WO2022022445A1 - Antibody that specifically binds to coronavirus or antigen-binding fragment thereof - Google Patents

Abstract

Disclosed in the present invention are an antibody that specifically binds to coronavirus or an antigen-binding fragment thereof, a nucleic acid molecule coding the antibody or the antigen-binding fragment thereof, a vector containing the nucleic acid molecule, and a host cell containing the vector. Also disclosed in the present invention are an application of the antibody or the antigen-binding fragment thereof in preparation of drugs for treating or preventing diseases caused by coronavirus, an application of the antibody or the antigen-binding fragment thereof in treatment or prevention of diseases caused by coronavirus, and an application of the antibody or the antigen-binding fragment thereof in test products.

Description

An antibody or antigen-binding fragment thereof that specifically binds to a coronavirus

This application claims the priority of a Chinese invention patent application (application number: 202010740319.3; invention title: an antibody that specifically binds to coronavirus or its antigen-binding fragment; application date: July 28, 2020), which is a Chinese invention patent application The entire contents of are incorporated herein by reference in their entirety.

technical field

The present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to a coronavirus, a nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof, a vector comprising the nucleic acid molecule, and a host cell comprising the vector; the present invention also relates to the antibody The application of its antigen-binding fragment in the preparation of medicines for treating or preventing diseases caused by coronavirus, the application in treating or preventing diseases caused by coronavirus, and the application in detection products belong to the field of biomedicine.

Background technique

Novel coronavirus pneumonia (2019-nCOV) is an acute respiratory infectious disease caused by SARS-COV-2 novel coronavirus. The virus has a very strong ability to spread and can be transmitted through respiratory tract, contact and other channels. Since the outbreak in December 2019, it has spread to all parts of the world, forming a worldwide pandemic. As of July 1, 2020, the SARS-CoV-2 coronavirus has caused more than 10 million infections worldwide, with more than 500,000 deaths, posing severe challenges to public health security around the world.

The SARS-CoV-2 virus belongs to the family Coronaviridae, and belongs to the β-coronavirus genus as the SARS coronavirus that broke out in 2003. The main envelope protein of the SARS-CoV-2 virus is its spike protein (also called Spike protein, or S protein for short). In the process of SARS-CoV-2 virus infection, its spike protein is hydrolyzed into two subunits S1 and S2 by proteases in the host cell, where S2 is a transmembrane protein and S1 has the ability to recognize and bind to the cellular receptor angiotensin Converting enzyme-2 (ACE-2) receptor binding domain (Receptor Binding domain, referred to as RBD). The spike protein composed of S1 and S2 mediates the invasion process of SARS-CoV-2 virus, specifically, it specifically recognizes and binds to host cell receptors, and mediates the virus invasion into host cells and the fusion process with host cells . Therefore, the spike protein of the SARS-CoV-2 virus is also a target for scientists in the field to develop neutralizing antibodies for the SARS-CoV-2 virus.

Studies have shown that the clinical use of virus-specific convalescent human plasma can effectively neutralize the virus, prevent the virus from spreading in various organs in the body, and also play an important role in the outcome of the patient's disease course. However, polyclonal plasma is not only limited in source, but its clinical application is also limited by conditions such as difficulty in quality control, differences in blood types of donors and recipients, and potential infectious factors. Therefore, scientists in the field hope to isolate fully human monoclonal antibodies that can neutralize SARS-CoV-2 virus from patients who have recovered from new coronary pneumonia. This is also one of the main directions of the current new coronavirus drug development.

Several research teams at home and abroad have reported that fully human monoclonal antibodies, such as BD-368-2, B38, etc., which can bind to the S protein of SARS-CoV-2 virus, isolated from the peripheral blood of recovered patients with COVID-19, are produced by recombinant The expressed S protein or S protein receptor binding region (RBD) of the SARS-CoV-2 virus was used as bait to screen and isolate B cells (memory B cells) that could bind to them from the peripheral blood of recovered patients. Methods The paired genes of the heavy chain and light chain of the antibody expressed by a single B cell were obtained, and the ability to neutralize the virus was verified after the antibody was expressed by in vitro recombination. Because this method uses marker proteins (the S protein or the receptor binding region of the S protein of the recombinantly expressed SARS-CoV-2 virus called bait) to screen and enrich B cells before antibody gene sequencing , therefore, only antibodies that specifically bind to the labeled protein can be screened.

Regarding the method of isolating fully human monoclonal antibodies from the peripheral blood of recovered patients with new coronary pneumonia, the in vitro monoclonal culture of human B cells and high-throughput antibody screening pioneered by Dr. Huang Jinghe (one of the inventors of this application) in 2013 can be used Technology (Huang J et al.Nature Protocols 2013), the general process is: first, use the SARS-CoV-2 and SARS-CoV pseudovirus neutralization system to detect the neutralizing antibodies in the serum of recovered patients with new coronary pneumonia, and screen out the SARS-CoV-2 neutralizing antibodies. -2 and SARS-CoV recovered patients with high neutralizing activity at the same time; then collected peripheral blood lymphocytes of the screened recovered patients, and used flow cytometry to sort out memory B lymphocytes; single B cells were inoculated into 384 In the well plate, cytokines and feeder cells were added for culture, and the culture supernatant was obtained (B cells secreted antibodies into the supernatant after in vitro expansion and differentiation), and then the in vitro high-throughput neutralization assay was used to detect the supernatant in the supernatant. The ability of antibodies to neutralize SARS-CoV-2 and SARS-CoV viruses, screened out positive clones that can neutralize both viruses at the same time, and cloned the variable regions of heavy and light chains of antibodies by RT-PCR. And constructed into an expression vector, the antibody was expressed by transfecting 293T cells, and the monoclonal antibody was finally purified.

Although most of the reported antibodies have good neutralizing ability to the tested SARS-CoV-2 virus strains, they are not suitable for other coronaviruses with similar gene sequences to SARS-CoV-2 virus, such as SARS-CoV, SARS-like viruses, etc. lack the ability to bind and neutralize, indicating that these antibodies may specifically bind to non-conserved regions of SARS-CoV-2 virus. Since the SARS-CoV-2 virus is an RNA virus, the genome sequence of the virus is prone to mutation during the epidemic. If the non-conserved regions recognized by these antibodies are mutated, it may lead to the generation of more infectious epidemic strains, causing existing antibodies to lose the neutralizing effect of the mutant virus strains. For example, the British mutant strain B.1.1.7, the Beta mutant strain B.1.351, the Brazilian mutant strain P1, the Delta mutant strain B.1.617.2, and the Nigerian mutant strain B.1.525, which have recently appeared around the world, all have a negative impact on the existing The 2019-nCoV neutralizing antibodies showed neutralization escape phenomenon; the currently used 2019-nCoV vaccines have greatly weakened the protective ability of these mutant strains.

