Monoclonal antibodies targeting SARS-CoV-2 receptor binding motifs and their recognized antigen epitopes and applications

Abstract

The present invention relates to a monoclonal antibody targeting a SARS-CoV-2 receptor binding motif, and its recognized antigenic epitope and application. The present invention provides a monoclonal antibody targeting a SARS-CoV-2 RBM, which is different from the existing commercialized monoclonal antibodies and can specifically bind to a new antigenic epitope located in the SARS-CoV-2 RBM. 473 YQ 479 , the antigen epitope partially overlaps with the ACE2 recognition site; the monoclonal antibody of the present invention can inhibit the binding of the virus to ACE2, is an effective neutralizing antibody, can be used for the development of drugs to prevent and / or treat diseases caused by SARS-CoV-2 infection, and has good clinical application value. The monoclonal antibody of the present invention has a clear recognition epitope, high affinity, strong specificity, and good stability, providing a new tool for the prevention, detection, and treatment of SARS-CoV-2.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunology, and specifically relates to a monoclonal antibody targeting a SARS-CoV-2 receptor binding motif, and its recognized antigen epitope and application. Background Art

[0002] The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses an unprecedented threat to global public health, and safe and effective preventive measures and therapies are urgently needed to combat the disease. The binding between the receptor binding domain (RBD) of the SARS-CoV-2 viral spike (S) protein and its cellular receptor angiotensin-converting enzyme 2 (ACE2) plays the most important role in viral attachment, fusion, and entry, and is therefore considered a major target for blocking viral entry. The RBD is also recognized as a prime candidate for antibody, inhibitor, and vaccine design. The receptor binding motif (RBM) is the major functional motif in the RBD, forming the interface between the S protein and hACE2. It is well known that the interaction interface between the RBD and ACE2 plays a crucial role in their binding activity. The most potent neutralizing antibodies directly target the RBM, either by competing for binding to the ACE2 binding site or by blocking the formation of the RBM and ACE2 binding interface through steric hindrance. The RBM, especially those residues involved in ACE2 recognition, is the main site of recognition by neutralizing antibodies.

[0003] Passive and active immunization approaches against viruses generally focus on the action of potent neutralizing antibodies. Passive immunization with plasma from patients who have recovered from viral infections has a long history of success. This passive immunization was widely used to prevent influenza virus infection during 1995 and was used during the limited Ebola virus epidemic in 2014-2015. This passive immunization approach has also been used to treat COVID-19, the disease caused by the SARS-CoV-2 coronavirus. Several reports have described significant benefits of infusing convalescent plasma into patients infected with SARS-CoV-2 without adverse side effects. The U.S. Food and Drug Administration recently approved plasma immunotherapy for this purpose and outlined safety standards. In recent years, highly specific neutralizing monoclonal antibodies against viruses have been developed as a more advanced alternative to convalescent patient plasma. The main goal of viral vaccine development is to induce antibodies with potent neutralizing activity, and this goal also applies to SARS-CoV-2.

[0004] Understanding the antibody response to the main SARS-CoV-2 candidates, especially the RBM, will provide useful information for the development of plasma- or monoclonal antibody-based immunotherapy and vaccines. Neutralizing monoclonal antibodies targeting the SARS-CoV-2 RBM can block the binding of the virus to hACE2 and are therefore promising antiviral drugs. However, rapidly spreading mutant SARS-CoV-2 strains may cause individual antibodies to escape and reduce vaccine efficacy. The combination of multiple antibodies targeting non-overlapping epitopes can provide enhanced protection and can greatly reduce the resistance of the virus through escape mutations. Therefore, new monoclonal antibodies targeting different antigenic epitopes are still needed to deal with new mutant strains. Summary of the invention

[0005] The purpose of the present invention is to provide a monoclonal antibody targeting the SARS-CoV-2 receptor binding motif, which can specifically recognize the antigenic epitope in the SARS-CoV-2 receptor binding motif 473 YQ 479 .

[0006] The second object of the present invention is to provide an antigen epitope that specifically binds to the above monoclonal antibody and its application.

