A tetravalent neutralizing antibody against novel coronavirus and application thereof

Abstract

The application relates to the field of biological medicine, and provides a tetravalent antibody for neutralization of a novel coronavirus, which comprises an antibody monomer nAb and an scFv structure coupled at the Fc end of the heavy chain of the antibody monomer through a linker 1. Compared with the nAb antibody, the tetravalent antibody has higher antiviral capacity for various epidemic strains of the novel coronavirus, and has good application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine; in particular, the present application provides a tetravalent antibody for neutralizing the novel coronavirus. BACKGROUND

[0002] The novel coronavirus SARS-CoV-2 can cause acute respiratory disease, pneumonia and acute respiratory distress syndrome, etc. The virus can rapidly mutate, and has caused extensive drug resistance to various prevention and treatment methods. Although there are currently various prevention and treatment programs, the potential risk of drug resistance breakthrough caused by the extensive mutation of the virus still needs to be prevented, and effective measures need to be explored. SUMMARY

[0003] The purpose of the present application is to develop an antibody that can be effective against various mutant strains based on the previous research on the novel coronavirus, and the antibody is a tetravalent neutralizing antibody.

[0004] In a first aspect, the present application provides a tetravalent neutralizing antibody against the novel coronavirus, comprising an antibody monomer nAb and a scFv structure coupled at the Fc end of the heavy chain of the antibody monomer through a linker 1, wherein the antibody monomer nAb comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region, and the light chain comprises a light chain variable region; the three CDRs of the heavy chain variable region are respectively: nAb-VH CDR1 (the amino acid sequence shown in SEQ ID NO: 3), nAb-VH CDR2 (the amino acid sequence shown in SEQ ID NO: 4), and nAb-VH CDR3 (the amino acid sequence shown in SEQ ID NO: 5); the three CDRs of the light chain variable region are respectively: nAb-VL CDR1 (the amino acid sequence shown in SEQ ID NO: 8), nAb-VL CDR2 (the amino acid sequence shown in SEQ ID NO: 9), and nAb-VL CDR3 (the amino acid sequence shown in SEQ ID NO: 10); the scFv structure is a VL-linker 2-VH structure, wherein the three CDRs of VH are respectively: nAb-VH CDR1 (the amino acid sequence shown in SEQ ID NO: 3), nAb-VH CDR2 (the amino acid sequence shown in SEQ ID NO: 4), and nAb-VH CDR3 (the amino acid sequence shown in SEQ ID NO: 5); the three CDRs of VL are respectively: nAb-VL CDR1 (the amino acid sequence shown in SEQ ID NO: 8), nAb-VL CDR2 (the amino acid sequence shown in SEQ ID NO: 9), and nAb-VL CDR3 (the amino acid sequence shown in SEQ ID NO: 10), the amino acid sequence of the linker 1 is shown in SEQ ID NO: 16; and the amino acid sequence of the linker 2 is shown in SEQ ID NO: 18.

[0005] Further preferably, the amino acid sequence of the heavy chain variable region of the antibody monomer nAb or the VH of the scFv structure is shown in SEQ ID NO: 2, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2; and the amino acid sequence of the light chain variable region of the antibody monomer nAb or the VL of the scFv structure is shown in SEQ ID NO: 7, or has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7.

[0006] Further preferably, the amino acid sequence of the VL-linker 2-VH is shown in SEQ ID NO: 12.

[0007] Further preferably, the tetravalent neutralizing antibody further comprises one or more of a heavy chain constant region, a light chain constant region, an Fc region; preferably, the light chain constant region is a lambda chain or kappa chain constant region; further preferably, the antibody is of IgG1, IgG2, IgG3 or IgG4 type; more preferably, the antibody is a chimeric antibody or a humanized antibody or an antigen binding fragment thereof.

[0008] Further preferably, the heavy and light chain sequences of the antibody monomer nAb are as set forth in SEQ ID NO: 1 and SEQ ID NO: 6, respectively.