Additionally, the May 2021 discovery in Malaysia of an entirely new coronavirus that can be passed from dogs to children points to a disturbing trend: future coronavirus outbreaks are no longer an uncommon event, likely to occur every now and then. Comeback in a few years.

Therefore, the development of broad-spectrum vaccines and broad-spectrum drugs with broad protection against various coronaviruses and various new coronavirus mutant strains is the key to relieving the pressure of current epidemic prevention and control and preventing the next coronavirus pandemic; In the field, those skilled in the art hope to develop antibodies with binding ability and neutralizing ability for various coronaviruses and various new coronavirus mutant strains.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, one aspect of the present invention provides an antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein,

Condition a): The heavy chain variable region comprises the CDR1 sequence of the heavy chain variable region as shown in SEQ ID NO.1, the CDR2 sequence of the heavy chain variable region as shown in SEQ ID NO.2 and the CDR2 sequence of the heavy chain variable region as shown in SEQ ID NO. . the CDR3 sequence of the heavy chain variable region shown in 3;

Condition b): the light chain variable region comprises the CDR1 sequence of the light chain variable region as shown in SEQ ID NO.4, the CDR2 sequence of the light chain variable region as shown in SEQ ID NO.5 and the CDR2 sequence of the light chain variable region as shown in SEQ ID NO. . the CDR3 sequence of the light chain variable region shown in 6;

The heavy chain variable region of the antibody or antigen-binding fragment thereof satisfies condition a); or,

The light chain variable region of the antibody or antigen-binding fragment thereof satisfies condition b); or,

The antibody or antigen-binding fragment thereof satisfies both conditions a) and b).

In a preferred embodiment of the present invention, the sequence of the heavy chain variable region is shown in SEQ ID NO.7, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.7.

In a preferred embodiment of the present invention, the sequence of the light chain variable region is shown in SEQ ID NO.8, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.8.

In a more preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof satisfies both conditions a) and b);

The sequence of the variable region of the heavy chain is shown in SEQ ID NO.7, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.7; and,

The sequence of the light chain variable region is shown in SEQ ID NO.8, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.8.

The percentage of "sequence homology" with respect to amino acid sequences is generated by determining the number of amino acid residues present in the two sequences to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and dividing the result Multiply by 100 to yield the percent homology of the sequence.

In a specific embodiment of the present invention, the above-mentioned heavy chain variable region can perform deletion, insertion or amino acid mutation of a small amount of amino acids on the basis of the sequence shown in SEQ ID NO. 7 to obtain a homology of more than 80% amino acid sequence. Substitution of a small amount of amino acids (deletion or insertion, or amino acid mutation, or substitution of similar amino acids), especially the variant obtained by conservative amino acid substitution in the framework region, which is higher than the sequence shown in SEQ ID NO.7 homology (more than 80% homology), and retain the original properties and functions of the variable region of the heavy chain, that is, the properties and functions of antibodies that specifically bind to coronaviruses, then these variants also fall into the Within the scope of protection of the present invention; similarly, the above-mentioned light chain variable region can carry out a small amount of amino acid deletion, insertion or amino acid mutation on the basis of the sequence shown in SEQ ID NO. 8, especially in the conservative part of the framework region The variants obtained by the amino acid replacement of the obtained variants retain the original properties and functions of the light chain variable region, that is, the properties and functions of antibodies that specifically bind to the coronavirus, and these variants also all fall into the present invention. within the scope of protection.

The framework region refers to the amino acid sequence located between the CDRs, including the framework region of the heavy chain variable region and the framework region of the light chain variable region.

In a more preferred embodiment of the present invention, the heavy chain amino acid sequence of the antibody or its antigen-binding fragment is shown in SEQ ID NO.11, or it has more than 80% of the sequence shown in SEQ ID NO.11 Sequence homology; and, the light chain amino acid sequence of the antibody or its antigen-binding fragment is shown in SEQ ID NO.12, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.12 .

Similarly, the amino acid sequence of the heavy chain can be based on the sequence shown in SEQ ID NO.11 by deletion, insertion or amino acid mutation of a small number of amino acids; the amino acid sequence of the light chain can be based on the sequence shown in SEQ ID NO.12. Deletion, insertion or amino acid mutation of a small amount of amino acids; as long as the obtained variants still have high homology and retain their original properties and functions, that is, the properties and functions of antibodies that specifically bind to coronaviruses , then these variants also fall within the scope of protection of the present invention.

In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that specifically binds to coronavirus.

In a more preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof is a neutralizing antibody or antigen-binding fragment thereof of coronavirus.

The term "neutralizing antibody" is an antibody or antigen-binding fragment that specifically binds to a viral receptor protein, and the specific binding can inhibit the biological function of the viral membrane protein, such as preventing the viral membrane protein from binding to its target cell receptor, which can be Specifically reduces the ability of the virus to infect target cells; in this application, the neutralizing antibody or antigen-binding fragment thereof of a coronavirus refers to an antibody or an antigen-binding fragment thereof that binds to the S protein of the coronavirus.

In a preferred embodiment of the present invention, the antibody is a monoclonal antibody.

In a more preferred embodiment of the present invention, the antibody is a fully human monoclonal antibody.

In a preferred embodiment of the present invention, the antibody is any one or a combination of IgG1, IgG2, IgG3 or IgG4.

Preferably, the antibody may be an intact antibody selected from IgG1, IgG2, IgG3 or IgG4.

In a preferred embodiment of the present invention, the antigen-binding fragment is Fv, Fab, F(ab') ₂ , Fab', dsFv, scFv or sc(Fv) ₂ .

In a preferred embodiment of the present invention, the above-mentioned antibody, or antigen-binding fragment thereof, may be further chemically modified, eg, one or more chemical groups may be attached to the antibody to increase one or more functional properties of the antibody. For example, common chemical modifications include glycosylation and PEGylation. Among them, for example, glycosylation modification can be carried out in the variable region of the heavy chain or light chain, and one or more glycosylation sites can be added to improve part of the function of the antibody, such as enhancing the immunogenicity of the antibody or improving the drug of the antibody dynamics, etc. For example, PEGylation can be achieved by subjecting the antibody or antigen-binding fragment thereof to an acylation reaction or an alkylation reaction with an active polyethylene glycol (eg, an active ester or aldehyde derivative of polyethylene glycol) under suitable conditions Modification to improve part of the function of the antibody, such as increasing the biological (eg serum) half-life of the antibody, etc. The above-mentioned chemical modification does not significantly change the basic functions and properties of the antibody of the present invention or its antigen-binding fragment, that is, the function and property of specific binding with the coronavirus; these chemically modified variants also fall within the scope of protection of the present invention. Inside.