[0007] The third object of the present invention is to provide a nucleic acid molecule comprising the above monoclonal antibody encoding gene, and an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium comprising the nucleic acid molecule.

[0008] A fourth object of the present invention is to provide applications of the above-mentioned monoclonal antibody, nucleic acid molecule, expression cassette, recombinant vector, recombinant cell or recombinant bacteria.

[0009] The fifth object of the present invention is to provide a pharmaceutical composition comprising the above monoclonal antibody.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] A monoclonal antibody targeting the SARS-CoV-2 receptor binding motif, comprising VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 1-3, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 4-6.

[0012] Preferably, the monoclonal antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.8.

[0013] It is obvious to those skilled in the art that, based on the amino acid sequences of the heavy and light chain variable regions of the monoclonal antibodies specifically disclosed in the present invention, one or more amino acids can be added, deleted, substituted, or modified by conventional protein engineering methods to obtain conservative variants or fragments thereof while still maintaining specific binding to the SARS-CoV-2 RBM.

[0014] Preferably, the heavy chain constant region of the monoclonal antibody is of IgG1 type, and the light chain constant region is of Kappa type.

[0015] Preferably, the monoclonal antibody specifically binds to an antigenic epitope located at the SARS-CoV-2 RBM, the antigenic epitope partially overlaps with the ACE2 recognition site, and its amino acid sequence is shown in SEQ ID NO.9 or SEQ ID NO.10.

[0016] An antigenic epitope of SARS-CoV-2 RBM, the amino acid sequence of the antigenic epitope is shown in SEQ ID NO.9 or SEQ ID NO.10; or the antigenic epitope is a truncated peptide of SEQ ID NO.9 containing a YQAGSTP core sequence.

[0017] The application of antigen epitopes is any of the following:

[0018] 1) Application in the preparation of SARS-CoV-2 antiserum or monoclonal antibodies;

[0019] 2) Application in the preparation of SARS-CoV-2 vaccines;

[0020] 3) Application in the preparation of SARS-CoV-2 immunoassay reagents or kits.

[0021] A nucleic acid molecule comprising a gene sequence encoding the above monoclonal antibody. Preferably, the gene sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.11, and the gene sequence of the light chain variable region is shown in SEQ ID NO.12.

[0022] The antibody nucleic acid molecules involved in the present invention can be obtained by genetic engineering recombination technology or chemical synthesis method. It is obvious to those skilled in the art that the variant sequence of the heavy chain variable region nucleotide sequence and / or the light chain variable region nucleotide sequence obtained after one or more nucleotide addition, deletion, substitution, modification, etc. of the above-mentioned nucleic acid molecules provided by the present invention, the single-chain antibody or chimeric monoclonal antibody or modified monoclonal antibody or other forms of monoclonal antibody or antibody fragment composed of the amino acid sequence encoded by it still retains the ability to specifically bind to SARS-CoV-2 RBM.

[0023] An expression cassette, recombinant vector, recombinant cell or recombinant bacteria containing the above nucleic acid molecule.

[0024] Specifically, the recombinant vector is selected from a prokaryotic or eukaryotic expression vector, and further, the recombinant vector is selected from a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus or other vectors. The expression system is a bacteria, a yeast, a filamentous fungus, a mammalian cell, an insect cell, a plant cell or a cell-free expression system.

[0025] The use of the above monoclonal antibody, nucleic acid molecule or the above expression cassette, recombinant vector, recombinant cell or recombinant bacteria is any of the following:

[0026] 1) Use in the preparation of drugs for preventing and / or treating diseases caused by SARS-CoV-2 infection;

[0027] 2) Application in the preparation of SARS-CoV-2 immunoassay reagents or kits.

[0028] A pharmaceutical composition comprises the above monoclonal antibody and a pharmaceutically acceptable carrier, diluent or excipient.