[0009] Further preferably, the tetravalent neutralizing antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is as set forth in SEQ ID NO: 17, and the amino acid sequence of the light chain is as set forth in SEQ ID NO: 6. It is understood by those skilled in the art that the tetravalent neutralizing antibody of the present application comprises two identical heavy chains and two identical light chains, wherein the heavy chain is a fusion heavy chain composed of the Fc terminal of the heavy chain of the antibody monomer nAb and the scFv structure connected by linker 1, and the light chain is identical to the light chain structure of the antibody monomer nAb.

[0010] In a second aspect, the present application provides a composition comprising the tetravalent neutralizing antibody of the present application; preferably, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0011] In a third aspect, there is provided use of the tetravalent neutralizing antibody or the composition of the present application in the manufacture of a medicament for preventing and / or treating novel coronavirus infection.

[0012] In a fourth aspect, there is provided use of the tetravalent neutralizing antibody or the composition of the present application in combination with one or more other therapeutic agents in the manufacture of a medicament for preventing and / or treating novel coronavirus infection; preferably, the other therapeutic agents include but are not limited to antiviral drugs, hormone drugs or biological macromolecular drugs.

[0013] In a fifth aspect, there is provided a biological material, which is:

[0014] (1) a nucleic acid molecule encoding the tetravalent neutralizing antibody of the present application; preferably, the nucleic acid molecule encodes the heavy chain and / or the light chain of the tetravalent neutralizing antibody;

[0015] (2) a vector comprising the nucleic acid molecule of (1);

[0016] (3) a cell comprising the nucleic acid molecule of (1) or the vector of (2).

[0017] Those skilled in the art can easily obtain and prepare the nucleic acid molecule of the antibody, the vector comprising the nucleic acid molecule and the cell according to the amino acid sequence of the antibody, which are all common knowledge in the art.

[0018] In a sixth aspect, there is provided a method for preparing the tetravalent neutralizing antibody of the present application, which comprises:

[0019] (1) chemical synthesis: synthesis according to the amino acid sequence of the tetravalent neutralizing antibody of the present application; or

[0020] (2) biological synthesis: culturing the above-mentioned cells to express the tetravalent neutralizing antibody; preferably, further purifying the tetravalent neutralizing antibody from the cell culture.

[0021] Both chemical synthesis and cell expression of antibodies are conventional techniques in the art; one skilled in the art can prepare the antibody of the present application without any doubt according to the information in the present application.

[0022] The tetravalent antibody of the present application retains the binding function to multiple mutant epidemic strains consistent with the antibody nAb, exhibits higher target binding affinity and pseudovirus neutralization activity, and has potential application value in the prevention or treatment of coronavirus infection alone or in combination with one or more other therapeutic agents. The tetravalent antibody of the present application has higher antiviral capacity than the nAb antibody to multiple coronavirus epidemic strains, and has good application value. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 : Schematic diagram of candidate Fc fusion scFv structure and SEC-HPLC detection results.

[0024] Figure 2 : ELISA binding experiment results of candidate Fc fusion scFv structure to coronavirus Spike S1 protein.

[0025] Figure 3 : BLI binding experiment results of candidate Fc fusion scFv structure to coronavirus Spike S1 protein.

[0026] Figure 4 : Neutralization experiment results of candidate Fc fusion scFv structure to wild type pseudovirus.

[0027] Figure 5 : Schematic diagram of tetravalent neutralizing antibody nAb-VLVH and SEC-HPLC detection results.

[0028] Figure 6 : ELISA binding experiment results of tetravalent neutralizing antibody nAb-VLVH to multiple epidemic strain subtypes of Spike S1 protein.

[0029] Figure 7 : Neutralization experiment results of tetravalent neutralizing antibody nAb-VLVH to multiple epidemic strain subtypes of pseudovirus. Detailed Implementation

[0030] definition

[0031] In this invention, the term "antibody" refers to an immunoglobulin that specifically recognizes and binds to an antigen, encompassing a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, or antibody fragments.

[0032] The term "variable region" refers to the structural domain of the antibody heavy or light chain that recognizes and specifically binds to antigenic epitopes.