In a preferred embodiment of the present invention, the above-mentioned antibodies, or antigen-binding fragments thereof, can be conjugated to other factors by chemical methods or genetic engineering methods; for example, these factors can provide the effect of targeting the antibody to a desired functional site or other properties; the above-mentioned antibodies, or complexes formed by conjugation of antigen-binding fragments thereof and other factors, fall within the protection scope of the present invention.

Another aspect of the present invention provides a nucleic acid molecule, wherein the nucleic acid molecule encodes the above-mentioned antibody, or an antigen-binding fragment thereof.

In a preferred embodiment of the present invention, in the nucleic acid molecule, the nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO. % sequence homology.

In a preferred embodiment of the present invention, in the nucleic acid molecule, the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO. % sequence homology.

In a more preferred embodiment of the present invention, in the nucleic acid molecule, the nucleic acid sequence encoding the heavy chain is as shown in SEQ ID NO.13, or it has more than 80% sequence with the sequence shown in SEQ ID NO.13 and, in the nucleic acid molecule, the nucleic acid sequence encoding the light chain is shown in SEQ ID NO.14, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.14.

Similarly, with regard to homology of nucleic acid sequences, deletions or insertions of a small number of nucleotides, or nucleotide mutations, as long as they do not affect the properties and functions of the encoded antibodies, or antigen-binding fragments thereof, fall within the scope of within the protection scope of the present invention.

Another aspect of the present invention provides a vector comprising the above-mentioned nucleic acid molecule.

In a preferred embodiment of the present invention, the vector further comprises an expression control sequence linked to the above-mentioned nucleic acid molecule.

The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can be transformed, transduced or transfected into a host cell so that the elements of genetic material it carries are expressed in the host cell. Vectors may contain various elements to control expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, reporter genes, and the like. Additionally, the vector may also contain an origin of replication site. The carrier may also include components to assist its entry into the cell, such as viral particles, liposomes or protein coats, but not only these substances. In embodiments of the present invention, the vector may be selected from, but is not limited to, plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes YAC, bacterial artificial chromosomes BAC or P1-derived artificial chromosomes PAC), phage (such as lambda phage or M13 phage) and animal viruses used as vectors, for example, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (eg, herpes simplex virus), poxviruses, baculoviruses, Papillomavirus, papillomavirus (eg SV40).

Another aspect of the present invention provides a host cell comprising the above-mentioned vector.

Regarding the "host cell", one can choose, but is not limited to: prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, S2 fruit fly cells or insect cells such as Sf9, or fibroblasts, CHO cells, COS Cells, NSO cells, HeLa cells, BHK cells, HEK293 cells and other animal cell models.

Preferably, the host cells are HEK293 cells.

Another aspect of the present invention provides a method for producing the above-mentioned antibody, or an antigen-binding fragment thereof, wherein the above-mentioned host cell is cultured to produce the above-mentioned antibody, or an antigen-binding fragment thereof.

Another aspect of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the above-mentioned antibody, or an antigen-binding fragment thereof.

In a preferred embodiment of the present invention, the pharmaceutical composition comprises a therapeutically effective amount of the antibody, or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier or diluent. Those skilled in the art can use a suitable pharmaceutical carrier or diluent, in combination with a therapeutically effective amount of the antibody, or an antigen-binding fragment thereof, to administer to a patient for the treatment or prevention of diseases caused by coronavirus.

Another aspect of the present invention provides the use of the above-mentioned antibody, or an antigen-binding fragment thereof, or the above-mentioned pharmaceutical composition, in the preparation of a medicine for treating or preventing diseases caused by coronavirus.

In a preferred embodiment of the present invention, the use refers to the use in the preparation of a medicament for the treatment or prevention of diseases caused by SARS-CoV-2, SARS-CoV or SARS-like coronavirus.

Another aspect of the present invention provides the use of the above-mentioned antibody, or antigen-binding fragment thereof, or the above-mentioned pharmaceutical composition, in the treatment or prevention of diseases caused by coronavirus.

In a preferred embodiment of the present invention, the use refers to the use in the treatment or prevention of diseases caused by SARS-CoV-2, SARS-CoV or SARS-like coronaviruses.

The SARS-CoV-2 virus, also known as Severe Acute Respiratory Syndrome Coronavirus 2 (Severe Acute Respiratory Syndrome Coronavirus 2), belongs to the family Coronaviridae and belongs to the genus Betacoronavirus, which can cause severe respiratory infections. Since the outbreak in 2019, a pandemic has formed around the world, and many mutant strains have been formed as the virus spreads widely and rapidly in the population. In addition to the original SARS-CoV-2 virus that appeared in 2019, the SARS-CoV-2 virus also includes, but is not limited to, Alpha mutant strain B.1.1.7, Beta mutant strain B.1.351, Gamma mutant strain P1, Kappa mutant B.1.617.1, Delta mutant B.1.617.2, Delta mutant derivative B.1.617.2.1, Iota mutant B.1.526, Epsilon mutant B.1.427, Epsilon mutant derivative B.1.429 , Eta mutant strain B.1.525, and Zeta mutant strain P.2 and so on.

The SARS-CoV virus, also known as Severe Acute Respiratory Syndrome Coronavirus (Severe Acute Respiratory Syndrome Coronavirus), belongs to the same genus of betacoronavirus as the SARS-CoV-2 virus; the SARS-CoV virus includes, but is not limited to Tor2 , SZ3, SZ1, BJ01, WH20, GZ-C, GZ0402, Sin852, Sin01-11, Urbani, HGZ8L1-A, GD01, PC4-127, PC4-13 and PC4-137, etc.

The SARS-like coronavirus includes, but is not limited to, GD-Pangolin, RaTG13, GX-Pangolin, GD03T0013, LYRa11, WIVI, Rs7327, Rs4231, RsSHC014 and Rs4084, etc.

One aspect of the present invention also provides a method for treating or preventing a disease caused by a coronavirus, the method comprising administering to a patient a therapeutically effective amount of the above-mentioned antibody, or an antigen-binding fragment thereof; A pharmaceutical composition comprising an amount of the above-mentioned antibody, or an antigen-binding fragment thereof. Preferably, the disease caused by the coronavirus is a disease caused by SARS-CoV-2, SARS-CoV or SARS-like coronavirus.

Another aspect of the present invention provides a detection product, wherein the detection product comprises the above-mentioned antibody, or an antigen-binding fragment thereof; the detection product is used to detect the presence or level of coronavirus in a sample.