[0029] Beneficial effects achieved by the present invention:

[0030] The present invention provides a monoclonal antibody targeting SARS-CoV-2 RBM, which can specifically bind to the antigen epitope located at SARS-CoV-2 RBM 473 YQ 479 , the antigen epitope partially overlaps with the ACE2 recognition site. Experiments have shown that the monoclonal antibody of the present invention can inhibit the binding of the virus to ACE2, is an effective neutralizing antibody, and can be used for the development of drugs to prevent and / or treat diseases caused by SARS-CoV-2 infection, and has good clinical application value. The monoclonal antibody of the present invention has a clear recognition epitope, excellent affinity and high specificity, and good stability, providing a new tool for the prevention, detection and treatment of SARS-CoV-2.

[0031] The present invention provides amino acid sequences of the variable regions of the heavy and light chains of a monoclonal antibody targeting the SARS-CoV-2 RBM. On this basis, conventional antibody engineering methods can be used to obtain the monoclonal antibody of the present invention. Furthermore, modifications such as the addition, deletion, and substitution of one or more amino acids can be used to obtain active fragments or conservative variants thereof, thereby laying a foundation for further improving the specificity and affinity of the antibody.

[0032] The present invention also provides a newly discovered antigenic epitope located on the SARS-CoV-2 RBM. The epitope peptide can specifically recognize SARS-CoV-2 antibodies and can also induce animals to produce effective humoral immune responses. It has strong immunoreactivity and immunogenicity, and provides an important antigenic target for SARS-CoV-2 vaccine design, antibody development, and antibody detection kit development. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the result of the reaction between the positively reacted polypeptide (SEQ ID NO.9) and the antiserum;

[0034] In the figure, S1-S6 are pig sera obtained by immunization with RBD antigen; M1-M6 are mouse sera obtained by immunization with RBD antigen; NC is a non-RBD region polypeptide, which serves as a negative control.

[0035] Figure 2 This is a graph showing the titer results of the serum of mice immunized with the positive reaction polypeptide (SEQ ID NO.9);

[0036] In the figure, mouse 1 to mouse 5 are the numbers of five mice immunized with positive peptides; BSA is a negative control.

[0037] Figure 3 This is the result of IFA identification of monoclonal antibody reactivity;

[0038] In the figure, 10D2 is the name of the monoclonal antibody of the present invention; Alexa Fluor488 is goat anti-mouse IgG labeled with Alexa Fluor488; DAPI is a nuclear dye, indicating the cell nucleus; NC is a monoclonal antibody against other viruses, serving as a negative control.

[0039] Figure 4 This is a diagram showing the Western-blotting identification results of the monoclonal antibody 10D2 of the present invention;

[0040] In the figure, M is a protein marker, and SARS-CoV-2, SARS-CoV, and MERS-CoV represent the S proteins of the corresponding viruses in the corresponding lanes.

[0041] Figure 5 The figure is a result diagram of the identification of the neutralization ability of the monoclonal antibody;

[0042] In the figure, 10D2 is the monoclonal antibody of the present invention, and NC is the monoclonal antibody of other viruses, which serves as a negative control.

[0043] Figure 6 Figure 1 shows the results of the identification of the minimal epitope peptide required for epitope activity;

[0044] In the figure, the left side shows the reaction results of N-terminal truncated peptides; the right side shows the reaction results of C-terminal truncated peptides.

[0045] Figure 7 The figure shows the spatial location and structural analysis of the minimum epitope (SEQ ID NO.10). DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with specific implementation methods, but the protection scope of the present invention is not limited thereto; the instruments and equipment involved in the following embodiments are all conventional instruments and equipment unless otherwise specified; the reagents involved are all commercially available conventional reagents unless otherwise specified; the test methods involved are all conventional methods unless otherwise specified.

[0047] Example 1 Analysis of B cell epitope peptides using sera from animals immunized with antigens that fully expose RBM

[0048] 1. Antigen design

[0049] Using a strategy similar to that previously reported (A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS, Dai, L, etc.), the antigen was engineered based on the SARS-CoV-2 RBD. The engineered antigen was in the form of a stable RBD-dimer that fully exposed the dual receptor binding motif without introducing any exogenous sequences, thereby retaining the antigen potency and improving the neutralizing antibody titer.