[0033] The CDR region, also known as the "complementarity-determining region," refers to the region in the antibody variable region that is highly variable in sequence and forms a structurally defined loop and / or contains antigen-contacting amino acid residues. The CDR is primarily responsible for the binding of the antibody to the antigen epitope, determining the antibody's specificity. In a given heavy or light chain variable region amino acid sequence, the specific amino acid sequence of each CDR is determined using any one or a combination of many well-known numbering rules, including, for example, Kabat, Contact, AbM, and Chothia.

[0034] The antibody of the present invention

[0035] This invention provides a quadrivalent monoclonal neutralizing antibody (also known as a quadrivalent antibody or neutralizing antibody) with high affinity for the Spike S1 protein of the novel coronavirus. Compared with nAb antibodies, it exhibits stronger target protein binding ability and pseudovirus neutralization ability against a variety of circulating strains of the novel coronavirus in in vitro experiments.

[0036] The antibody monomer nAb of this invention is a combination of SEQ ID NO:22 and SEQ ID NO:50 of WO2021248279A1. The entire contents of that patent document are incorporated herein by reference.

[0037] The tetravalent neutralizing antibody or its antigen-binding fragment of the present invention comprises substitution, insertion, or deletion. The antibody of the present invention includes modifications to the light chain variable region, the heavy chain variable region, the light chain, or the heavy chain, such that the modified amino acid sequence differs from the amino acid sequence from which the antibody is derived. For example, the amino acid sequence derived from the same specified protein can be similar to the starting sequence, for example, having a certain percentage of identity, such as 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the percentage identity with the starting sequence.

[0038] In the present invention, "identity" refers to the percent of amino acid (or base) identity between two sequences or between two nucleic acid molecules when aligned for maximum sequence comparison. The alignment and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. (2007) in Current Protocols in Molecular Biology. Alignments are preferably performed using default parameters. One such alignment program is BLAST using default parameters. In particular, the programs are BLASTN and BLASTP.

[0039] In certain embodiments, amino acid modifications can be introduced into the Fc region of the antibodies provided herein, either at one or more amino acid positions, to thereby generate Fc variants. Fc variants can comprise a human Fc region sequence comprising an amino acid modification at one or more amino acid positions.

[0040] "Antibodies and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, multispecific, recombinant, heterologous, chimeric, humanized, de-immunized antibodies, or Fab fragments, Fab' fragments, F(ab')2 fragments, single chain antibodies scFv, nanobodies, and epitope-binding fragments of any of the above. 2 "Antibodies and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, multispecific, recombinant, heterologous, chimeric, humanized, de-immunized antibodies, or Fab fragments, Fab' fragments, F(ab')2 fragments, single chain antibodies scFv, nanobodies, and epitope-binding fragments of any of the above.

[0041] In certain embodiments, the antibodies can be further modified to add functional components, moieties suitable for antibody derivatization include, but are not limited to, the following examples, PEG, dextran, proteins, lipids, therapeutic agents, or toxins. The antibodies can be modified by phosphorylation, acetylation, glycosylation, pegylation, amidation, or linkage to other proteins, etc.

[0042] Use of novel coronavirus neutralizing antibodies

[0043] The tetravalent anti-novel coronavirus monoclonal neutralizing antibodies and antigen-binding fragments thereof and pharmaceutical compositions comprising the same provided by the present invention can be used for the prevention or treatment of novel coronavirus infection. The present invention relates to methods of preventing and / or treating a subject from a novel coronavirus infection by administering the antibodies or fragments thereof of the present invention to a subject in need thereof.

[0044] The antibodies (and any additional therapeutic agents) of the present invention can be administered by any appropriate mode, including, but not limited to, intraperitoneal, intravenous, subcutaneous, intranasal, intramuscular injection. The antibodies and variants or compositions thereof can be administered by any convenient route, for example, by injection, either subcutaneously, intradermally, intramuscularly, intraperitoneally, intravenously, or intrathecally, by absorption through epithelial or mucocutaneous membranes, by inhalation, or by insufflation.