In a specific embodiment of the present invention, the detection products include, but are not limited to, detection reagents, detection kits, detection chips or test strips, and the like.

The above-mentioned antibodies or antigen-binding fragments thereof of the present invention can be labeled by chemical methods or genetic engineering methods, and the labeled antibodies or antigen-binding fragments thereof can be used for detection; the labeled antibodies or antigen-binding fragments thereof fall within the scope of the present invention within the scope of protection.

The specific detection method can adopt the following steps: 1) providing a sample; 2) contacting the sample with the antibody or its antigen-binding fragment that specifically binds to the coronavirus of the present invention; 3) detecting the sample and the antibody or its antigen Immunoreactivity between binding fragments.

The inventors of the present invention have obtained an antibody that specifically binds to coronavirus and an antigen-binding fragment thereof by using in vitro monoclonal culture of B cells and high-throughput antibody screening technology. Coronaviruses including SARS, for SARS-CoV-2 new coronavirus mutants including SARS-CoV-2 British mutant B.1.1.7, Delta mutant B.1.617.2 and Beta mutant B.1.351 Both have broad-spectrum and potent neutralizing ability, and have good prospects for clinical application in the future.

Description of drawings

Figure 1 shows the results of SDS-PAGE detection of the expressed and purified antibody;

Figure 2 shows the detection results of monoclonal antibody GW01 binding to S1 and RBD proteins of SARS-CoV-2 virus;

Figure 3 shows the detection results of monoclonal antibody GW01 binding to S1 and RBD proteins of SARS-CoV virus;

Fig. 4 is the affinity detection result of monoclonal antibody GW01 binding to the RBD protein of SARS-CoV2 virus;

Figure 5 is a fitting curve diagram of the inhibition rate of different concentrations of monoclonal antibody GW01 on the live virus of SARS-CoV-2 new coronavirus;

Figure 6 is a fitting curve diagram of the inhibition rate of different concentrations of mAb GW01 on the live virus of the Alpha mutant strain B.1.1.7 of SARS-CoV-2;

Figure 7 is a fitting curve diagram of the inhibition rate of different concentrations of monoclonal antibody GW01 on the live virus of SARS-CoV-2 Beta mutant strain B.1.351;

Figure 8 is a fitting curve diagram of the inhibition rate of different concentrations of mAb GW01 on the live virus of the Delta mutant strain B.1.617.2 of SARS-CoV-2;

Figure 9. Fitting curve diagram of the inhibition rate of different concentrations of mAb GW01 on the live virus of Eta mutant strain B.1.525 of SARS-CoV-2.

detailed description

The present invention will be described in detail below with reference to the specific embodiments shown in the accompanying drawings. However, these embodiments do not limit the present invention, and structural, method, or functional changes made by those skilled in the art according to these embodiments are all included in the protection scope of the present invention.

Example embodiments will now be described more fully with reference to the accompanying drawings. Example embodiments, however, can be embodied in various forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of example embodiments to those skilled in the art.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. Those who do not indicate specific technology or conditions in the embodiment, according to the technology or conditions described in the literature in this area (for example, refer to J. Sambrook et al., "Molecular Cloning Experiment Guide" third edition translated by Huang Peitang et al. Press) or follow the product manual.

Example 1: Screening and detection of antibodies that specifically bind to coronaviruses

The inventor conducted pseudovirus analysis on the plasma of patients with novel coronavirus pneumonia (follow-up two weeks after recovery and discharge) who were admitted to the inventor's unit (Shanghai Public Health Clinical Center) from January 20, 2020 to February 26, 2020. and experimental screening, it was found that the serum of one of the mild patients had strong neutralizing activity against SARS-CoV-2 pseudovirus. With the written consent of the ethics committee of the inventor's unit and the patient himself, the peripheral blood was drawn for research.

1. Sorting of peripheral blood memory B cells

1) Separation of peripheral blood lymphocytes: After extracting the peripheral blood of the above-mentioned patients during the recovery period and mixing with an equal amount of normal saline, the lymphocyte separation liquid Lymphoprep (Stemcell Technologies, Item No. 07851) was used to separate peripheral blood lymphocytes. For the operation process, see Lymphocyte Separation liquid manual.

2) Sorting peripheral blood memory B cells: The peripheral blood lymphocytes isolated in the above step 1) were stained with an antibody mixture at 4°C and in the dark for 30 min, wherein the antibody mixture was anti-CD19-PE-Cy7 (BD Bioscience), IgA - Mixture consisting of APC (Jackson Immunoresearch), IgD-FITC (BD Bioscience) and IgM-PE (Jackson Immunoresearch); after staining, washed with 10 ml PBS-BSA buffer and resuspended in 500 μl PBS-BSA; finally with FACSAria The III cell sorter (Becton Dickinson) sorted CD19+IgA-IgD-IgM-memory B cells.

2. Incubation of peripheral blood memory B cells

The above-sorted CD19+IgA-IgD-IgM-memory B cells were resuspended in cultures containing 10% FBS and 100U/ml IL-2, 50ng/ml IL-21 and irradiated 3T3-msCD40L feeder cells memory B cells were seeded in a 384-well microtiter plate at a density of 4 cells/well (final volume was 50 μl), and incubated for 13 days; growth factors IL-2 and IL-21 stimulated the division and growth of memory B cells, Antibodies are secreted into the incubated medium. For specific culture methods, see the reference Huang J et al. Nature Protocols 2013, 8(10): 1907-15.

3. Production of SARS-CoV-2 and SARS-CoV pseudoviruses

SARS-CoV-2 and SARS-CoV pseudoviruses are non-replication-defective retroviral particles with SARS-CoV-2 and SARS-CoV spike membrane proteins (Spike, S), respectively, on the surface and carrying a luciferase reporter gene , they can simulate the infection process of SARS-CoV-2 and SARS-CoV virus to host cells (such as human liver cancer cell line Huh-7, 293T cell line 293T-ACE2 stably expressing human ACE2 receptor), and in the infected cells Internally expressed luciferase reporter gene. Since pseudovirus infection cannot produce mature virus particles, it can be safely performed in a biosafety secondary laboratory.

SARS-CoV-2 and SARS-CoV pseudoviruses were obtained by co-transfecting 293T cells with their respective S protein expression plasmids and an HIV Env-deficient backbone plasmid (pNL4-3.Luc.R-E-) with a luciferase reporter gene, respectively. The S gene sequences of SARS-CoV-2 and SARS-CoV were designed according to NCBI GenBank sequences NC_045512 and ABD72979.1. After codon optimization, the gene sequences were synthesized by Nanjing GenScript and connected to the pcDNA3.1 eukaryotic expression vector. Constructed into SARS-CoV-2 and SARS-CoV S protein expression plasmids. The pNL4-3.Luc.R-E-backbone plasmid was derived from the NIH AIDS Reagent Program in the United States. All plasmids were amplified by transforming DH5α competent cells, and purified by using the plasmid purification kit produced by MegiBio. The purification operation process refers to the kit instructions.

