Antibodies or antigen-binding fragments thereof to coronavirus

Abstract

The present invention relates to antibodies or antigen-binding fragments thereof against coronaviruses, nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, vectors comprising the nucleic acid molecules, host cells comprising the vectors, and the use of the antibodies or antigen-binding fragments thereof in preparing medicaments for treating or preventing diseases caused by coronaviruses, as well as their use in detection products. The inventors have obtained a series of antibodies and antigen-binding fragments thereof against coronaviruses using in vitro monoclonal culture of B cells and high-throughput antibody screening technology. These antibodies and antigen-binding fragments thereof have potent binding and neutralizing abilities for the SARS-CoV-2 virus, and are all able to recognize and bind to the S1 protein and RBD of the SARS-CoV-2 virus with very strong affinity. It can be inferred that these antibodies may also have binding and neutralizing abilities for other coronaviruses, as well as coronaviruses that may appear in the future, and have good prospects for clinical application in the future.

Description

Technical Field

[0001] The present invention relates to a coronavirus antibody or an antigen-binding fragment thereof, a nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof, a vector comprising the nucleic acid molecule, a host cell comprising the vector, and the use of the antibody or the antigen-binding fragment thereof in preparing a medicament for treating or preventing diseases caused by coronavirus, as well as the use of the antibody or the antigen-binding fragment thereof in detection products, and belongs to the field of biomedicine. Background Art

[0002] Novel coronavirus pneumonia (2019-nCoV) is an acute respiratory infectious disease caused by the novel coronavirus SARS-CoV-2. The virus is highly transmissible, spreading through multiple routes, including respiratory and contact, posing a severe challenge to global public health security.

[0003] The SARS-CoV-2 virus belongs to the Coronaviridae family and is a beta coronavirus, the same as the SARS coronavirus that broke out in 2003. The amino acid homology is as high as 77.2%. The main envelope protein of the SARS-CoV-2 virus is its spike protein (also known as Spike protein, referred to as S protein), which is hydrolyzed by intracellular proteases into two parts, S1 and S2, during the viral infection process. S2 is a transmembrane protein, and S1 has a receptor binding domain (RBD) that recognizes and binds to the cell receptor angiotensin-converting enzyme-2 (ACE-2). The spike protein composed of S1 and S2 is the viral receptor that the SARS-CoV-2 virus specifically recognizes, binds to the target cell receptor, and mediates viral infection. It is also the recognition target of the neutralizing antibodies to be developed.

[0004] To date, there are no effective drugs or vaccines in the world to treat and prevent SARS-CoV-2 virus infection. Clinically, only supportive symptomatic treatment can be adopted for patients with COVID-19. Studies have shown that the clinical use of virus-specific recovered human plasma can effectively neutralize the virus, prevent the virus from spreading to various organs in the body, and also play an important role in the patient's disease course. However, not only is the source of polyclonal plasma limited, but its clinical application is also restricted by conditions such as difficulty in quality control, differences in blood types between donors and recipients, and potential infectious factors. Isolating fully human monoclonal antibodies that can neutralize the SARS-CoV-2 virus from recovered COVID-19 patients can effectively overcome the above problems and is currently one of the main directions of new coronavirus drug development.

[0005] To date, multiple research teams, both domestic and international, have reported the isolation of fully human monoclonal antibodies (mAbs) that bind to the SARS-CoV-2 S protein from the peripheral blood of recovered COVID-19 patients, such as BD-368-2 and B38. These antibodies are currently in the experimental development stage. The method employed by these research teams uses recombinantly expressed SARS-CoV-2 S protein or its receptor-binding domain (RBD) as bait. B cells (memory B cells) that bind to these proteins are then isolated from the peripheral blood of recovered patients. Cell-based or single-cell sequencing methods are then used to obtain the heavy and light chain pairs of the antibodies expressed by individual B cells. After the antibodies are recombinantly expressed in vitro, their ability to neutralize the virus is then verified. Because this method pre-selects and enriches B cells with a marker protein (the recombinantly expressed SARS-CoV-2 S protein or its RBD, referred to as bait) before antibody gene sequencing, only antibodies that specifically bind to the marker protein are screened.

[0006] Dr. Huang Jinghe (one of the inventors of this application) pioneered the human B cell in vitro monoclonal culture and high-throughput antibody screening technology in 2013 (Huang J et al. Nature Protocols 2013), which isolated fully human monoclonal antibodies from the peripheral blood of patients recovered from COVID-19. The process is as follows: first, the neutralizing antibodies in the serum of patients recovered from COVID-19 are detected using the SARS-CoV-2 and SARS-CoV pseudovirus neutralization system, and patients with high neutralizing activity against both SARS-CoV-2 and SARS-CoV are screened; then, peripheral blood lymphocytes are collected from the recovered patients, and memory B lymphocytes are sorted out by flow cytometry; single B cells are inoculated into 384-well plates, and cytokines and feeder cells are added for culture. After the cultured B cells are amplified and differentiated in vitro, they secrete antibodies into the supernatant. Then, an in vitro high-throughput neutralization experiment was used to detect the neutralizing ability of the antibodies in the supernatant against SARS-CoV-2 and SARS-CoV viruses, and positive clones that could simultaneously neutralize both viruses were screened. The heavy and light chain variable regions of the antibodies were cloned using RT-PCR, constructed into antibody heavy chain and light chain expression vectors, and then transfected into 293T cells to express and purify monoclonal antibodies.