[0045] Anti-novel coronavirus tetravalent monoclonal antibody sequences under the present invention

[0046] Table 1: Amino acid sequence numbers and sequences. Included: heavy chain HC, heavy chain variable region VH, nAb-VH CDR1, nAb-VH CDR2, nAb-VH CDR3 of antibody monomer nAb used in the present application; light chain LC, light chain variable region VL, nAb-VL CDR1, nAb-VL CDR2, nAb-VL CDR3 of antibody monomer nAb; scFv structure (including VH- linker2-VL (abbreviated as VHVL) and VL-linker2-VH (abbreviated as VLVH)); human IgG1 Fc sequence; Fc fusion protein Fc-VHVL and Fc-VLVH; connecting peptide linker1 connecting the scFv structure and the intramolecularly coupled antibody structure of tetravalent antibody nAb-VLVH; fusion heavy chain and light chain of nAb-VLVH (identical to the light chain of nAb).

[0047]

[0048]

[0049]

[0050]

[0051] Examples

[0052] Example 1: Preparation of Fc fusion scFv structure

[0053] VH-linker2-VL and VL-linker2-VH (sequences are SEQ ID NO: 11 and SEQ ID NO: 12) were respectively chimerized with human IgG1 Fc constant region gene (Fc constant region sequence is SEQ ID NO: 13) and cloned into expression vectors, named Fc-VHVL and Fc-VLVH (sequences are SEQ ID NO: 14 and SEQ ID NO: 15) respectively. After one week of transient expression in HEK293 cells, the harvested culture supernatant was purified by Protein A (Bestchrom, AA0275) affinity, and the sample purity was greater than 98% (by SEC-HPLC detection). Figure 1 ).

[0054] Example 2: ELISA binding experiment of Fc fusion scFv structure

[0055] ELISA experiment was used to detect the affinity of related samples to wild type Spike S1 protein (Acrobiosystems, S1N-C52H4). The negative control hIgG1, antibody nAb, Fc-VHVL and Fc-VLVH were diluted to 10 nM with PBS, and after incubating the coated enzyme-labeled plate at 4°C overnight, they were washed with PBST for three times. 100 μl of BSA blocking solution (2% BSA in PBST solution) was added to each well, and incubated at 37°C for 1 h. After washing with PBST for three times, 100 μl of gradient concentration diluted wild type S1 protein was added, with an initial concentration of 5 μg / ml, 4-fold gradient dilution, and a total of 8 concentration gradients. After incubation at 37°C for 1.5 h, they were washed with PBST for three times, and anti-HIS tag-HRP antibody (KPL, A00612, 1:600 dilution) was added, and incubated at 37°C for 1 h. After washing with PBST for three times, TMB developing solution (TGEN, TMB-S-003) was added, and incubated at 37°C for 0.5 h in the dark. 100 μl of stop solution was added, and after color development, the OD450nm value was determined by an enzyme-labeled instrument.

[0056] The ELISA results are shown in Figure 2 As shown in the table, the negative control hIgG1 has no binding signal, the EC50 of the neutralizing antibody nAb is 0.737 nM, and only Fc-VLVH maintains high affinity among the two Fc-scFv fusion proteins, with an EC50 value of 0.740 nM.

[0057] Example 3 BLI binding experiment of Fc fusion scFv structure

[0058] To further clarify the affinity results, BLI (Biolayer interferometry) antigen binding kinetics experiment was carried out on Octet Red96 (manufacturer: ForteBio) instrument to detect the affinity of related samples to wild type Spike S1 protein (Acrobiosystems, S1N-C52H4). In the BLI detection, the biosensor (manufacturer: ForteBio) was pre-wetted in PBST for 10 minutes, and then the gradient diluted (initial concentration of 400 nM, 2-fold gradient dilution) candidate molecules (nAb, Fc-VHVL, Fc-VLVH) were fixed to the surface of the biosensor. Then gradient diluted wild type Spike S1 protein (initial concentration of 400 nM, 2-fold gradient dilution, a total of 5 concentration gradients) was combined on the biosensor, followed by protein dissociation step. The experimental data was analyzed by Octet Data Analysis (version 7.0) system software, and the kinetic data was fitted by 1:1 binding model.