293T cells were cultured in DMEM medium containing 10% fetal bovine serum (Gibco) and seeded into 10 cm cell dishes before transfection. After 24 hours of culture, the backbone plasmid (pNL4-3.Luc.RE-) was co-transfected with the plasmid expressing SARS-CoV or SARS-CoV-2 at a ratio of 3:1 using EZ Trans cell transfection reagent (Liji Biotechnology). 293T cells were transfected. For the detailed transfection method, please refer to the instruction manual of EZ Trans cell transfection reagent. 48 hours after transfection, the supernatant containing pseudovirus was collected, centrifuged at 1500 rpm for 10 minutes to remove cell debris, and then aliquoted and stored in a -80°C refrigerator for the detection of neutralizing antibodies.

4. Neutralization screening

After peripheral blood memory B cells were cultured for 13 days in vitro, 40 μl of culture supernatant was collected from each well for the detection of SARS-CoV-2 and SARS-CoV neutralizing antibodies. The detection method is as follows: take 20 μl of the culture supernatant and 20 μl of the pseudovirus supernatant obtained from the above production, mix them in a 384-well cell culture plate, and incubate at room temperature for 30 minutes, add 50 μl of 5000 293T-ACE2 cells to each well and continue to culture the cells. Cultured in a cell incubator. After 48 hours, use a luciferase detection kit (Luciferase Assay System, Promega Cat. #E1500) to lyse the cells and detect the luciferase activity in each well. The specific detection method refers to the kit instructions. The chemiluminescence RLU value of each well was detected by a multifunctional microplate reader (Perkin Elmer). According to the ratio of the culture supernatant to the virus control RLU value, the neutralization inhibition percentage of the culture supernatant to the pseudovirus was calculated, and the wells with the inhibition percentage greater than 90% were screened as virus neutralization positive wells.

5. RT-PCR amplification of heavy and light chain genes

For the virus neutralization-positive well B cells obtained by the above screening, RT-PCR was used to amplify the variable regions of the heavy and light chains of immunoglobulin genes.

For the primer design and specific operation process of RT-PCR, see the reference Tiller, T. et al. J. Immunol Methods 2018, 329: 112–124. Primers include forward and reverse primers specific for the leader and constant regions of IgG.

The amplified antibody heavy chain variable region DNA and light chain variable region DNA were purified and recovered by agarose gel electrophoresis, and then cloned into the PMD19-T vector using the PMD19-T vector cloning kit (Takara 6013). Refer to the kit instructions for the process, and select single clones for gene sequencing.

After sequencing, the heavy chain variable region DNA sequence is shown in SEQ ID NO.9; the light chain variable region DNA sequence is shown in SEQ ID NO.10.

6. Expression and purification of monoclonal antibodies

The correctly sequenced antibody heavy chain variable region DNA and pCMV/R-10E8 heavy chain gene (NIH AIDS Reagent Program Cat 12290) were digested with Age I and Sal I respectively, and the recovered target fragments were ligated and purified and transformed into DH5α receptors. Construct antibody expression heavy chain plasmids in live cells;

The correctly sequenced antibody light chain variable region DNA and pCMV/R-10E8 light chain gene (NIH AIDS Reagent Program Cat 12291) were digested with Age I and Xho I respectively, and the recovered target fragments were ligated and purified and transformed into DH5α receptors Construct the antibody expression light chain plasmid in the state cell;

The antibody heavy chain and light chain plasmids were purified by the plasmid purification kit (MegiBio) (see Figure 1 for the SDS-PAGE detection results of the expressed and purified antibody), and EZ Trans cell transfection reagent (Liji Bio) was used to 1: A ratio of 1 was expressed in co-transfected 293T cells. After 72 hours, the cell transfection supernatant was collected, and the antibody IgG in the supernatant was purified using a protein-G column (Tiandiren Biotechnology Co., Ltd., Changzhou). The purification method was based on the instructions for use of the protein-G column. Absorbance at 280 nm was measured using Nanodrop 2000 (Thermo Fisher) and antibody concentration was calculated. The obtained antibody IgG (designated as mAb GW01) was purified.

The antibody heavy chain and light chain plasmids were handed over to Beijing Liuhe Huada Gene Technology Co., Ltd. for nucleic acid sequence sequencing. After determination, the amino acid sequence of the heavy chain of the monoclonal antibody GW01 is shown in SEQ ID NO.11, and the amino acid sequence of the light chain is shown in SEQ ID NO.11. ID NO.12. Correspondingly, the nucleic acid sequence of the heavy chain of monoclonal antibody GW01 is shown in SEQ ID NO.13, and the nucleic acid sequence of the light chain is shown in SEQ ID NO.14.

According to the IMGT numbering system scheme, the amino acid sequence of the heavy chain variable region of monoclonal antibody GW01 is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

Regarding the heavy and light chain variable regions of the obtained monoclonal antibody GW01, CDR positions can be determined using the CDR numbering scheme used by those skilled in the art (eg, Kabat, Chothia or IMGT numbering scheme). Specifically, in this example, the IMGT numbering system scheme was used to determine the heavy chain variable region CDR1-3 and the light chain variable region CDR1-3 of the monoclonal antibody GW01.

The amino acid sequence of heavy chain variable region CDR1 is shown in SEQ ID NO.1, the amino acid sequence of heavy chain variable region CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of heavy chain variable region CDR3 is shown in SEQ ID NO.2. 3 shown.

The amino acid sequence of light chain variable region CDR1 is shown in SEQ ID NO.4, the amino acid sequence of light chain variable region CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of light chain variable region CDR3 is shown in SEQ ID NO.5. 6 shown.

In addition, those skilled in the art can, according to the above-mentioned CDR numbering system scheme, to the framework region in the heavy chain variable region or the light chain variable region in the monoclonal antibody GW01, for the heavy chain variable region or the light chain variable region in the framework region, In the CDR region, conservative amino acid substitutions (deletion or insertion of a small number of amino acids, or amino acid mutation, or substitution of similar amino acids) are performed, as long as the variant retains the original properties and functions of the heavy chain variable region or light chain variable region, These variants also fall within the scope of the present invention.