[0007] While antibodies reported by other teams have demonstrated strong neutralizing activity against the tested SARS-CoV-2 strains, SARS-CoV-2 is an RNA virus, and its genome is susceptible to mutations during transmission. Mutations in the non-conserved regions recognized by these antibodies, resulting in new circulating strains, can lead to the antibodies losing their protective effect against the mutated virus.

[0008] Therefore, those skilled in the art still hope to develop new antibodies that have binding and neutralizing capabilities against coronaviruses, including SARS-CoV-2 virus. Summary of the Invention

[0009] To solve the above technical problems, the present invention provides, on one hand, a coronavirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises three light chain complementary determining regions LCDR1, LCDR2, and LCDR3; wherein:

[0010] The general sequence formula of the HCDR1 is: GX1TVSSNY, wherein X1 is any one of L, I or F amino acids;

[0011] The general sequence formula of the HCDR2 is: X2YSGGSX3, wherein X2 is any one of L and I, and X3 is any one of A and T.

[0012] Preferably, the general sequence formula of the HCDR3 is: ARDLIX4YGMDV, wherein X4 is any amino acid of D or T;

[0013] The sequence of the LCDR1 is QGISSY, and the sequence of the LCDR2 is AAS;

[0014] The general sequence formula of the LCDR3 is: QQLNSYPPX5T, wherein X5 is any amino acid of L or Y.

[0015] In a preferred embodiment of the present invention, the sequence of the HCDR1 is shown as SEQ ID NO.1, the sequence of the HCDR2 is shown as SEQ ID NO.2, and the sequence of the HCDR3 is shown as SEQ ID NO.3; and the sequence of the LCDR1 is shown as SEQ ID NO.5, the sequence of the LCDR2 is shown as SEQ ID NO.6, and the sequence of the LCDR3 is shown as SEQ ID NO.7; or,

[0016] The sequence of the HCDR1 is shown as SEQ ID NO.11, the sequence of the HCDR2 is shown as SEQ ID NO.12, and the sequence of the HCDR3 is shown as SEQ ID NO.13; and the sequence of the LCDR1 is shown as SEQ ID NO.15, the sequence of the LCDR2 is shown as SEQ ID NO.16, and the sequence of the LCDR3 is shown as SEQ ID NO.17.

[0017] In another preferred embodiment of the present invention, the heavy chain variable region has the sequence shown in SEQ ID NO. 4 or a sequence having more than 80% sequence homology to the sequence shown in SEQ ID NO. 4, and the light chain variable region has the sequence shown in SEQ ID NO. 8 or a sequence having more than 80% sequence homology to the sequence shown in SEQ ID NO. 8; or,

[0018] The heavy chain variable region has a sequence as shown in SEQ ID NO.14 or a sequence with more than 80% sequence homology to the sequence shown in SEQ ID NO.14, and the light chain variable region has a sequence as shown in SEQ ID NO.18 or a sequence with more than 80% sequence homology to the sequence shown in SEQ ID NO.18.

[0019] In a preferred embodiment of the present invention, the sequence of the HCDR1 is shown as SEQ ID NO.21, the sequence of the HCDR2 is shown as SEQ ID NO.22, and the sequence of the HCDR3 is shown as SEQ ID NO.23; and the sequence of the LCDR1 is shown as SEQ ID NO.25, the sequence of the LCDR2 is shown as SEQ ID NO.26, and the sequence of the LCDR3 is shown as SEQ ID NO.27; or,

[0020] The sequence of the HCDR1 is shown in SEQ ID NO.31, the sequence of the HCDR2 is shown in SEQ ID NO.32, and the sequence of the HCDR3 is shown in SEQ ID NO.33; and the sequence of the LCDR1 is shown in SEQ ID NO.35, the sequence of the LCDR2 is shown in SEQ ID NO.36, and the sequence of the LCDR3 is shown in SEQ ID NO.37; or,

[0021] The sequence of the HCDR1 is shown in SEQ ID NO.41, the sequence of the HCDR2 is shown in SEQ ID NO.42, and the sequence of the HCDR3 is shown in SEQ ID NO.43; and the sequence of the LCDR1 is shown in SEQ ID NO.45, the sequence of the LCDR2 is shown in SEQ ID NO.46, and the sequence of the LCDR3 is shown in SEQ ID NO.47; or,

[0022] The sequence of the HCDR1 is shown in SEQ ID NO.51, the sequence of the HCDR2 is shown in SEQ ID NO.52, and the sequence of the HCDR3 is shown in SEQ ID NO.53; and the sequence of the LCDR1 is shown in SEQ ID NO.55, the sequence of the LCDR2 is shown in SEQ ID NO.56, and the sequence of the LCDR3 is shown in SEQ ID NO.57; or,

[0023] The sequence of the HCDR1 is shown as SEQ ID NO.61, the sequence of the HCDR2 is shown as SEQ ID NO.62, and the sequence of the HCDR3 is shown as SEQ ID NO.63; and the sequence of the LCDR1 is shown as SEQ ID NO.65, the sequence of the LCDR2 is shown as SEQ ID NO.66, and the sequence of the LCDR3 is shown as SEQ ID NO.67.