[0059] As can be seen from the results, Figure 3The binding kinetics data obtained from BLI showed a consistent trend with the ELISA results. The affinity of Fc-VLVH and nAb for wild-type Spike S1 protein was very close, with EC50 values ​​of 5.04 nM and 4.17 nM, respectively.

[0060] Example 4: Sham poison neutralization experiment of Fc fused scFv structure

[0061] To verify the biological activity of the Fc-scFv fusion structure, pseudotoxic neutralizing activity assays were performed on related molecules. First, HEK293T cells (GM-C09233, stably transfected with human ACE2) were injected at a concentration of 1×10⁻⁶ cells. 4 Cells were seeded at a density of 400 nM in 96-well plates and cultured for 24 h. The fusion protein or antibody was diluted to 400 nM as the first well, followed by 8 concentration points using a 4-fold serial dilution, and then diluted with a titer of 2 × 10⁻⁶. 5 An equal volume of TU / ml wild-type SARS-CoV-2 pseudovirus (GM-0220PV07 from Jiman Biotechnology) was mixed and incubated at room temperature for 1 hour. The cell culture medium was discarded, and the incubated virus solution was added. After 6 hours of infection, the medium was replaced with fresh medium and cultured for another 48 hours. After culture, 100 μl of fluorescent detection reagent (Promega, E2620) was added to each well, and luciferase activity (RLU) was measured using a microplate reader. Neutralization inhibition rate % = 100 - [(Treatment group RLU - Blank group RLU) × 100 / (Antibody-free group RLU - Blank group RLU)]. The inhibition rate of each treatment group was simulated using GrapPad Prism 6.0, and the IC50 was calculated. 50 Numerical value.

[0062] Depend on Figure 4 It can be seen that in the neutralization experiment of the wild-type pseudovirus of COVID-19, the IC50 of nAb was... 50 The concentration was 3.54 nM, while Fc-VLVH maintained good pseudotoxicity neutralizing activity, with an IC50 of 3.54 nM. 50 The concentration was 4.39 nM. Corresponding to the aforementioned binding activity assay, the neutralizing activity of the low-affinity Fc-VHVL was significantly reduced, with an IC50 of 4.39 nM. 50 The molecular weight was 178.20 nM. This indicates that the scFv structure of the VL-linker 2-VH maintains good target binding activity and neutralizing biological activity against the wild-type SARS-CoV-2 virus. This structure will be used as a candidate structure for subsequent tetravalent antibody development.

[0063] Example 5: Preparation of tetravalent antibody nAb-VLVH

[0064] In previous studies, it was found that nAb can bind to the relatively conserved epitope in the Spike S1 protein of the new coronavirus, promote the dimerization of the Spike trimer on the virus surface, and thus inhibit the recognition of the virus and the receptor, thereby playing a virus neutralization role. This embodiment plans to fuse scFv to the heavy chain Fc end of nAb to form a single-target four-valent antibody with an IgG1-(scFv)2 structure Figure 5 ), in order to further enhance the virus neutralization effect.

[0065] The foregoing experiments found that the Fc-VLVH structure has similar target affinity and pseudovirus neutralization activity to the parent antibody. In this embodiment, the VLVH structure is fused to the heavy chain Fc end of nAb and cloned into an expression vector, named nAb-VLVH, with heavy and light chain sequences of SEQ ID NO: 17 and NO: 6 (consistent with the nAb light chain). After one week of expression by HEK293 cells, the culture supernatant was purified by Protein A affinity to obtain the relevant sample. SEC-HPLC detection showed that the sample purity can reach 96.3% Figure 5 )。

[0066] Example 6 ELISA binding experiment of four-valent antibody nAb-VLVH to Spike S1 protein of various epidemic strain subtypes

[0067] The ELISA experiment was used to detect the affinity of nAb-VLVH to the Spike S1 protein of various epidemic strains of the new coronavirus. The relevant target proteins were purchased from Acrobiosystems, and the material information is as follows: wild strain (S1N-C52H4), Alpha strain (B.1.1.7, S1N-C52Hr), Beta strain (B.1.351, S1N-C52Hm), Gamma strain (P.1, S1N-C52Hp), Delta strain (B.1.617.2, S1N-C52Hu), Epsilon strain (B.1.429, S1N-C52Hs), Kappa strain (B.1.617.1, S1N-C52Ht), Lambda strain (C.37, SPN-C52H), Omicron strain (B.1.1.529, S1N-C52Ha).