Similarly, conservative amino acid substitutions (deletion or insertion of a small number of amino acids, or amino acid mutation, or substitution of similar amino acids) are performed on the amino acid sequences of the heavy and light chains of mAb GW01, as long as the variant retains the variable heavy chain The original properties and functions of the light chain variable region or light chain variable region, these variants also fall within the scope of the present invention.

performance data

1. The monoclonal antibody GW01 of this application recognizes the detection of S1 and RBD proteins of SARS-CoV-2 and SARS-CoV virus

The recognition of S1 and RBD proteins of SARS-CoV-2 and SARS-CoV virus by the monoclonal antibody GW01 obtained by the above purification was detected by enzyme-linked immunosorbent assay (ELISA).

The detection method was as follows: 1 μg/ml of antigenic protein (Yiqiao Shenzhou) was coated in a 96-well ELISA plate, overnight at 4°C. The plate was washed 5 times with PBS-T solution (0.2% Tween-20), and 300 μl of blocking solution (PBS, 1% FBS, 5% milk) was added to each well to block for 1 hour at room temperature. The plate was washed three times with PBS-T, and the monoclonal antibody GW01 was serially diluted 5 times with PBS diluent (PBS, 5% FBS, 2% BSA, 1% Tween-20), and 100 μl samples were added to the ELISA plate, 37 Incubate for 1 hour. The plate was washed 5 times with PBS-T, and 100 μl of horseradish peroxidase-labeled goat anti-human IgG antibody (Jackson Immunoresearch) diluted with PBS diluent 1:2500 was added to each well, and incubated at room temperature for 1 hour. The plate was washed 5 times with PBS-T, 150 μl of ABTS chromogenic substrate (Thermo Fisher) was added, and after 30 minutes of color development in the dark at room temperature, the absorbance value at 405 nm wavelength was read by a microplate reader.

The detection results are shown in Figure 2 and Figure 3, where Figure 2 shows the detection results of the monoclonal antibody GW01 binding to the S1 and RBD proteins of the SARS-CoV-2 virus, and Figure 3 is the detection results of the monoclonal antibody GW01 binding to the S1 and RBD proteins of the SARS-CoV-2 virus. Test results.

It can be seen from Figure 2 that the monoclonal antibody GW01 can bind to the conserved RBD region of the S1 protein of the SARS-CoV-2 virus; it can be seen from Figure 3 that GW01 can bind to the conserved region of the RBD of the S1 protein of the SARS-CoV virus; it can be inferred from this So, the monoclonal antibody GW01 of this application has broad-spectrum neutralization ability for coronavirus.

2. The binding ability of the monoclonal antibody GW01 of this application to the RBD protein of SARS-CoV-2 virus was detected by biofilm layer interference technology

In order to detect the interaction between the monoclonal antibody GW01 of this application and the SARS-CoV-2 RBD protein, the biofilm layer interference technology was used to detect the binding kinetics between them, and the detection process was carried out on an OctetRED96 (Fortebio) instrument.

The detection method is as follows: soak the AHC probe in sterile water for 10 minutes to equilibrate, and the detection process is all carried out under the reaction conditions of 30 °C. It can be divided into the following five steps: 1) Zero adjustment, immerse the probe in the Activate in sterile water for 60 seconds to obtain the detection baseline; 2) immerse the probe in 10 μg/ml GW01 antibody solution and act for 200 seconds to capture the antibody; 3) adjust to zero again, immerse the probe in buffer (add 0.02% Tween20 PBS solution) for 120 seconds to remove unbound antibodies; 4) To bind RBD, immerse the probe into RBD protein solution with an initial concentration of 111.1 nM and 3-fold serial dilution, and act for 300 seconds to obtain mAb GW01 that binds to RBD 5) Binding and dissociation, put the probe into the buffer for 300 seconds. The binding of the protein causes the change of the thickness of the biofilm, which causes the relative displacement of the interference light wave, which is detected by the spectrometer and forms an interference spectrum, which is displayed as the real-time displacement (nm) of the interference spectrum. In this way, the dynamic curve of the binding and dissociation of RBD to the monoclonal antibody GW01 of the present application was detected. During data analysis, the data of the sample wells were subtracted from the data of the buffer control wells, the non-specific interference of the buffer solution was deducted, and the 1:1 binding model was used to perform the overall curve fitting for the binding of GW01 at different dilution concentrations of RBD to obtain Average association constant _Kon , dissociation constant _Koff and affinity constant _KD values.

The detection results are shown in Figure 4, and five curves represent the dynamic binding and dissociation curves of the monoclonal antibody GW01 of the present application and five different concentrations of RBD, showing that the binding of the monoclonal antibody GW01 of the present application and RBD is concentration gradient dependent; the monoclonal antibody GW01 of the present application After binding with RBD, it dissociates, the dissociated RBD is very small, and the K _D value is (0.65±0.02) nM, which shows that the monoclonal antibody GW01 of this application has a very strong affinity with the conserved region of RBD of SARS-CoV-2 . From this, it can be inferred that the fact that the monoclonal antibody GW01 of the present application has a very strong neutralizing activity against the conserved RBD region of the SARS-CoV-2 virus is due to the fact that the monoclonal antibody GW01 of the present application has a very high affinity for the conserved region of the RBD of the coronavirus. . Combined with the results of Figure 2 and Figure 3, it is further verified that the monoclonal antibody GW01 of the present application has broad-spectrum neutralization ability for coronavirus.

3. Detection of the neutralizing activity of the monoclonal antibody GW01 of this application to SARS-CoV-2, SARS-CoV and bat SARS-like coronaviruses

Inhibition of pseudovirus infection of Huh-7 and 293T-Ace2 cells by different concentrations of mAb GW01 in 96-well cell plates -CoV-WIV1) and RS3367 viruses.

The detection method is as follows: 1) Huh-7 or 293T-ACE2 cells were seeded in a 96-well cell plate, 1×10 ⁴ cells were seeded in each well, and cultured in a cell incubator at 37°C, 5% CO ₂ for 24 hours; 2) Monoclonal antibody GW01 was inoculated with The cell culture medium was diluted to different concentrations, mixed with an equal volume of pseudovirus dilution containing 100 TCID50, and incubated at 37°C for 1 hour; 3) Discard the cell culture medium, add 50 μl of virus-antibody complex to each well, set up duplicate wells, and set at the same time Antibody-free group, virus-free group and positive serum control group; 4) After culturing for 12 hours, add 150 μl of maintenance solution to each well, and continue to culture at 37°C for 48 hours; 5) Use a luciferase detection kit (Luciferase Assay System, Promega Cat. #E1500) Lyse the cells and detect the luciferase activity of each well, the specific detection method refers to the kit instructions; use a multi-function microplate reader (Perkin Elmer) to detect the chemiluminescence RLU value of each well; 6) According to the antibody and virus control RLU value The percentage of neutralization inhibition of different concentrations of antibodies against pseudoviruses was calculated from the ratio of , and the half-inhibitory dose IC50 of the antibody against the virus was calculated using PRISM7 software (GraphPad).

