Targeting RBD anti-SARS-CoV and SARS-CoV-2 fully human monoclonal antibody 1E4 and application thereof

Abstract

The invention provides an RBD-targeted high-neutralizing-activity anti-SARS-CoV and SARS-CoV-2 fully human monoclonal antibody 1E4 and application thereof, the monoclonal antibody 1E4 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has three complementary determining regions, and the amino acid sequences of the three complementary determining regions are respectively: GYSFVDYW, IYPGDSNI and AR; the amino acid sequences of three complementary determining regions of the light chain variable region are respectively as follows: QSISRW, EAS and QHYNSFS. The monoclonal antibody 5E4 has efficient and specific anti-SARS-CoV and SARS-CoV-2 neutralizing activity, is high in expression, completely human-derived and good in stability, and can be used for preparing new coronavirus detection products or drugs for preventing and treating new coronavirus diseases.

Description

Technical Field

[0001] The present invention belongs to the fields of microbiology and immunology, and relates to a fully human monoclonal antibody 1E4 targeting RBD with high neutralizing activity against SARS-CoV and SARS-CoV-2, and its application. Background Art

[0002] Coronaviruses (CoVs) are a class of enveloped, positive-strand RNA viruses that are widespread in nature. They can infect a variety of hosts, including humans, bats, and birds, causing respiratory, gastrointestinal, and even neurological diseases. To date, seven coronaviruses are known to infect humans, four of which (HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1) are prevalent but have low pathogenicity, typically causing only common cold symptoms.

[0003] SARS-CoV-2 shares a high degree of evolutionary homology with SARS-CoV, both relying on the spike protein (S protein) to bind to the host cell ACE2 receptor to mediate infection. The receptor binding domain (RBD) of the S protein is the primary target for neutralizing antibodies. Monoclonal antibodies targeting this region can effectively block viral invasion and have important therapeutic and preventive value. Although multiple vaccines are currently available, continuous viral mutations have led to frequent immune escape, challenging the effectiveness of vaccines. Furthermore, effective antiviral drugs remain scarce, and clinical treatment still relies primarily on supportive care. There is an urgent need to develop highly effective, broad-spectrum, and safe antibody drugs.

[0004] Fully human monoclonal antibodies have become an important strategy for antiviral therapy due to their high specificity, low immunogenicity, and favorable safety profile. The highly neutralizing, fully human monoclonal antibody 5E4, developed in this study, targets the RBD region of SARS-CoV and SARS-CoV-2, demonstrating potent neutralization and excellent stability. This antibody is expected to provide new solutions for the prevention and treatment of SARS-CoV, SARS-CoV-2, and their variants, and to provide an effective biomedical weapon for future responses to emerging coronavirus outbreaks. Summary of the Invention

[0005] In order to solve the technical problem, the present invention provides a fully human monoclonal antibody 1E4 with high neutralizing activity against SARS-CoV and SARS-CoV-2 targeting RBD and its application. The fully human monoclonal therapeutic antibody has good protective effect against SARS-CoV and SARS-CoV-2.

[0006] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect of the present invention, a highly neutralizing, fully human anti-SARS-CoV and SARS-CoV-2 monoclonal antibody 1E4 targeting RBD is provided. The monoclonal antibody 1E4 comprises a heavy chain variable region and a light chain variable region. The amino acid sequences of the three complementarity determining regions in the heavy chain variable region are GYSFVDYW, IYPGDSNI, and AR, respectively; and the amino acid sequences of the three complementarity determining regions in the light chain variable region are QSISRW, EAS, and QHYNSFS, respectively.

[0007] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1E4 is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the monoclonal antibody 1E4 is shown in SEQ ID NO: 3.

[0008] Furthermore, the amino acid sequence of the heavy chain constant region of the monoclonal antibody 1E4 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain constant region of the monoclonal antibody 1E4 is shown in SEQ ID NO: 7.