[0024] In another preferred embodiment of the present invention, the heavy chain variable region has the sequence shown in SEQ ID NO. 24 or a sequence having more than 80% sequence homology to the sequence shown in SEQ ID NO. 24, and the light chain variable region has the sequence shown in SEQ ID NO. 28 or a sequence having more than 80% sequence homology to the sequence shown in SEQ ID NO. 28; or,

[0025] The heavy chain variable region has the sequence shown in SEQ ID NO. 34 or a sequence having more than 80% sequence homology to the sequence shown in SEQ ID NO. 34, and the light chain variable region has the sequence shown in SEQ ID NO. 38 or a sequence having more than 80% sequence homology to the sequence shown in SEQ ID NO. 38; or,

[0026] The heavy chain variable region has the sequence shown in SEQ ID NO.44 or a sequence having more than 80% sequence homology to the sequence shown in SEQ ID NO.44, and the light chain variable region has the sequence shown in SEQ ID NO.48 or a sequence having more than 80% sequence homology to the sequence shown in SEQ ID NO.48; or,

[0027] The heavy chain variable region has the sequence shown in SEQ ID NO. 54 or a sequence having a sequence homology of more than 80% to the sequence shown in SEQ ID NO. 54, and the light chain variable region has the sequence shown in SEQ ID NO. 58 or a sequence having a sequence homology of more than 80% to the sequence shown in SEQ ID NO. 58; or,

[0028] The heavy chain variable region has a sequence as shown in SEQ ID NO.64 or a sequence with more than 80% sequence homology to the sequence shown in SEQ ID NO.64, and the light chain variable region has a sequence as shown in SEQ ID NO.68 or a sequence with more than 80% sequence homology to the sequence shown in SEQ ID NO.68.

[0029] With respect to percentages of "sequence homology," the number of matching positions is determined by determining the number of amino acid residues present in the two sequences, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying that number by 100 to yield the percent identity of the sequences.

[0030] In a specific embodiment of the present invention, the heavy chain variable region can be subjected to amino acid addition, deletion or substitution based on the first amino acid sequence, and the light chain variable region can be subjected to amino acid addition, deletion or substitution based on the second amino acid sequence, such as substitution of similar amino acids or addition or subtraction of a small number of amino acids, especially addition, subtraction or substitution of amino acids in the conserved sequence portion. The obtained antibody variants have a high homology (more than 80% homology) and retain the original antibody function, i.e., the function and property of specific binding to coronavirus. These variants also fall within the scope of protection of the present invention.

[0031] In a preferred embodiment of the present invention, the heavy chain amino acid sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.9, and the light chain amino acid sequence is shown in SEQ ID NO.10; or,

[0032] The heavy chain amino acid sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.19, and the light chain amino acid sequence is shown in SEQ ID NO.20; or

[0033] The heavy chain amino acid sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.29, and the light chain amino acid sequence is shown in SEQ ID NO.30; or

[0034] The heavy chain amino acid sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.39, and the light chain amino acid sequence is shown in SEQ ID NO.40; or

[0035] The heavy chain amino acid sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.49, and the light chain amino acid sequence is shown in SEQ ID NO.50; or

[0036] The heavy chain amino acid sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.59, and the light chain amino acid sequence is shown in SEQ ID NO.60; or

[0037] The heavy chain amino acid sequence of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.69, and the light chain amino acid sequence is shown in SEQ ID NO.70.

[0038] In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof is a neutralizing antibody or antigen-binding fragment thereof against coronavirus.

[0039] The term "neutralizing antibody" refers to an antibody or antigen-binding fragment that specifically binds to a viral receptor protein. This specific binding can inhibit the biological function of the viral receptor protein, such as preventing the receptor protein from binding to its target cell receptor, and can specifically reduce the ability of the virus to infect target cells. In this application, a neutralizing antibody or antigen-binding fragment thereof for coronavirus refers to an antibody or antigen-binding fragment thereof that binds to the S protein of the coronavirus.

[0040] The term "antibody" is used herein in the broadest sense to cover natural antibodies and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, intact antibodies, and antibody fragments, antigen-binding proteins, fusion proteins, etc. that exhibit the desired antigen-binding activity.

[0041] In a preferred embodiment of the present invention, the antibody is a monoclonal antibody.

[0042] In a more preferred embodiment of the present invention, the antibody is a fully human monoclonal antibody.

[0043] In a preferred embodiment of the present invention, the Fc domain of the antibody is derived from the Fc domain of an immunoglobulin, including a native sequence Fc domain or a variant Fc domain.

[0044] In a preferred embodiment of the present invention, the antibody is any one of IgG1, IgG2, IgG3 or IgG4, or a combination of several of them.

[0045] Preferably, the antibody may be a complete antibody selected from IgG1, IgG2, IgG3 or IgG4.

[0046] In a preferred embodiment of the present invention, the antigen-binding fragment is Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 or a single-chain antibody.

[0047] In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof can be combined with other antibodies or antigen-binding fragments thereof to form an antibody or antigen-binding fragment thereof having at least two antigen-binding sites, i.e., a multispecific antibody or antigen-binding fragment thereof; the two antigen-binding sites can be different epitopes of the same antigen or different epitopes of different antigens.

[0048] In a preferred embodiment of the present invention, the above-mentioned antibody, or its antigen-binding fragment, can be further chemically modified, for example, one or more chemical groups can be attached to the antibody to increase one or more functional properties of the antibody. For example, common chemical modifications include glycosylation modification and pegylation modification. Among them, for example, glycosylation modification can be performed on the heavy chain or light chain variable region to add one or more glycosylation sites to improve some functions of the antibody, such as enhancing the immunogenicity of the antibody or improving the pharmacokinetics of the antibody. For example, under appropriate conditions, the antibody or its antigen-binding fragment is subjected to an acylation reaction or an alkylation reaction with active polyethylene glycol (such as an active ester or aldehyde derivative of polyethylene glycol) to achieve pegylation modification to improve some functions of the antibody, such as increasing the biological (such as serum) half-life of the antibody. The above-mentioned chemical modification does not significantly change the basic functions and properties of the antibody or its antigen-binding fragment of the present invention, that is, the function and properties of specific binding to coronavirus; these chemically modified variants also fall within the scope of protection of the present invention.