[0068] The negative control hIgG1, nAb, and nAb-VLVH were diluted to 10 nM with PBS and coated on the enzyme-labeled plate overnight, and then the method consistent with Example 2 was used to detect the ELISA affinity of the candidate molecules to the target proteins of each strain. After the reaction, the OD450 nm value was measured by an enzyme-labeled instrument. The relevant results are as follows: Figure 6As shown, the negative control hlgGl had no binding to the relevant target proteins; the neutralizing antibody nAb showed good binding activity except for no binding to the S1 protein of the Omicron strain; the target recognition properties of the tetravalent antibody nAb-VLVH were consistent with those of the parent nAb, and it had no binding to the protein of the Omicron strain, but the binding to the proteins of other strains was significantly enhanced. The relevant data showed that the tetravalent antibody structure helped to improve its affinity to the S1 proteins of multiple strains.

[0069] Example 7 Neutralization experiment of tetravalent antibody nAb-VLVH on multiple epidemic strain subtypes of pseudovirus

[0070] To further clarify the biological activity of the tetravalent antibody nAb-VLVH, the neutralization activity detection of pseudovirus against multiple subtypes of the new crown epidemic strain was carried out. The relevant experimental steps were consistent with those of Example 4, and each strain subtype pseudovirus was purchased from Jiman Biological, and the relevant material information was as follows: wild strain (GM-0220PV07), Alpha strain (GM-0220PV33), Beta strain (GM-0220PV32), Gamma strain (GM-0220PV47), Delta strain (GM-0220PV45), Epsilon strain (GM-0220PV50), Kappa strain (GM-0220PV43), Lambda strain (GM-0220PV53), Omicron strain (GM-0220PV84).

[0071] The relevant results are shown in Figure 7 As shown, the negative control hlgGl had no neutralizing effect on each subtype strain; the neutralizing antibody nAb had good neutralizing activity on the pseudovirus of each subtype except for the Omicron strain; compared with nAb, the tetravalent antibody nAb-VLVH effectively improved the neutralizing activity on multiple pseudovirus subtypes except for the Lambda strain, and had no neutralizing effect on the Omicron strain. This experiment showed that the tetravalent antibody structure had better biological activity than the parent neutralizing antibody, and had good potential application value.

Claims

1. A tetravalent neutralizing antibody against novel coronavirus, comprising an antibody monomer nAb and a scFv structure coupled at the Fc end of the heavy chain of the antibody monomer through a linker 1, wherein, The antibody monomer nAb comprises a heavy chain comprising a heavy chain variable region and a light chain comprising a light chain variable region; the three CDRs of the heavy chain variable region are respectively: nAb-VH CDR1 with an amino acid sequence as shown in SEQ ID NO: 3, nAb-VH CDR2 with an amino acid sequence as shown in SEQ ID NO: 4, and nAb-VH CDR3 with an amino acid sequence as shown in SEQ ID NO: 5; the three CDRs of the light chain variable region are respectively: nAb-VL CDR1 with an amino acid sequence as shown in SEQ ID NO: 8, nAb-VL CDR2 with an amino acid sequence as shown in SEQ ID NO: 9, and nAb-VL CDR3 with an amino acid sequence as shown in SEQ ID NO: 10; the scFv structure is a VL-Linker2-VH structure, wherein the three CDRs of the VH are respectively: nAb-VH CDR1 with an amino acid sequence as shown in SEQ ID NO: 3, nAb-VH CDR2 with an amino acid sequence as shown in SEQ ID NO: 4, and nAb-VH CDR3 with an amino acid sequence as shown in SEQ ID NO: 5; the three CDRs of the VL are respectively: nAb-VL CDR1 with an amino acid sequence as shown in SEQ ID NO: 8, nAb-VL CDR2 with an amino acid sequence as shown in SEQ ID NO: 9, and nAb-VL CDR3 with an amino acid sequence as shown in SEQ ID NO: 10, the amino acid sequence of the Linker1 is as shown in SEQ ID NO: 16; and the amino acid sequence of the Linker2 is as shown in SEQ ID NO:

18.

2. The tetravalent neutralizing antibody of claim 1, wherein, The antibody monomer nAb comprises a heavy chain comprising a heavy chain variable region and a light chain comprising a light chain variable region; the three CDRs of the heavy chain variable region are respectively: nAb-VH CDR1 with an amino acid sequence as shown in SEQ ID NO: 3, nAb-VH CDR2 with an amino acid sequence as shown in SEQ ID NO: 4, and nAb-VH CDR3 with an amino acid sequence as shown in SEQ ID NO: 5; the three CDRs of the light chain variable region are respectively: nAb-VL CDR1 with an amino acid sequence as shown in SEQ ID NO: 8, nAb-VL CDR2 with an amino acid sequence as shown in SEQ ID NO: 9, and nAb-VL CDR3 with an amino acid sequence as shown in SEQ ID NO: 10; the scFv structure is a VL-Linker2-VH structure, wherein the three CDRs of the VH are respectively: nAb-VH CDR1 with an amino acid sequence as shown in SEQ ID NO: 3, nAb-VH CDR2 with an amino acid sequence as shown in SEQ ID NO: 4, and nAb-VH CDR3 with an amino acid sequence as shown in SEQ ID NO: 5; the three CDRs of the VL are respectively: nAb-VL CDR1 with an amino acid sequence as shown in SEQ ID NO: 8, nAb-VL CDR2 with an amino acid sequence as shown in SEQ ID NO: 9, and nAb-VL CDR3 with an amino acid sequence as shown in SEQ ID NO: 10, the amino acid sequence of the Linker1 is as shown in SEQ ID NO: 16; and the amino acid sequence of the Linker2 is as shown in SEQ ID NO:

18.

3. The tetravalent neutralizing antibody of claim 2, wherein, The antibody monomer nAb comprises a heavy chain comprising a heavy chain variable region and a light chain comprising a light chain variable region; the three CDRs of the heavy chain variable region are respectively: nAb-VH CDR1 with an amino acid sequence as shown in SEQ ID NO: 3, nAb-VH CDR2 with an amino acid sequence as shown in SEQ ID NO: 4, and nAb-VH CDR3 with an amino acid sequence as shown in SEQ ID NO: 5; the three CDRs of the light chain variable region are respectively: nAb-VL CDR1 with an amino acid sequence as shown in SEQ ID NO: 8, nAb-VL CDR2 with an amino acid sequence as shown in SEQ ID NO: 9, and nAb-VL CDR3 with an amino acid sequence as shown in SEQ ID NO: 10; the scFv structure is a VL-Linker2-VH structure, wherein the three CDRs of the VH are respectively: nAb-VH CDR1 with an amino acid sequence as shown in SEQ ID NO: 3, nAb-VH CDR2 with an amino acid sequence as shown in SEQ ID NO: 4, and nAb-VH CDR3 with an amino acid sequence as shown in SEQ ID NO: 5; the three CDRs of the VL are respectively: nAb-VL CDR1 with an amino acid sequence as shown in SEQ ID NO: 8, nAb-VL CDR2 with an amino acid sequence as shown in SEQ ID NO: 9, and nAb-VL CDR3 with an amino acid sequence as shown in SEQ ID NO: 10, the amino acid sequence of the Linker1 is as shown in SEQ ID NO: 16; and the amino acid sequence of the Linker2 is as shown in SEQ ID NO:

18.