The results are shown in Table 1 below.

Table 1

It can be seen from Table 1 that at the concentration of ng/ml, the monoclonal antibody GW01 can not only neutralize SARS-CoV-2 virus well, but also has good neutralization effect on SARS coronavirus and bat SARS-like coronavirus. and activity; this shows that the monoclonal antibody GW01 of this application has a strong broad-spectrum neutralization ability against coronavirus.

4. The monoclonal antibody GW01 of this application is for SARS-CoV-2 live virus , SARS-CoV-2 Beta mutant strain B.1.351, Alpha mutant strain B.1.1.7, Delta mutant strain B.1.617.2, Eta mutant strain B .1.525 Results of Neutralizing Activity of Live Toxins

The experiment involved live virus, so it was done in the Biosafety Level 3 Facility (BSL-3) of the State Key Laboratory of Respiratory Diseases at the Wuhan Institute of Virology, Chinese Academy of Sciences and the National Clinical Research Center for Respiratory Diseases, Guangzhou Institute of Respiratory Health.

In this experiment, the neutralization effect of the monoclonal antibody GW01 of the present application on the live virus of SARS-CoV-2 and the live virus of SARS-CoV-2 mutant was analyzed by measuring the plaque reduction on Vero-E6 cells. The experimental operation is roughly as follows: serially diluted monoclonal antibody GW01 and 100 plaque forming units (pfu) of live virus were incubated at 37°C for 30 minutes; the mixture was added to the monolayer of Vero-E6 cells at 37°C. After 1 hour of adsorption, the supernatant was removed, and a 0.9% methylcellulose overlay was added. After 3 days of incubation, plaques were visualized and counted by fixation with 4% formaldehyde and stained with 0.5% crystal violet. Calculate the percentage of live virus of different concentrations of mAb GW01 against SARS-CoV-2 new coronavirus and the inhibition percentage of live virus of mutant strains.

The results are shown in Figure 5 (inhibition rate of different concentrations of mAb GW01 on the live virus of SARS-CoV-2), Figure 6 (different concentrations of mAb GW01 on Alpha mutant strain B.1.1.7 of SARS-CoV-2) Inhibition rate of live virus), Figure 7 (inhibition rate of different concentrations of mAb GW01 on the live virus of SARS-CoV-2 Beta mutant B.1.351), Figure 8 (different concentrations of mAb GW01 on SARS-CoV-2 Inhibition rate of Delta mutant strain B.1.617.2 of 2), Figure 9 (inhibition rate of different concentrations of monoclonal antibody GW01 on the live virus of Eta mutant strain B.1.525 of SARS-CoV-2).

Using PRISM7 software (GraphPad), the IC50 value calculated from the results in Figure 5 was 0.519 μg/mL; the IC50 value calculated from the results in Figure 6 was 0.398 μg/mL; the IC50 value calculated from the results in Figure 7 was 0.576 μg/mL mL; the IC50 value calculated from the results in Figure 8 was 0.350 μg/mL; the IC50 value calculated from the results in Figure 9 was 0.281 μg/mL.

It can be seen from the data obtained in Figures 5-9 that the monoclonal antibody GW01 of the present application is effective against the live virus of SARS-CoV-2 and SARS-CoV-2 mutant strains B.1.1.7, The live toxins of B.1.351, B.1.617.2 and B.1.525 all have good neutralizing activity.

In particular, the Beta mutant B.1.351 of the SARS-CoV-2 new coronavirus is a recently reported immune escape strain that cannot be neutralized by a variety of known neutralizing antibodies (such as REGN10989, which is currently FDA-approved for clinical treatment of COVID-19, etc. Antibodies); the Delta mutant B.1.617.2 of the SARS-CoV-2 new coronavirus is a strain with both fast transmission and immune escape. However, it can be seen from the results in Figures 7 and 8 that the monoclonal antibody GW01 of the present application has significant neutralizing activity against both the Beta mutant strain B.1.351 and the Delta mutant strain B.1.617.2.

In summary, the monoclonal antibody GW01 of the present application is suitable for coronaviruses including SARS-CoV-2, SARS-CoV or SARS-like, and for SARS-CoV-2 Alpha mutant strain B.1.1.7, Delta mutant strain B. SARS-CoV-2 mutant strains including 1.617.2 and Beta mutant strain B.1.351 have broad-spectrum and potent neutralizing ability, and have good clinical application prospects in the future.

Application example

This application example describes a method that can be used to treat diseases caused by coronaviruses, including SARS-CoV-2, by administering the coronavirus-specific monoclonal antibodies of the present application.

Although specific methods, dosages and modes of administration are provided, those skilled in the art will understand that changes may be made without materially affecting treatment. Based on the guidelines disclosed herein, a coronavirus infection can be treated or prevented by administering a therapeutically effective amount of one or more of the antibodies described herein, thereby reducing or eliminating a coronavirus infection.

The specific application method is as follows:

1) Screening subjects: Subjects are first screened to determine whether they are infected with coronaviruses such as SARS-CoV-2. The method used to screen for SARS-CoV-2 infection can use nucleic acid detection of respiratory specimens combined with clinical CT diagnosis. (Note: The antibodies and their antigen-binding fragments that specifically bind to coronaviruses claimed in this application can also be used for detection products, that is, detection products for screening SARS-CoV-2 coronavirus infection, such as detection kits).

Detection of SARS-CoV-2 coronavirus nucleic acid or viral S protein in a subject's respiratory specimen indicates that the subject is infected with SARS-CoV-2.

2) Pre-treatment of the subject: In particular embodiments, the subject is treated prior to administration of a therapeutic agent comprising one or more antiviral drug therapies known to those of skill in the art. However, such pre-treatment is not always required and can be determined by the skilled clinician.

3) Administration of the therapeutic composition

After screening the subject, the above-mentioned therapeutically effective dose of the coronavirus-specific monoclonal antibody of the present application is administered to the subject (such as an adult at risk of being infected with SARS-CoV-2 coronavirus or known to be infected with SARS-CoV-2 coronavirus. or newborn babies). Additional drugs, such as antiviral agents, can be administered to the subject at the same time as, before, or after administration of the disclosed agents. Administration is accomplished by any method known in the art such as oral administration, inhalation, intravenous, intramuscular, intraperitoneal or subcutaneous. To prevent, reduce, inhibit and/or treat the condition of a subject, the amount of the composition administered will depend on the subject being treated, the severity of the disorder and the mode of administration to the subject being treated. Ideally, a therapeutically effective amount of an agent is an amount sufficient to prevent, reduce, and/or inhibit, and/or treat a condition in a subject without causing substantial cytotoxic effects in the subject. An effective amount can be readily determined by one of skill in the art, eg, using routine experiments to establish dose-response curves. Likewise, these compositions can be formulated with inert diluents or pharmaceutically acceptable carriers. In a specific example, the antibody is administered at 5 mg/kg every two weeks or 10 mg/kg every two weeks, depending on the particular stage of SARS-CoV-2 viral infection. In one example, the antibody is administered continuously. In another example, the antibody or antibody fragment is administered at 50 μg per kg twice a week for 2-3 weeks. Therapeutic compositions can be administered chronically (eg, over a period of months or years).