[0049] In a preferred embodiment of the present invention, the aforementioned antibodies or antigen-binding fragments thereof can be conjugated to other factors through chemical or genetic engineering methods; for example, these factors can provide the ability to target the antibody to a desired functional site or other properties; for example, these factors can be one or more heterologous molecules, preferably, the heterologous molecules are cytotoxic agents. Complexes formed by conjugating the aforementioned antibodies, or antigen-binding fragments thereof, to other factors fall within the scope of protection of the present invention.

[0050] 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.

[0051] 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 as SEQ ID NO.71, and the nucleic acid sequence encoding the light chain variable region is shown as SEQ ID NO.72; or,

[0052] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO.75, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO.76; or,

[0053] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO.79, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO.80; or,

[0054] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO.83, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO.84; or,

[0055] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO.87, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO.88; or,

[0056] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO.91, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO.92; or,

[0057] The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO.95, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO.96.

[0058] In a more preferred embodiment of the present invention, in the nucleic acid molecule,

[0059] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO.73, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO.74; or,

[0060] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO.77, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO.78; or,

[0061] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO.81, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO.82; or,

[0062] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO.85, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO.86; or,

[0063] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO.89, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO.90; or,

[0064] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO.93, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO.94; or,

[0065] The nucleic acid sequence encoding the heavy chain is shown in SEQ ID NO.97, and the nucleic acid sequence encoding the light chain is shown in SEQ ID NO.98.

[0066] In another aspect, the present invention provides a vector comprising the above nucleic acid molecule.

[0067] In a preferred embodiment of the present invention, the vector further comprises an expression regulatory sequence connected to the above nucleic acid molecule.

[0068] The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. A vector can be used to transform, transduce, or transfect host cells, allowing the genetic material it carries to be expressed in host cells. A vector may contain a variety of elements that control expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication origin site. A vector may also include components that facilitate its entry into cells, such as viral particles, liposomes, or protein coats, but is not limited to these. In embodiments of the present invention, a vector may be selected from, but is not limited to, plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes YACs, bacterial artificial chromosomes BACs, or P1-derived artificial chromosomes PACs), bacteriophages (such as lambda phage or M13 phage), and animal viruses used as vectors, such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40).

[0069] In another aspect, the present invention provides a host cell comprising the above vector.

[0070] Regarding "host cells", 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, insect cells such as S2 Drosophila cells or Sf9, or animal cell models such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, etc.

[0071] Preferably, the host cell is a HEK293 cell.

[0072] In another aspect, the present invention provides a method for producing the above-mentioned antibody or antigen-binding fragment thereof, wherein the above-mentioned host cell is cultured to produce the antibody or antigen-binding fragment thereof.

[0073] In another aspect, the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the above-mentioned antibody, or an antigen-binding fragment thereof.

[0074] In a preferred embodiment of the present invention, the pharmaceutical composition comprises a therapeutically effective amount of a neutralizing antibody, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier or diluent. Those skilled in the art can use an appropriate pharmaceutical carrier or diluent in combination with a therapeutically effective amount of the neutralizing antibody, or an antigen-binding fragment thereof, and administer the composition to a patient for the treatment or prevention of a disease caused by a coronavirus.

[0075] In another aspect, the present invention provides the use of the above-mentioned antibody, or its antigen-binding fragment, or the above-mentioned pharmaceutical composition in the preparation of a drug for treating or preventing diseases caused by coronavirus.

[0076] In a preferred embodiment of the present invention, the use refers to use in the preparation of drugs for treating or preventing diseases caused by SARS-CoV-2, SARS-CoV or SARS-like coronaviruses.

[0077] In one aspect, the present invention also provides a method for treating or preventing a disease caused by a coronavirus, comprising administering to a patient a therapeutically effective amount of the aforementioned antibody, or an antigen-binding fragment thereof; or administering to a patient a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned 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 a SARS-like coronavirus.

[0078] In another aspect, the present invention provides a detection product, wherein the detection product comprises the above-mentioned antibody, or an antigen-binding fragment thereof.

[0079] The detection product is used to detect the presence or level of coronavirus in a sample.

[0080] In a specific embodiment of the present invention, the detection product includes, but is not limited to, a detection reagent, a detection kit, a detection chip or a test paper, etc.

[0081] 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 protection of the present invention.

[0082] The specific detection method can adopt the following steps: 1) providing a sample; 2) contacting the sample with the above-mentioned coronavirus antibody or antigen-binding fragment thereof of the present invention; 3) detecting the immune reaction between the sample and the antibody or antigen-binding fragment thereof.