4. The tetravalent neutralizing antibody of claim 2, wherein, ​ 5. The tetravalent neutralizing antibody of claim 2, wherein, the amino acid sequence of the VH of the heavy chain variable region or scFv structure of the antibody monomeric nAb has at least 97% sequence identity to SEQ ID NO: 2; and the amino acid sequence of the VL of the light chain variable region or scFv structure of the antibody monomeric nAb has at least 97% sequence identity to SEQ ID NO:

7.

6. The tetravalent neutralizing antibody of claim 2, wherein, the amino acid sequence of the VH of the heavy chain variable region or scFv structure of the antibody monomeric nAb has at least 98% sequence identity to SEQ ID NO: 2; and the amino acid sequence of the VL of the light chain variable region or scFv structure of the antibody monomeric nAb has at least 98% sequence identity to SEQ ID NO:

7.

7. The tetravalent neutralizing antibody of claim 2, wherein, the amino acid sequence of the VH of the heavy chain variable region or scFv structure of the antibody monomeric nAb has at least 99% sequence identity to SEQ ID NO: 2; and the amino acid sequence of the VL of the light chain variable region or scFv structure of the antibody monomeric nAb has at least 99% sequence identity to SEQ ID NO:

7.

8. The tetravalent neutralizing antibody of claim 2, wherein, the amino acid sequence of the VH of the heavy chain variable region or scFv structure of the antibody monomeric nAb is set forth in SEQ ID NO: 2; and the amino acid sequence of the VL of the light chain variable region or scFv structure of the antibody monomeric nAb is set forth in SEQ ID NO:

7.

9. The tetravalent neutralizing antibody of claim 1, wherein, the amino acid sequence of the VL-Linker2-VH is set forth in SEQ ID NO:

12.

10. The tetravalent neutralizing antibody of any one of claims 1-9, wherein, the tetravalent neutralizing antibody further comprises one or more of a heavy chain constant region, a light chain constant region, an Fc region.

11. The tetravalent neutralizing antibody of claim 10, wherein, the light chain constant region is a lambda chain or a kappa chain constant region.

12. The tetravalent neutralizing antibody of claim 10, wherein, the antibody is of an IgG1, IgG2, IgG3, or IgG4 type.

13. The tetravalent neutralizing antibody of claim 10, wherein, the antibody is a chimeric antibody or a humanized antibody.

14. The tetravalent neutralizing antibody of claim 10, wherein, the heavy and light chain sequences of the antibody monomeric nAb are set forth in SEQ ID NO: 1 and SEQ ID NO: 6, respectively.

15. The tetravalent neutralizing antibody of claim 14, wherein, the tetravalent neutralizing antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 17, and the amino acid sequence of the light chain is set forth in SEQ ID NO:

6.

16. A composition comprising the tetravalent neutralizing antibody of any one of claims 1-15.

17. The composition of claim 16, wherein, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.

18. Use of the tetravalent neutralizing antibody of any one of claims 1 to 15 or the composition of claim 16 or 17 for the manufacture of a medicament for the prevention and / or treatment of a novel coronavirus infection, wherein, the novel coronavirus is a wild-type strain, an Alpha strain, a Beta strain, a Gamma strain, a Delta strain, an Epsilon strain, a Kappa strain, or a Lambda strain.

19. A biological material, which is: (1) a nucleic acid molecule encoding the tetravalent neutralizing antibody of any one of claims 1-15; the nucleic acid molecule encodes a heavy chain and a light chain of the tetravalent neutralizing antibody; (2) a vector comprising the nucleic acid molecule of (1); and (3) a cell comprising the nucleic acid molecule of (1) or the vector of (2).

20. A method for preparing the tetravalent neutralizing antibody of any one of claims 1-15, which is: (1) a chemical synthesis method: synthesizing according to the amino acid sequence of the tetravalent neutralizing antibody; or (2) a biological synthesis method: culturing the cell of claim 19 to express the tetravalent neutralizing antibody.

21. The method of making according to claim 20, wherein, The biosynthetic method also includes further purifying the tetravalent neutralizing antibody from the cell culture.

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