[0050] 2. Obtaining Animal Serum

[0051] The above antigen (5 μg per mouse, 50 μg per pig) was used to immunize mice and pigs by intramuscular injection. After the initial injection, all animals were boosted on the 14th day and the 28th day after the initial immunization, for a total of 2 booster immunizations. Blood samples were collected 14 days after the last booster immunization and used for B cell epitope peptide analysis experiments.

[0052] 3. Analysis of B cell epitope peptides

[0053] According to the reference sequence of SARS-CoV-2S protein (access number: YP_009724390), 22 overlapping peptides were synthesized to cover the entire RBD, with a length of 20 amino acids and adjacent peptides overlapping by 5 amino acids.

[0054] Each peptide segment was coated with a 96-well ELISA plate and subjected to indirect enzyme-linked immunosorbent assay (ELISA) with the obtained animal serum to determine the positive reaction peptide segment. The amino acid sequence of the positive reaction peptide segment is shown in SEQ ID NO.9. The reaction of the positive reaction polypeptide with the immunized animal serum is shown in Figure 1 .

[0055] The steps of indirect ELISA are as follows:

[0056] (1) Dilute the above peptide fragments with CBS solution to a concentration of 2.5 μg / mL and coat the ELISA plate at 100 μl / well at 37°C

[0057] Incubate for 2 hours;

[0058] (2) Discard the coating solution, wash the plate with PBST, and block with 5% skim milk at 4°C overnight;

[0059] (3) Mouse serum samples (primary antibody) diluted 1:100 and pig serum samples (primary antibody) diluted 1:1000 were added to the ELISA plate in sequence, 50 μl / well, and incubated at 37°C for 30 min;

[0060] (4) Discard the primary antibody, wash the plate with PBST, clean it, and pat it dry;

[0061] (5) Add the diluted HRP-labeled goat anti-mouse IgG (secondary antibody) or goat anti-pig IgG (secondary antibody) into the reaction wells, 50 μl / well, and incubate at 37°C for 30 min;

[0062] (6) Discard the secondary antibody, rinse with PBST, and pat dry;

[0063] (7) Add 100 μl of freshly prepared TMB colorimetric solution to each well and react in the dark for 5 min;

[0064] (8) Add 50 μl 2M H2SO4 to each well to terminate the reaction;

[0065] (9) Read the OD of each well using an ELISA reader 450 value.

[0066] Example 2 Preparation of monoclonal antibodies

[0067] 1. Preparation of immunogens - peptide-coupled carrier protein BSA (bovine serum albumin)

[0068] The coupling was performed using the water-soluble amino-thiol crosslinker Sulfo-SMCC. Sulfo-SMCC has two reactive groups, sulfo-NHS ester and maleimide, which can react between primary amino groups and thiol groups. First, under the condition of pH 7-9, Sulfo-SMCC reacts with the primary amine group of the carrier protein BSA to form a stable amide bond to obtain an activated carrier protein BSA. Secondly, the activated BSA is dialyzed against PBS (pH 7.2-7.4), and the dialysate is changed three times, each time at an interval of 6 hours. The dialyzed solution is collected and the protein concentration is adjusted to 5 mg / ml with PBS. Finally, under the condition of pH 6.5-7.5, the activated BSA reacts with the thiol group of the positive reaction peptide SEQ ID NO.9 to form a stable thioether bond, forming an immunogenic carrier protein BSA and a positive reaction peptide conjugate for antibody preparation.

[0069] 2. Animal Immunization

[0070] (1) adding Freund's complete adjuvant to the above conjugate, emulsifying it and using it for the first immunization;

[0071] (2) Five female BALB / c mice aged 4 to 8 weeks were immunized by multiple subcutaneous injections at the back, with an immunization dose of 20 μg / mouse;

[0072] (3) BALB / c mice were boosted twice every two weeks with the same method and dose after emulsifying the above conjugate with Freund's incomplete adjuvant;

[0073] (4) After 2 weeks, blood was collected from the tail vein to determine the titer of specific antibodies against the SARS-CoV-2 S protein, and mice with higher titers were selected ( Figure 2 ), 4 days before cell fusion, BALB / c mice were super-immunized with the above-mentioned conjugate without adjuvant by tail vein injection, and the immunization dose was 40 μg / mouse.