[0009] Furthermore, the monoclonal antibody further comprises: An antibody with the same function obtained by replacing, deleting and / or adding one or more amino acids in the amino acid sequence of the monoclonal antibody; or comprising a heavy chain variable region having an amino acid sequence that is at least 80% homologous to the heavy chain variable region; and a light chain variable region having an amino acid sequence that is at least 80% homologous to the light chain variable region; Or an antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

[0010] In other embodiments, V H and / or V L The amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the above sequence. H and V L V H and V L Antibodies to the regions can be obtained by mutagenizing (eg, site-directed mutagenesis or PCR-mediated mutagenesis) nucleic acid molecules encoding SEQ ID NOs: 1-6 and then testing the encoded altered antibodies for retained function using functional assays described herein.

[0011] In other embodiments, the variable region gene can be converted into a scFv gene. Once the V encoding H and V LThe fragmented DNA fragments can be further manipulated by standard recombinant DNA technology, for example, the variable region gene is converted into a full-length antibody chain gene, a Fab fragment gene or a scFv gene.

[0012] In these operations, the encoding V L or V H The DNA fragment of the present invention is operably linked to another DNA fragment encoding another protein such as an antibody constant region or a flexible linker. As used herein, the term "operably linked" means that the two DNA fragments are linked together so that the amino acid sequences encoded by the two DNA fragments remain in the reading frame.

[0013] In the second aspect of the present invention, the present invention provides a nucleic acid molecule encoding the monoclonal antibody, wherein the nucleic acid molecule comprises a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region.

[0014] Furthermore, the polynucleotide sequences encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody 1E4 are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0015] Furthermore, the polynucleotide sequence encoding the heavy chain constant region of the monoclonal antibody 1E4 is shown in SEQ ID NO: 6, and the polynucleotide sequence encoding the light chain constant region is shown in SEQ ID NO: 8.

[0016] In a third aspect of the present invention, an expression vector comprising the nucleic acid is provided, wherein the expression vector is capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell.

[0017] The vector can be a plasmid vector, a phage vector, a viral vector or a mammalian expression vector. The present invention specifically uses a mammalian expression vector.

[0018] In the fourth aspect of the present invention, an engineered bacterium or eukaryotic host cell comprising the expression vector is provided.

[0019] In the fifth aspect of the present invention, the use of the monoclonal antibody in the preparation of SARS-CoV and SARS-CoV-2 therapeutic drugs or new coronavirus detection products is provided.

[0020] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: The present invention provides a fully human monoclonal antibody 1E4 targeting RBD with high neutralizing activity against SARS-CoV and SARS-CoV-2, demonstrating excellent neutralizing protection against SARS-CoV and SARS-CoV-2 infected cells. The results of the present invention demonstrate that the antibody has broad application prospects in the preparation of therapeutic drugs for SARS-CoV and SARS-CoV-2. Specifically, the monoclonal antibody disclosed in the present invention also has the following technical advantages: (1) Fully human, no humanization modification is required for clinical application.

[0021] (2) High neutralizing activity. In the SARS-CoV infected cell model, the half effective concentration (EC50) of monoclonal antibody 1E4 against SARS-CoV wild type was 344.4 ng / mL; in the SARS-CoV infected cell model, the half effective concentration (EC50) of monoclonal antibody 1E4 against SARS-CoV-2 strain was 97.07 ng / mL.

[0022] (3) The mechanism of action is clear: Monoclonal antibody 1E4 binds to RBD with high specificity, indicating that the monoclonal antibody targets the receptor binding region and exerts an antiviral effect by specifically blocking the binding of the virus to the receptor.

[0023] (4) Good stability: Because the monoclonal antibody genes come from the same cell in the human body, they are naturally paired. It is known that the half-life of IgG1 antibodies in the human body is 21 to 28 days. In theory, the publicly available monoclonal antibodies have a consistent half-life in the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is a diagram of single-cell sorting by flow cytometry; Figure 2 Automatic nucleic acid electrophoresis instrument for detecting the pattern of monoclonal antibody variable region gene amplification; Figure 3 Figure 2 is a diagram for detecting the binding activity between 1E4 and RBD protein; Figure 4 This is the EC50 determination curve of the antibody in the pseudovirus cell model; the half-maximal effective concentration of monoclonal antibody 1E4 against SARS-CoV and SARS-CoV-2. DETAILED DESCRIPTION

[0026] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0027] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0028] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0029] The monoclonal antibody of the present application, its preparation method and application effect will be described in detail below with reference to the examples and experimental data.