[0083] The inventors used B cell in vitro monoclonal culture and high-throughput antibody screening technology to obtain a series of coronavirus antibodies and antigen-binding fragments thereof. These antibodies and antigen-binding fragments thereof have strong binding and neutralizing abilities for the SARS-CoV-2 virus, and are all able to recognize and bind to the S1 protein and RBD of the SARS-CoV-2 virus with very strong affinity. It can be inferred that the series of coronavirus antibodies and antigen-binding fragments thereof of the present invention may also have binding and neutralizing abilities for other coronaviruses, as well as coronaviruses that may appear in the future, and have good prospects for clinical application in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 The results of the monoclonal antibody 4L12 recognizing the S1 protein and its RBD, and the S2 protein of the SARS-CoV-2 virus;

[0085] Figure 2 The results of the monoclonal antibody 12F5 recognizing the S1 protein and its RBD, and the S2 protein of the SARS-CoV-2 virus;

[0086] Figure 3 The results of the monoclonal antibody 3D13 recognizing the S1 protein and its RBD, and the S2 protein of the SARS-CoV-2 virus;

[0087] Figure 4 The test results of monoclonal antibody 10C2 recognizing the S1 protein and its RBD, and S2 protein of the SARS-CoV-2 virus;

[0088] Figure 5 The results of the monoclonal antibody 16L9 recognizing the S1 protein and its RBD, and the S2 protein of the SARS-CoV-2 virus;

[0089] Figure 6 The detection results of monoclonal antibody 20E21 recognizing the S1 protein and its RBD, and S2 protein of the SARS-CoV-2 virus;

[0090] Figure 7 The results of the monoclonal antibody 22H22 recognizing the S1 protein and its RBD, and the S2 protein of the SARS-CoV-2 virus;

[0091] Figure 8 This is the affinity test result of monoclonal antibody 4L12 binding to the RBD of the S1 protein of the SARS-CoV-2 virus;

[0092] Figure 9 The results of affinity test of monoclonal antibody 12F5 binding to the RBD of the S1 protein of SARS-CoV-2 virus;

[0093] Figure 10This is the affinity test result of monoclonal antibody 3D13 binding to the RBD of the S1 protein of the SARS-CoV-2 virus;

[0094] Figure 11 This is the affinity test result of monoclonal antibody 10C2 binding to the RBD of the S1 protein of the SARS-CoV-2 virus;

[0095] Figure 12 This is the affinity test result of monoclonal antibody 16L9 binding to the RBD of the S1 protein of the SARS-CoV-2 virus;

[0096] Figure 13 This is the affinity test result of monoclonal antibody 20E21 binding to the RBD of the S1 protein of the SARS-CoV-2 virus;

[0097] Figure 14 This is the affinity test result of monoclonal antibody 22H22 binding to the RBD of the S1 protein of the SARS-CoV-2 virus. DETAILED DESCRIPTION

[0098] 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 any structural, methodological, or functional changes made by those skilled in the art based on these embodiments are all within the scope of protection of the present invention.

[0099] Example embodiments will now be described more fully with reference to the accompanying drawings. However, example embodiments can be embodied in many 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 the example embodiments to those skilled in the art.

[0100] Unless otherwise specified, the materials and reagents used in the following examples were commercially available. Where specific techniques or conditions are not specified in the examples, the experiments were performed according to those described in the literature in the field (e.g., Molecular Cloning: A Laboratory Manual, 3rd edition, by J. Sambrook et al., translated by Huang Peitang et al., Science Press) or according to the product instructions.

[0101] Example 1: Screening and detection of coronavirus antibodies

[0102] The inventors conducted pseudovirus neutralization experiment screening on the plasma of patients with novel coronavirus pneumonia who were admitted to the inventor's unit (Shanghai Public Health Clinical Center) from January 20, 2020 to February 26, 2020 (followed up two weeks after recovery and discharge). It was found that the serum of three 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 patients themselves, their peripheral blood was drawn for research.

[0103] 1. Isolation of peripheral blood memory B cells

[0104] 1) Isolation of peripheral blood lymphocytes: Peripheral blood was drawn from the patients in the convalescent phase and mixed with an equal amount of normal saline. Peripheral blood lymphocytes were isolated using Lymphoprep (Stemcell Technologies, Catalog No. 07851), a lymphocyte separation medium. For the procedure, refer to the instructions for the lymphocyte separation medium.

[0105] 2) Isolation of Peripheral Blood Memory B Cells: The peripheral blood lymphocytes isolated in step 1) above were stained with an antibody mixture consisting of anti-CD19-PE-Cy7 (BD Bioscience), IgA-APC (Jackson Immunoresearch), IgD-FITC (BD Bioscience), and IgM-PE (Jackson Immunoresearch) at 4°C in the dark for 30 min. After staining, the cells were washed with 10 ml of PBS-BSA buffer and resuspended in 500 μl of PBS-BSA. Finally, CD19+IgA-IgD-IgM- memory B cells were sorted using a FACSAria III cell sorter (Becton Dickinson).

[0106] 2. Incubation of peripheral blood memory B cells

[0107] The sorted CD19+IgA-IgD-IgM- memory B cells were resuspended in culture medium containing 10% FBS, 100 U / ml IL-2, 50 ng / ml IL-21, and irradiated 3T3-msCD40L feeder cells. Memory B cells were seeded at a density of 4 cells / well in a 384-well microtiter plate (final volume of 50 μl) and incubated for 13 days. Growth factors IL-2 and IL-21 stimulated the memory B cells to divide and grow, secreting antibodies into the incubated culture medium. For specific culture methods, see Huang J et al. Nature Protocols 2013, 8(10):1907-15.