[0074] 3. Cell Fusion and Monoclonal Antibody Preparation

[0075] The spleen cells of immunized mice were fused with mouse myeloma cells SP2 / 0 at a ratio of 8:1 using the polyethylene glycol method, and the fused cells were screened using HAT selection medium; 12 days after fusion, SARS-CoV-2 S protein and positive reaction peptide SEQ ID NO.9 were used as coating antigens, respectively, and positive hybridoma cells were preliminarily screened by indirect ELISA method;

[0076] The steps of the indirect ELISA method were carried out as in Example 1, with different primary and secondary antibodies. The primary antibody was a 1:100 diluted hybridoma supernatant, and the secondary antibody was HRP-labeled goat anti-mouse IgG (secondary antibody). Other operations were the same as in Example 1.

[0077] 4. Subcloning of hybridoma cells by limiting dilution

[0078] The positive hybridoma cells were diluted to about 1.5 cells / ml with 1640 / 10 complete medium, and 100 μl per well was added to a 96-well plate pre-coated with 100 μl feeder cells, and cultured in a 37°C, 5% CO2 incubator for 6 to 8 days; the positive hybridoma cells were further screened by indirect ELISA and indirect immunofluorescence assay (IFA); 2 to 3 subclonings were performed until a hybridoma cell line that stably secreted monoclonal antibodies against SARS-CoV-2S protein and positive polypeptide SEQ ID NO.9 was obtained, and the target hybridoma cells were obtained. The positive monoclonal clones obtained by screening were expanded and cultured according to the cell number of 1 to 2×10 6 / tube for cryopreservation.

[0079] 5. Stability identification of monoclonal hybridoma cell lines

[0080] The established monoclonal hybridoma cell line was cultured continuously for 3 months and repeatedly cryopreserved and revived in liquid nitrogen to identify the stability of the hybridoma cells; the results showed that the monoclonal hybridoma cell line had good stability.

[0081] 6. Preparation of monoclonal antibodies by inducing ascites in vivo

[0082] Multiparous female Balb / c mice were selected and intraperitoneally injected with 500 μl sterilized paraffin. One week later, the monoclonal hybridoma cells were intraperitoneally injected at a volume of 2 × 10 5 After the mouse abdomen was swollen, the ascites was extracted and the supernatant was obtained after centrifugation. The ascites was purified using the saturated ammonium sulfate method.

[0083] Example 3 Purification and identification of antibodies

[0084] 1. Antibody purification was performed by saturated ammonium sulfate precipitation method. The operation method is as follows:

[0085] (1) Take 5 ml of monoclonal antibody ascites, add 5 ml of PBS buffer, and then add 2.5 ml of saturated ammonium sulfate solution dropwise to make a final concentration of 20% ammonium sulfate solution. Stir while adding, mix thoroughly, and let stand for 30 minutes.

[0086] (2) Centrifuge at 8000 r / min for 20 min and discard the precipitate to remove fibrin.

[0087] (3) Add 12.5 ml of saturated ammonium sulfate solution to the supernatant, mix thoroughly, and let stand for 30 minutes.

[0088] (4) Centrifuge at 8000 r / min for 20 min and discard the supernatant.

[0089] (5) Add 10 ml of PBS buffer to the precipitate to dissolve it, then add 5 ml of saturated ammonium sulfate solution to make it a 33% ammonium sulfate solution. After thoroughly mixing, let it stand for 30 minutes.

[0090] (6) Centrifuge at 8000 r / min for 20 min and discard the supernatant to remove albumin.

[0091] (7) Repeat step 5 2 to 3 times.

[0092] (8) Dissolve the precipitate in 5 ml of PBS buffer, place it in a dialysis bag, and dialyze it against PBS buffer at 4°C, changing the buffer four times.