[0030] Example 1. Screening and Preparation of Human Anti-SARS-CoV and SARS-CoV-2 Monoclonal Antibodies 1. Prepare a 96-well plate: 20 μl of RNAse-free water + 20 U of RNAse inhibitor per well, cover with a sealing film, and place at 4°C for use. 2. Prepare samples: (1) Resuscitate cells: Take out the frozen PBMC cells isolated from the peripheral blood of patients who recovered from pneumonia caused by the new coronavirus infection from -80°C and quickly place them in warm water at 37°C. After the cells thaw, centrifuge at 800 rpm for 5 minutes and discard the supernatant; resuspend in 2-3 ml of FPBS into a flow tube, balance and centrifuge at 800 rpm for 5 minutes, discard the supernatant; resuspend in 2-3 ml of FPBS, centrifuge, and discard the supernatant. Finally, add 100 μl of FPBS to resuspend, and aspirate 2 μl to dilute 10 times for cell counting.

[0031] (2) Single staining tubes: 7 tubes (FVS-780, CD3-BV510, CD4-BV510, CD8-BV510, CD19-PE, IgD-BB700, CD20-BV421, CD38-FITC), 3 × 10 cells per tube 6 cells, add the dye to the flow cytometry tubes containing cells according to the antibody concentration recommended in the instructions, and use FPBS to make up each reaction volume to 50 μl. (3) Bare cell control: 1 tube, 3× 10 6 cells, and make up to 50 μl with FPBS. (4) Cells for sorting: 1 tube, first determine the number of cells, and the final concentration is 100 μl system (1 × 10 6cells), and FVS-780, CD3-BV510, CD4-BV510, CD8-BV510, CD19-PE, IgD-BB700, CD20-BV421, CD38-FITC, and Biotin-S1 fluorescent dyes were added at the same time.

[0032] (5) Incubate the sample at 4°C in the dark for 1 h.

[0033] (6) Add 3 ml of FPBS to each tube, centrifuge at 800 g for 5 min at 4°C, discard the supernatant, and repeat the washing process twice.

[0034] (7) After resuspending in 400 μl FPBS, remove the cell clumps using a 40 μm cell sieve and store in the dark at 4°C for sorting.

[0035] 3. Flow cytometry sorting: select CD19 + , CD3 - , CD4 - , CD8 - , IgD - , CD38 + , CD20 - , S1 + The results of flow cytometry sorting are shown in Figure 1 First, lymphocytes were selected, then non-adherent single cells were selected, then living cells were selected, and then CD19 + 、CD3 － 、CD4 － 、CD8 － B cells, and then select IgD － mature B cells, and finally select S1 + The mature B cells are our target cells.

[0036] 4. Amplify the variable region gene of fully human monoclonal antibody using single cell PCR technology 4.1 Reverse Transcription PCR (QIAGEN, 210212) was performed using the following protocol: 94 cells were sorted by flow cytometry. All primers specific for each subtype of the heavy chain (H), kappa light chain (κ), and lambda light chain (λ) were added to each reaction (primer sequences are shown in Table 1).

[0037] Primers: H: 5′L-VH 1, L-VH 3, L-VH 4 / 6, 5′L-VH 5, Hu IgG-const-anti, 3′CμCH1 κ: 5′L Vκ1 / 2, 5′L Vκ3, 5′L Vκ4, 3′Cκ543–566λ: 5′L Vλ1, 5′L Vλ2, 5′L Vλ3, 5′LVλ4 / 5, 5′L Vλ6, 5′L Vλ7, 5′L Vλ8, 3′Cλ Table 1 - Reverse transcription PCR primers

[0038] The PCR reaction system contained: 6 μL of 5× buffer, 1.2 μL of dNTP, 1.2 μL of reverse transcriptase (Takara Biotechnology Co., Ltd., RR036A), primers as above, single cell template, and the volume was filled up to 30 μL with water.