[0108] 3. Production of SARS-CoV-2 and SARS-CoV pseudoviruses

[0109] SARS-CoV-2 and SARS-CoV pseudoviruses are non-replication-defective retroviral particles that bear the SARS-CoV-2 and SARS-CoV spike (S) proteins on their surfaces, respectively, and carry a luciferase reporter gene. They can mimic the infection process of SARS-CoV-2 and SARS-CoV viruses in host cells (such as the human hepatoma cell line Huh-7 and the 293T cell line 293T-ACE2, which stably expresses the human ACE2 receptor) and express the luciferase reporter gene in infected cells. Because pseudovirus infection cannot produce mature viral particles, related operations can be safely performed in a biosafety level 2 laboratory.

[0110] SARS-CoV-2 and SARS-CoV pseudoviruses were generated by co-transfecting 293T cells with their respective S protein expression plasmids and an HIV Env-deficient backbone plasmid (pNL4-3.Luc.RE-) carrying a luciferase reporter gene. The S gene sequences of SARS-CoV-2 and SARS-CoV were designed based on NCBI GenBank sequences NC_045512 and ABD72979.1, respectively. After codon optimization, the gene sequences were synthesized by Nanjing GenScript and ligated into the pcDNA3.1 eukaryotic expression vector to construct SARS-CoV-2 and SARS-CoV S protein expression plasmids. The pNL4-3.Luc.RE- backbone plasmid was obtained from the NIH AIDS Reagent Program. All plasmids were amplified by transformation into DH5α competent cells and purified using a plasmid purification kit manufactured by Meiji Biotechnology. Purification procedures were performed according to the kit instructions.

[0111] 293T cells were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco) and seeded into 10 cm dishes before transfection. After 24 hours of culture, the backbone plasmid (pNL4-3.Luc.RE-) and a plasmid expressing SARS-CoV or SARS-CoV-2 were co-transfected into the 293T cells at a 3:1 ratio using EZ Trans Cell Transfection Reagent (Liji Biotechnology). For detailed transfection procedures, refer to the EZ Trans Cell Transfection Reagent user manual. Forty-eight hours after transfection, the pseudovirus-containing supernatant was harvested, centrifuged at 1500 rpm for 10 minutes to remove cell debris, and aliquots were stored frozen at -80°C for neutralizing antibody testing.

[0112] 4. Neutralization screening

[0113] After 13 days of in vitro culture of peripheral blood memory B cells, 40 μl of culture supernatant was collected from each well for testing for neutralizing antibodies against SARS-CoV-2 and SARS-CoV. The assay was performed as follows: 20 μl of culture supernatant was mixed with 20 μl of pseudovirus supernatant produced above in a 384-well cell culture plate. After incubation at room temperature for 30 minutes, 5000 293T-ACE2 cells (50 μl) were added to each well and cultured in a cell culture incubator. After 48 hours, cells were lysed using a luciferase assay kit (Luciferase Assay System, Promega Cat. #E1500) and luciferase activity was measured in each well. The specific assay method was described in the kit instructions. Chemiluminescence (RLU) values ​​were measured in each well using a multi-function microplate reader (Perkin Elmer). The percentage of neutralization inhibition of the pseudovirus by the culture supernatant was calculated based on the ratio of the RLU values ​​of the culture supernatant to the virus control. Wells with an inhibition percentage greater than 90% were selected as virus neutralization-positive wells.

[0114] 5. RT-PCR amplification of heavy and light chain genes

[0115] B cells from virus neutralization-positive wells were used to amplify the variable regions of the heavy and light chains of immunoglobulin genes using RT-PCR. For primer design and detailed RT-PCR procedures, see Tiller, T. et al. J. Immunol Methods 2018, 329:112–124. The amplified heavy and light chain variable region genes were purified by agarose gel electrophoresis and cloned into the PMD19-T vector using the PMD19-T vector cloning kit (Takara 6013). Detailed procedures are described in the kit instructions. Single clones were then selected for gene sequencing.

[0116] 6. Expression and purification of monoclonal antibodies

[0117] The correctly sequenced antibody heavy chain variable region gene and pCMV / R-10E8 heavy chain gene (NIHAIDS Reagent Program Cat 12290) were digested with Age I and Sal I, respectively, and the target fragments recovered by gel purification were ligated and transformed into DH5α competent cells to construct the antibody expression heavy chain plasmid; the correctly sequenced antibody lambda or kappa light chain variable region gene and pCMV / R-10E8 lambda light chain gene expression plasmid (NIHAIDS Reagent Program Cat 12291) or pCMV / R-N6 kapp light chain gene expression plasmid (NIH AIDS Reagent Program Cat 12966) were digested with Age I and Xho I or Age I and Bsiw I, respectively, and the target fragments recovered by gel purification were ligated and transformed into DH5α competent cells to construct the antibody expression light chain plasmid; the antibody heavy chain and light chain plasmids were purified using a plasmid purification kit (Meiji Biotechnology) (see Figure 1 Purified antibodies were expressed (SDS-PAGE results) and co-transfected into 293T cells at a 1:1 ratio using EZ Trans Cell Transfection Reagent (Liji Biotechnology). After 72 hours, the transfection supernatant was collected and purified using a Protein-G column (Tiandi Renhe Biotechnology Co., Ltd., Changzhou) according to the Protein-G column instructions. The purified IgG was measured for absorbance at 280 nm using a Nanodrop 2000 (Thermo Fisher), and the antibody concentration was calculated.

[0118] Through the above sections 1-6, the inventors of the present application obtained several IgG antibodies, and this application discloses 7 of them (named in order: 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22).