[0093] (9) Centrifuge at 8000 r / min for 20 min, discard the precipitate, and the supernatant is the purified antibody. Measure the antibody concentration, aliquot, and store at -20°C.

[0094] 2. Monoclonal Antibody Titer Determination

[0095] The indirect ELISA assay method was carried out in accordance with Example 1, with a slightly different primary antibody: the purified monoclonal antibody was diluted 2-fold with 5% skim milk starting from 1:200, and added to the ELISA plate in sequence, 50 μl / well, and incubated at 37°C for 30 min; the other steps were carried out in accordance with Example 1, and the ELISA test results showed that the monoclonal antibody titer was 1:4.096×10 5 .

[0096] 3. Subtype identification

[0097] The subtype of the monoclonal antibody was identified using a mouse monoclonal antibody subtype identification kit (Sigma, Mouse Monoclonal Antibody Isotyping Kit). The identification results showed that the monoclonal antibody belonged to IgG1 and the light chain type was Kappa.

[0098] 4. Monoclonal antibody specificity identification

[0099] The above monoclonal antibodies were diluted in a certain ratio and added to 293T cells transiently transfected with the eukaryotic expression vector pLVX-SARS-CoV-2-S. The results were determined by IFA detection method. Figure 3As shown in Figure 2, the monoclonal antibody reacts specifically with the cell-derived SARS-CoV-2S protein, but does not react with untransfected cells; the Western-blotting results are shown in Figure 2. Figure 4 As shown, the monoclonal antibody specifically recognizes and reacts with the S protein of SARS-CoV-2, but does not react with the S protein of SARS-CoV and MERS-CoV.

[0100] 5. Neutralizing activity of monoclonal antibodies

[0101] Through the surrogate virus neutralization test (sVNT), specifically using cPass TM The SARS-CoV-2 neutralizing antibody kit (GenScript, Nanjing, China) was used to evaluate the neutralizing ability of the monoclonal antibody. The specific operation steps were carried out according to the product instructions. The negative control was a monoclonal antibody against African swine fever virus. The results are as follows Figure 5 As shown, Inhibition (inhibition value) = (1-sample OD value / negative control OD value) × 100%. When the inhibition value is ≥ 20%, it indicates that the tested monoclonal antibody is a neutralizing antibody. Figure 5 It can be seen that the monoclonal antibody is a neutralizing antibody.

[0102] Example 4 Monoclonal Antibody Variable Region Gene Amplification and Sequencing

[0103] 1. Primer Design

[0104] Design the heavy chain variable region primer sequence based on the sequence characteristics of mouse monoclonal antibodies

[0105] P1: 5'-AGGTSMARCTGCAGSAGTCWGG-3';

[0106] P2: 5'-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC-3'.

[0107] Design of light chain variable region primer sequences

[0108] P3: 5'-ACTAGTCGACATGGAGWCAGACACACTSCTGYTATGGGT-3';

[0109] P4: 5'-CCCAAGCTTACTGGATGGTGGGAAGATGGA-3'.

[0110] 2. Polymerase chain reaction (PCR) amplification

[0111] The variable region sequences of monoclonal antibodies were obtained through molecular cloning technology and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.

[0112] Total RNA from hybridoma cells was isolated using TRIzol and analyzed using PrimeScript TM cDNA was synthesized using the Takara Biomedical Technology (Beijing) Co. II kit. The variable light chain region (VL) and variable heavy chain region (VH) of the monoclonal antibody were amplified using the above primers in two separate PCR reactions.

[0113] The sequencing results are as follows: the amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody of the present invention are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. Further analysis revealed that the amino acid sequences of the heavy chain variable region VHCDR1-3 of the monoclonal antibody are shown in SEQ ID NO.1-3, respectively; the amino acid sequences of the light chain variable region VLCDR1-3 of the monoclonal antibody are shown in SEQ ID NO.4-6, respectively.