[0039] The PCR reaction conditions were as follows: reverse transcription at 50°C for 30 min; followed by pre-denaturation at 95°C for 15 min, 40 cycles of 95°C for 40 s, 55°C for 30 s, and 72°C for 1 min, and a final extension at 72°C for 10 min.

[0040] 4.2 Nested PCR 1 μl of the reverse transcription product was used as a template for PCR amplification of the variable regions of H, κ, and λ: the primers for amplifying the heavy chain variable region, kappa light chain variable region, and λ light chain variable region are shown in Table 2 below.

[0041] Table 2. Nested PCR primers

[0042] Note: The single underlined part is used for fusion with the upstream fragment, and the underlined bold part is used for fusion with the downstream fragment.

[0043] The PCR reaction system included: 12.5 μL of DNA polymerase mixture (CW2849, Kangwei Century Biotechnology Co., Ltd.), primers as above, 1 μL of reverse transcription product as template, and water to 25 μL.

[0044] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 4 min, followed by 40 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 45 min, and a final extension at 72°C for 10 min.

[0045] 4.3 Automatic nucleic acid electrophoresis instrument for detection Some results of automatic nucleic acid electrophoresis detection are shown in Figure 2 , only the 1E4 heavy and light chain variable region bands are shown in the figure.

[0046] 5. Synthesis of polynucleotide sequences of variable regions Suzhou GeneWeiZ Biotechnology Co., Ltd. was commissioned to synthesize the variable region DNA sequence of the antibody based on the above polynucleotide sequence. A signal peptide sequence and a restriction enzyme cleavage site (5'-GAATTCGCCACCATGGAGACAGACACCCTGCTCCTGTGGGTGCTGCTG-3') were added before the antibody heavy and light chain variable regions, and a restriction enzyme cleavage site (5'-GGTACC-3') was added to the tail.

[0047] 6. Construction of plasmid expression vector Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the antibody heavy chain constant region DNA sequences and light chain constant region DNA sequences with restriction enzyme cleavage sites (5'-GAGCTCGGTACC-3') added before and at the end of the heavy and light chain constant region DNA sequences. (The heavy chain constant region sequence is represented by SEQ ID NO:5, and the DNA coding sequence is represented by SEQ ID NO:6; the kappa light chain constant region sequence is represented by SEQ ID NO:7, and the DNA coding sequence is represented by SEQ ID NO:8). The pCAGGS empty plasmid (available from Shanghai Yuanmu Biotechnology Co., Ltd., Cat. No. P0165) and the antibody heavy chain constant region DNA sequence were digested with restriction endonucleases Nhe I and Sac I. The digested plasmid and antibody constant region DNA fragments were ligated using T4 DNA ligase to generate a plasmid vector containing the antibody heavy chain constant region. The pCAGGS empty plasmid and the antibody light chain constant region DNA sequence were digested using restriction endonucleases Nhe I and Sac I. The digested plasmid and the antibody constant region DNA fragments were ligated using T4 DNA ligase to obtain a plasmid vector containing the antibody light chain constant region.

[0048] The constructed plasmid vector containing the antibody heavy chain constant region and the antibody heavy chain variable region synthesized in step 5 were digested with restriction endonucleases EcoR I and Kpn I, and the digestion products were ligated to obtain a eukaryotic expression vector for expressing the 1E4 antibody heavy chain. The constructed plasmid vector containing the antibody light chain constant region and the antibody light chain variable region synthesized in step 5 were digested with restriction endonucleases EcoR I and Kpn I, and the digestion products were ligated with T4 DNA ligase to obtain a eukaryotic expression vector for expressing the 1E4 antibody light chain. These two vectors can then be transfected into eukaryotic cells for antibody expression.

[0049] The amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of monoclonal antibody 1E4 are shown in SEQ ID NO: 1, amino acid sequences 27-32, 50-52, and 89-95, respectively; the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the light chain variable region are shown in SEQ ID NO: 3, amino acid sequences 26-33, 51-58, and 97-98, respectively. Details are shown in Table 3.