[0119] The amino acid sequence numbering information of the 7 antibodies is shown in Table 1 below:

[0120] Table 1

[0121]

[0122]

[0123] The nucleotide sequence numbering information of the 7 antibodies is shown in Table 2 below:

[0124] Table 2

[0125]

[0126] 7. Detection of the neutralizing activity of the seven monoclonal antibodies (4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22) against SARS-CoV-2 coronavirus

[0127] Different concentrations of monoclonal antibodies were tested in 96-well cell plates to inhibit pseudovirus infection of Huh-7 cells to detect the neutralizing ability of monoclonal antibodies against SARS-CoV-2 coronavirus.

[0128] The detection method is as follows: 1) Huh-7 cells were seeded in a 96-well cell plate, with 1×10 4 The cells were cultured in a 37°C, 5% CO2 cell culture incubator for 24 hours; 2) the monoclonal antibody was diluted to different concentrations in cell culture medium, mixed with an equal volume of pseudovirus dilution containing 100 TCID50, and incubated at 37°C for 1 hour; 3) the cell culture medium was discarded, 50 μl of the virus-antibody complex was added to each well, and duplicate wells were set up. A no-antibody group, a no-virus group, and a positive serum control group were also set up; 4) after 12 hours of incubation, 150 μl of maintenance solution was added to each well, and the cells were cultured for a further 48 hours at 37°C; 5) the cells were lysed using a luciferase assay kit (Luciferase Assay System, Promega Cat. #E1500) and the luciferase activity in each well was measured. The specific detection method was referred to the kit instructions; the chemiluminescence (RLU) value of each well was measured using a multi-function microplate reader (Perkin Elmer); 6) the neutralization inhibition percentage of the different concentrations of the monoclonal antibody against the pseudovirus was calculated based on the ratio of the RLU values ​​of the monoclonal antibody to the virus control, and the half-maximal inhibitory dose (IC50) of the monoclonal antibody for virus inhibition was calculated using PRISM7 software (GraphPad).

[0129] The results are shown in Table 3 below.

[0130] Table 3

[0131] IC50 (ng / mL) 4L12 4.5 12F5 11.1 3D13 16.1 10C2 30.1 16L9 4.1 20E21 2.3 22H22 60.6

[0132] As shown in Table 3, the seven monoclonal antibodies (4L12, 12F5, 3D13, 10C2, 16L9, 20E21, and 22H22) can effectively neutralize SARS-CoV-2 at concentrations in the ng / ml range, demonstrating very strong neutralizing activity. The stronger the neutralizing activity, the less antibody is required, and the lower the cost. Therefore, these seven antibodies, 4L12, 12F5, 3D13, 10C2, 16L9, 20E21, and 22H22, have promising clinical application prospects.

[0133] 8. Detection of the seven monoclonal antibodies (4L12, 12F5, 3D13, 10C2, 16L9, 20E21, and 22H22) of this application for recognition of the S1 protein and RBD protein of the SARS-CoV-2 virus

[0134] The recognition of the S1 and RBD proteins of the SARS-CoV-2 virus by the 7 purified monoclonal antibodies was detected in turn by enzyme-linked immunosorbent assay (ELISA).

[0135] The assay method is as follows: 1 μg / ml antigen protein (Sino Biological) was coated in a 96-well ELISA plate overnight at 4°C. The plate was washed five times with PBS-T (0.2% Tween-20) and blocked at room temperature for 1 hour with 300 μl of blocking buffer (PBS, 1% FBS, 5% milk) added to each well. The plate was washed three times with PBS-T. After a five-fold serial dilution of the monoclonal antibody in PBS (PBS, 5% FBS, 2% BSA, 1% Tween-20), 100 μl of the sample was added to the ELISA plate and incubated at 37°C for 1 hour. The plate was washed five times with PBS-T and 100 μl of horseradish peroxidase-conjugated goat anti-human IgG antibody (Jackson Immunoresearch) diluted 1:2500 in PBS was added to each well and incubated at room temperature for 1 hour. The plate was washed five times with PBS-T, and 150 μl of ABTS color development substrate (ThermoFisher) was added. After color development at room temperature in the dark for 30 minutes, the absorbance at a wavelength of 405 nm was read using a microplate reader.

[0136] See also Figure 1 , the detection results of monoclonal antibody 4L12 recognizing the S1 protein and its RBD, and S2 protein of the SARS-CoV-2 virus;

[0137] See also Figure 2 , the detection results of monoclonal antibody 12F5 recognizing the S1 protein and its RBD, and S2 protein of the SARS-CoV-2 virus;

[0138] See also Figure 3 , the detection results of monoclonal antibody 3D13 recognizing the S1 protein and its RBD, and S2 protein of the SARS-CoV-2 virus;

[0139] See also Figure 4 , the detection results of monoclonal antibody 10C2 recognizing the S1 protein and its RBD, and S2 protein of the SARS-CoV-2 virus;

[0140] See also Figure 5 , the detection results of monoclonal antibody 16L9 recognizing the S1 protein and its RBD, and S2 protein of the SARS-CoV-2 virus;

[0141] See also Figure 6 , the detection results of monoclonal antibody 20E21 recognizing the S1 protein and its RBD, and S2 protein of the SARS-CoV-2 virus;

[0142] See also Figure 7 , the detection results of monoclonal antibody 22H22 recognizing the S1 protein and its RBD, and S2 protein of SARS-CoV-2 virus.