4) Evaluation

Subjects infected with SARS-CoV-2 are monitored for reduction in the level of SARS-CoV-2 virus, or reduction in one or more clinical symptoms associated with COVID-19 disease, following administration of one or more therapies. In certain instances, starting after 2 days of treatment, subjects are subjected to one or more analyses. Subjects are monitored using any method known in the art. For example, biological samples including throat swabs from subjects can be obtained and assessed for changes in SARS-CoV-2 virus levels.

5) Additional treatment

In specific embodiments, if the subject is stable or has a minor, mixed or partial response to treatment, additional treatment may be administered after re-evaluation with the same regimen and formulation of substances that they had previously received for the desired period of time.

It should be understood that although this specification is described in terms of embodiments, not every embodiment only includes an independent technical solution, and this description in the specification is only for the sake of clarity, and those skilled in the art should take the specification as a whole, and each The technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

The series of detailed descriptions listed above are only specific descriptions for the feasible embodiments of the present invention, and they are not used to limit the protection scope of the present invention. Changes should all be included within the protection scope of the present invention.

Claims (23)

1. An antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, characterized in that:

   Condition a): The heavy chain variable region comprises the CDR1 sequence of the heavy chain variable region as shown in SEQ ID NO.1, the CDR2 sequence of the heavy chain variable region as shown in SEQ ID NO.2 and the CDR2 sequence of the heavy chain variable region as shown in SEQ ID NO. . the CDR3 sequence of the heavy chain variable region shown in 3;

   Condition b): the light chain variable region comprises the CDR1 sequence of the light chain variable region as shown in SEQ ID NO.4, the CDR2 sequence of the light chain variable region as shown in SEQ ID NO.5 and the CDR2 sequence of the light chain variable region as shown in SEQ ID NO. . the CDR3 sequence of the light chain variable region shown in 6;

   The heavy chain variable region of the antibody or antigen-binding fragment thereof satisfies condition a); or,

   The light chain variable region of the antibody or antigen-binding fragment thereof satisfies condition b); or,

   The antibody or antigen-binding fragment thereof satisfies both conditions a) and b).
2. The antibody of claim 1, or an antigen-binding fragment thereof, characterized in that:

   The sequence of the heavy chain variable region is shown in SEQ ID NO.7, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.7.
3. The antibody of claim 1, or an antigen-binding fragment thereof, characterized in that:

   The sequence of the light chain variable region is shown in SEQ ID NO.8, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.8.
4. The antibody of claim 1, or an antigen-binding fragment thereof, characterized in that:

   The antibody or antigen-binding fragment thereof satisfies both conditions a) and b);

   The sequence of the variable region of the heavy chain is shown in SEQ ID NO.7, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.7; and,

   The sequence of the light chain variable region is shown in SEQ ID NO.8, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.8.
5. The antibody of claim 4, or an antigen-binding fragment thereof, characterized in that:

   The heavy chain amino acid sequence of the antibody or its antigen-binding fragment is shown in SEQ ID NO.11, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.11; and,

   The light chain amino acid sequence of the antibody or its antigen-binding fragment is shown in SEQ ID NO.12, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.12.
6. The antibody, or antigen-binding fragment thereof, according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that specifically binds to a coronavirus.
7. The antibody of claim 6, or an antigen-binding fragment thereof, characterized in that:

   The antibody or its antigen-binding fragment is a coronavirus neutralizing antibody or its antigen-binding fragment.
8. The antibody of claim 6, or an antigen-binding fragment thereof, characterized in that:

   The antibody is a monoclonal antibody.
9. The antibody of claim 8, or an antigen-binding fragment thereof, wherein the antibody is a fully human monoclonal antibody.
10. The antibody of claim 9, or an antigen-binding fragment thereof, wherein the antibody is any one or a combination of IgG1, IgG2, IgG3 or IgG4.
11. The antibody of claim 1, or an antigen-binding fragment thereof, wherein the antigen-binding fragment is Fv, Fab, F(ab') ₂ , Fab', dsFv, scFv or sc(Fv) ₂ .
12. A nucleic acid molecule, characterized in that: the nucleic acid molecule encodes the antibody according to any one of claims 1 to 11, or an antigen-binding fragment thereof.
13. The nucleic acid molecule of claim 12, wherein:

    In the nucleic acid molecule, the nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO.9, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.9.
14. The nucleic acid molecule of claim 12, wherein:

    In the nucleic acid molecule, the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO.10, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.10.
15. The nucleic acid molecule of claim 12, wherein:

    In the nucleic acid molecule, the nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO.13, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.13; and,

    In the nucleic acid molecule, the nucleic acid sequence encoding the light chain is shown in SEQ ID NO.14, or it has more than 80% sequence homology with the sequence shown in SEQ ID NO.14.
16. A vector comprising the nucleic acid molecule of any one of claims 12 to 15.
17. A host cell comprising the vector of claim 16.
18. The host cell of claim 17, wherein the host cell is a 293 cell.
19. A pharmaceutical composition, characterized in that: the pharmaceutical composition comprises the antibody according to any one of claims 1 to 11, or an antigen-binding fragment thereof.
20. Use of the antibody according to any one of claims 1 to 11, or an antigen-binding fragment thereof, or the pharmaceutical composition according to claim 19, in the preparation of a medicament for the treatment or prevention of diseases caused by coronavirus.
21. The use according to claim 20, characterized in that: the use refers to the use in the preparation of medicines for the treatment or prevention of diseases caused by SARS-CoV-2, SARS-CoV or SARS-like coronaviruses.
22. A detection product, characterized in that: the detection product comprises the antibody that specifically binds to coronavirus according to any one of claims 1 to 11, or an antigen-binding fragment thereof.
23. A kind of method of producing the antibody of specificity binding coronavirus as described in any one of claim 1 to 11, or its antigen-binding fragment, it is characterized in that: cultivate the host cell as claimed in claim 17, to produce all said antibody, or an antigen-binding fragment thereof.

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