[0050] Table 3

[0051] 7. Transient expression and affinity chromatography purification of monoclonal antibodies Using the Expi293 expression system, 15 μg of heavy chain and 15 μg of light chain were mixed and transfected into Expi 293F cells according to the instructions of the transfection reagent (Shanghai Liji Biotechnology Co., Ltd., EZ Trans Plus, AC04L011). After 5-6 days, the culture medium was harvested and the supernatant was about 50 ml after centrifugation. A 5 ml pre-packed Protein A affinity chromatography column was used and equilibrated with 20 mM PBS before loading. The sample was injected after the conductivity showed a baseline. After loading, the column was washed with 20 mM PBS until the baseline was stable. The target protein was eluted with 0.1 M glycine buffer (pH 3.0). When the OD280 was close to the baseline, the collection was stopped and the column was washed with at least 3 column volumes of 20 mM PBS until the baseline was stable. After the baseline was stable, the column was washed with 20% ethanol.

[0052] Example 2. Analysis of Binding Activity of Human Monoclonal Antibody 1E4 to SARS-CoV RBD and SARS-CoV-2 RBD 1. Antibody and Protein Dilution: Dilute 1E4 to a concentration of 5 μg / mL in kinetic buffer (1× PBS, 0.02% Tween-20). Dilute the SARS-CoV RBD and SARS-CoV-2 RBD proteins to 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, and 7.8125 nM in KB buffer.

[0053] 2. Take out the black low-adsorption 96-well plate and add the corresponding liquid to each column in the following order, with a volume of 200 μL.

[0054] Well A1-H1: KB buffer; Wells A2-G2: 5 μg / mL 1E4 antibody; Well H2: KB buffer; Well A3-H3: KB buffer; Wells A4-G4: SARS-CoV RBD protein or SARS-CoV-2 RBD protein at decreasing concentrations from 500 nM to 6.25 nM; Well H4: 100 nM SARS-CoV RBD protein or SARS-CoV-2 RBD protein; Well A5-H5: KB buffer; Wells A6-H6: regeneration buffer (10 mM glycine buffer, pH 2.0); Well A7-H7: KB buffer; Wells A8-H8: regeneration buffer; Well A9-H9: KB buffer; Well A10-H10: regeneration buffer.

[0055] 3. Soak the Protein A sensor (ForteBio, Cat. No. 18-5010) in KB buffer for 10 minutes in advance and prepare it for loading.

[0056] 4. Place the sensor and the black 96-well plate in the corresponding positions of the machine. Set the running program as follows: The sensor was equilibrated in KB buffer for 60 s.

[0057] The sensor was bound to 1E4 antibody at room temperature for 300 s until saturation.

[0058] The sensor was shaken in buffer for 30 s to determine the baseline.

[0059] The sensor was bound to different concentrations of SARS-CoV RBD protein or SARS-CoV-2 RBD protein for 400 s.

[0060] The sensor was dissociated in KB buffer for 400 s.

[0061] Sensor regeneration: The sensor was shaken in regeneration buffer for 30 s and then in KB buffer for 60 s, repeated three times.

[0062] 5. Set row H as the reference well, select a 1:1 binding model, and calculate the dissociation constant of 1E4 binding to SARS-CoV RBD protein or SARS-CoV-2 RBD protein from the binding curve, ensuring that R2 ≥ 0.98.

[0063] 6. Results: See Figure 3 . Figure 3 middle, Figure 3 Curve A shows the affinity test results of 1E4 and SARS-CoV RBD protein, KD=1.243 × 10 -8 M. Figure 3Curve B shows the affinity test results of 1E4 and SARS-CoV-2 RBD protein, and the results show KD=4.675 × 10 -9 M.

