L1~FR-C68-L4, wherein the amino acid sequence of FR-C68-L1 is shown as SEQ ID NO. 12, the amino acid sequence of FR-C68-L2 is shown as SEQ ID NO. 13, the amino acid sequence of FR-C68-L3 is shown as SEQ ID NO. 14, and the amino acid sequence of FR-C68-L4 is shown as 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) has at least 95% identity with SEQ ID NO. 1; and the amino acid sequence of the light chain variable region (1) has at least 95% identity with SEQ ID NO.

9.

4. The neutralizing antibody or antigen binding fragment thereof of claim 1, wherein The heavy chain variable region (2) further comprises FR-C74-H1-FR-C74-H4, wherein the amino acid sequence of FR-C74-H1 is shown as SEQ ID NO. 20, the amino acid sequence of FR-C74-H2 is shown as SEQ ID NO. 21, the amino acid sequence of FR-C74-H3 is shown as SEQ ID NO. 22, and the amino acid sequence of FR-C74-H4 is shown as SEQ ID NO. 23; and the light chain variable region (2) further comprises FR-C74-L1-FR-C74-L4, wherein the amino acid sequence of FR-C74-L1 is shown as SEQ ID NO. 27, the amino acid sequence of FR-C74-L2 is shown as SEQ ID NO. 28, the amino acid sequence of FR-C74-L3 is shown as SEQ ID NO. 29, and the amino acid sequence of FR-C74-L4 is shown as 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) has at least 95% identity with SEQ ID NO. 16; and the amino acid sequence of the light chain variable region (2) has at least 95% identity with SEQ ID NO.

24.

6. Use of the neutralizing antibody or antigen-binding fragment thereof according to any one of claims 1-5 in the preparation of a product for resisting infection by the novel coronavirus.

7. Use of the neutralizing antibody or antigen-binding fragment thereof according to any one of claims 1-5 in the preparation of a product for detecting the novel coronavirus.

8. A medicament for the treatment of a novel coronavirus infection, characterized in that, The product comprises the neutralizing antibody or antigen-binding fragment thereof according to any one of claims 1-5, and a pharmaceutically acceptable carrier.

9. The medicament according to claim 8, characterized in that, The carrier comprises an excipient.

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

11. Use of the biological material according to claim 10 in the preparation of an antibody or antigen-binding fragment thereof for resisting the novel coronavirus.

12. A method of producing an antibody or antigen-binding fragment thereof against a novel coronavirus, characterized by, The cell according to claim 10 is cultured under conditions that allow expression of the antibody or antigen-binding fragment thereof for resisting the novel coronavirus, and the antibody or antigen-binding fragment thereof for resisting the novel coronavirus is obtained from the culture of the cell.

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