[0143] from Figure 1-7 It can be seen that monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 can all recognize and bind to the S1 protein and its RBD (conserved region) of the SARS-CoV-2 virus. Given that the RBD of the S1 protein of the coronavirus is the region that binds to the ACE2 receptor and is highly conserved, it can be inferred that the monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 of the present application, in addition to having strong binding and neutralizing abilities against the SARS-CoV-2 virus, may also have binding and neutralizing abilities against other coronaviruses and coronaviruses that may appear in the future.

[0144] 8. Biofilm interferometry was used to detect the binding ability of the seven monoclonal antibodies (4L12, 12F5, 3D13, 10C2, 16L9, 20E21, and 22H22) of the present application to the RBD of the S1 protein of the SARS-CoV-2 virus

[0145] In order to detect the interaction between the 7 monoclonal antibodies of the present application and the RBD of the S1 protein of the SARS-CoV-2 virus, the binding kinetics between them was detected by biofilm interferometry. The detection process was carried out on an OctetRED96 (Fortebio) instrument.

[0146] The detection method is as follows: the AHC probe is pre-equilibrated by soaking it in sterile water for 10 minutes. The entire detection process is performed at 30°C and can be divided into the following five steps: 1) Zero adjustment: Immerse the probe in sterile water for 60 seconds to obtain a detection baseline; 2) Antibody capture: Immerse the probe in a 10 μg / ml monoclonal antibody solution for 200 seconds to capture the antibody; 3) Re-zero adjustment: Immerse the probe in a buffer solution (PBS with 0.02% Tween 20) for 120 seconds to remove unbound antibody; 4) RBD binding: Immerse the probe in a 3-fold serial dilution of RBD protein solution starting at 100 nM for 300 seconds to obtain a dynamic curve of monoclonal antibody binding to RBD; 5) Binding and dissociation: Place the probe in buffer for 300 seconds. Protein binding causes changes in the biofilm thickness, resulting in a relative shift of the interfering light waves, which is detected by the spectrometer and forms an interference spectrum, displayed as the real-time displacement of the interference pattern (nm). This was used to detect the dynamic curve of the binding and dissociation between RBD and the monoclonal antibody of the present application. During data analysis, the data of the sample wells were subtracted from the data of the buffer control wells to deduct the nonspecific interference of the buffer solution. A 1:1 binding model was used to fit the overall curve of the binding of RBD to the monoclonal antibody at different dilution concentrations to obtain the average binding constant K. on , dissociation constant K off and the affinity constant K D value.

[0147] Test results see Figure 8-14 , which are the affinity test results of monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 binding to the RBD of the S1 protein of the SARS-CoV-2 virus; each figure has five curves, which represent the dynamic binding and dissociation curves of the monoclonal antibody with five different concentrations of RBD.

[0148] from Figure 8-14 It can be seen that the 7 monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 of the present application all bind to the RBD of the S1 protein of the SARS-CoV-2 virus in a concentration gradient-dependent manner; after binding, they dissociate, and the dissociated RBD is very small; the K values ​​of the 7 monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 are DThe values ​​are (1.49±0.06)nM, (2.22±0.07)nM, (4.17±0.15)nM, (3.36±0.18)nM, (1.21±0.06)nM, (2.3±0.07)nM, and (5.07±0.2)nM, respectively; it shows that the 7 monoclonal antibodies of the present application have very strong affinity for the conserved RBD region of the S1 protein of SARS-CoV-2. It can be inferred that the 7 monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21, and 22H22 of the present application demonstrated in Section 7 above have strong neutralizing activity against the RBD of the S1 protein of the SARS-CoV-2 virus. This is because the 7 monoclonal antibodies of the present application have very strong affinity for the conserved RBD region of the S1 protein of the SARS-CoV-2 virus. Comprehensive Table 1 and Figure 1-14 The results further verified that the seven monoclonal antibodies 4L12, 12F5, 3D13, 10C2, 16L9, 20E21 and 22H22 of the present application not only have strong binding and neutralizing abilities against SARS-CoV-2, but may also have binding and neutralizing abilities against other coronaviruses and coronaviruses that may appear in the future.

[0149] It should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each implementation method can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0150] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Any equivalent implementation methods or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof against SARS-CoV-2 virus, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3; characterized in that: The sequence of the HCDR1 is shown as SEQ ID NO.31, the sequence of the HCDR2 is shown as SEQ ID NO.32, and the sequence of the HCDR3 is shown as SEQ ID NO.33; in addition, the sequence of the LCDR1 is shown as SEQ ID NO.35, the sequence of the LCDR2 is AAS, and the sequence of the LCDR3 is shown as SEQ ID NO.

37.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The heavy chain variable region has a sequence as shown in SEQ ID NO.34 or a sequence with more than 80% sequence homology to the sequence shown in SEQ ID NO.34, and the light chain variable region has a sequence as shown in SEQ ID NO.38 or a sequence with more than 80% sequence homology to the sequence shown in SEQ ID NO.

38.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: The antibody is a monoclonal antibody.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein: The antibody is a fully human monoclonal antibody.

5. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: The antibody is any one of IgG1, IgG2, IgG3 or IgG4, or a combination of several of them.

6. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: The antigen-binding fragment is Fv, Fab, F(ab')2, Fab', dsFv, scFv or sc(Fv)2.

7. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. A vector comprising the nucleic acid molecule according to claim 7.

9. A host cell comprising the vector according to claim 8.

10. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

11. A detection product, characterized in that: The detection product comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

12. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that: Cultivate the host cell of claim 9 to produce the antibody or antigen-binding fragment thereof.

13. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating or preventing diseases caused by SARS-CoV-2 virus.

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