[0064] Example 3: Neutralization activity analysis in pseudovirus cell model 1. Pseudovirus packaging: Different pseudoviruses were packaged based on the SARS-CoV and SARS-CoV-2 spike proteins (S proteins). The hACE2 gene was stably transfected into BHK-21 cells to construct hACE2-BHK-21 cells stably expressing human ACE2. Plasmids expressing different S proteins were transfected into Vero-E6 cells using lipofectamine 2000 (Biosharp, BL623B). After 24 h, 5 h-long-lived VSV-dG-fLuc (1×10 6 Transfected cells were inoculated with a 1:20% tissue culture infectious dose (TCID50) / mL of VSV-G monoclonal antibody (I1-hybridoma culture supernatant) in growth medium (DMEM, 10% fetal bovine serum). After 24 hours, pseudovirus supernatants were collected, centrifuged at 3000 rpm for 10 minutes, aliquoted, and stored frozen at 80°C. TCID50 values ​​of pseudovirus were calculated using a serial dilution method for infection of BHK-21-hACE2 cells using the Reed-Muench method.

[0065] 2. Diluted antibodies: SARS-CoV and SARS-CoV-2 pseudovirus (3×10 5 TCID50 / well) were incubated with diluted serum on a 96-well white plate at room temperature for 30 min, and then incubated with trypsin-treated BHK-21-hACE2 cells at 2×10 4 / hole density mix.

[0066] Luciferase assay and analysis: After 16 h of culture, the culture medium of the infected cells was removed and the cells were lysed with 1× Bright-Glo Luciferase Assay Reagent (Promega). Chemiluminescence detection was performed using a SpectraMax iD3 multiwell luminometer (Molecular Devices). The 50% neutralization dilution titer (NT50) was calculated using GraphPad Prism 7 software, and nonlinear regression curve fitting was performed.

[0067] See the results Figure 4 (The horizontal axis represents the antibody concentration, and the vertical axis represents the neutralization effect % relative to the negative control group). Figure 4 It can be seen that in the pseudovirus model, the half effective concentration (EC50 ) was 344.4 ng / mL; the half effective concentration (EC 50 ) was 97.07 ng / mL.

[0068] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0069] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0070] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A fully human monoclonal antibody 1E4 targeting RBD with high neutralizing activity against SARS-CoV and SARS-CoV-2, characterized in that: The monoclonal antibody 1E4 includes a heavy chain variable region and a light chain variable region. The amino acid sequences of the three complementarity determining regions of the heavy chain variable region are shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; the amino acid sequences of the three complementarity determining regions of the light chain variable region are shown in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively.

2. The fully human monoclonal antibody 1E4 targeting RBD with high neutralizing activity against SARS-CoV and SARS-CoV-2 according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1E4 is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the monoclonal antibody 1E4 is shown in SEQ ID NO:

3.

3. The fully human monoclonal antibody 1E4 targeting RBD with high neutralizing activity against SARS-CoV and SARS-CoV-2 according to claim 1, characterized in that: The amino acid sequence of the heavy chain constant region of the monoclonal antibody 1E4 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain constant region of the monoclonal antibody 1E4 is shown in SEQ ID NO:

7.

4. The fully human monoclonal antibody 1E4 targeting RBD with high neutralizing activity against SARS-CoV and SARS-CoV-2 according to claim 1, characterized in that The monoclonal antibody further comprises: An antibody with the same function obtained by replacing, deleting and / or adding one or more amino acids in the amino acid sequence of the monoclonal antibody; or comprising a heavy chain variable region having an amino acid sequence that is at least 80% homologous to the heavy chain variable region; and a light chain variable region having an amino acid sequence that is at least 80% homologous to the light chain variable region; Or an antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

5. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 4, characterized in that: The nucleic acid molecules include a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region.

6. The nucleic acid molecule according to claim 5, characterized in that The polynucleotide sequences encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody 1E4 are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

7. The nucleic acid molecule according to claim 5, characterized in that The polynucleotide sequences encoding the heavy chain constant regions of the monoclonal antibody 1E4 are shown in SEQ ID NO: 6, and the polynucleotide sequences encoding the light chain constant regions are shown in SEQ ID NO:

8.

8. An expression vector comprising the nucleic acid molecule according to any one of claims 5 to 7, characterized in that: The expression vector is capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell.

9. An engineered bacterium or eukaryotic host cell comprising the expression vector according to claim 8.

10. Use of the monoclonal antibody according to any one of claims 1 to 4 in the preparation of COVID-19 therapeutic drugs or novel coronavirus detection products.

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