[0114] Example 5 Identification of the Minimal Motif of the Epitope Recognized by the Specific Monoclonal Antibody Targeting the SARS-CoV-2 Receptor Binding Motif

[0115] A truncated library was established by systematically truncating the positive polypeptide R465 (SEQ ID NO.9) from both ends. The amino acid sequence of the truncated peptide is shown in Table 1. The reaction of the truncated polypeptide with the monoclonal antibody was identified by indirect ELISA. The indirect ELISA assay was performed with reference to Example 1. The primary antibody was different. The monoclonal antibody of the present invention was used as the primary antibody. The results are shown in FIG. Figure 6 As shown, the left side shows the reaction result of N-terminal truncated polypeptide and monoclonal antibody, and the right side shows the reaction result of C-terminal truncated polypeptide and monoclonal antibody. Figure 6 It can be seen that the minimum epitope peptide required for epitope activity is " 473 YQ 479 ” (SEQ ID NO.10).

[0116] Table 1 List of amino acid sequences of truncated peptides

[0117]

[0118]

[0119] Example 6 Spatial localization and structural analysis of antigenic epitopes

[0120] The spatial distribution and structural properties of the epitope (SEQ ID NO. 10) were analyzed by mapping to the SARS-CoV-2 RBD-ACE2 complex (PDB ID: 6M0J) and the SARS-CoV-2 S protein trimer (PDB ID: 7A95). Figure 7As shown, the epitope is located on a loop structure in the RBM region and overlaps with the ACE2 binding site, further indicating that it is a target for potential neutralizing antibodies that interfere with virus-receptor binding.

[0121] Example 7 Immunotherapy (cocktail therapy) comprising the antibody

[0122] The development of SARS-CoV-2 monoclonal antibodies that inhibit the binding of S protein to ACE2 by targeting epitopes on the receptor binding domain of S protein is the focus of therapeutic interventions for COVID-19. However, as the pandemic continues to spread among the population, it is possible that viral mutants with the ability to escape neutralization by a single antibody will emerge. The effect of treating the virus with a single antibody drug will decrease over time, but the "cocktail therapy" using a mixture of multiple antibodies remains effective in the face of viral mutations. The combination of two or more neutralizing antibodies with different epitopes can ensure protection against these mutants. The monoclonal antibody provided by the present invention targets a potential neutralizing epitope SEQID NO.10 located in the SARS-CoV-2 receptor binding motif, which can be used as a candidate antibody for "cocktail therapy". The specific implementation method can refer to the prior art (REGN-COV2, a Neutralizing Antibody Cocktail, in Outpatients with Covid-19, Weinreich DM, etc. and Structure-based development of human antibody cocktails against SARS-CoV-2, Wang, N, etc.), which will not be described in detail here. <110> Zhengzhou University <120> Monoclonal antibodies targeting SARS-CoV-2 receptor binding motifs and their recognized antigen epitopes and applications <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <221> VHCDR1 <400> 1 Gly Tyr Thr Phe Thr Lys Tyr Arg 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial sequence <221> VHCDR2 <400> 2 Ile Asn Thr Asn Thr Gly Glu Ser 1 5 <210> 3 <211> 6 <212> PRT <213> Artificial sequence <221> VHCDR3 <400> 3 Ala Ser Ser Met Asp Tyr 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial sequence <221> VLCDR1 <400> 4 Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr 1 5 10 <210> 5 <211> 3 <212> PRT <213> Artificial sequence <221> VLCDR2 <400> 5 Leu Val Ser 1 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <221> VLCDR3 <400> 6 Gln His Ile Arg Glu Leu Thr Arg Ser 1 5 <210> 7 <211> 108 <212> PRT <213> Artificial sequence <221> Heavy chain variable region <400> 7 Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser 1 5 10 15 Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr Arg Met His Trp Val 20 25 30 Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr 35 40 45 Asn Thr Gly Glu Ser Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala 50 55 60 Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn 65 70 75 80 Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Ser Ser Met Asp 85 90 95 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 100 105 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <221> Light Chain Variable Region <400> 8 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg 85 90 95 Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp 100 105 <210> 9 <211> 20 <212> PRT <213> Artificial sequence <221> SARS-CoV-2 RBM epitopes <400> 9 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 1 5 10 15 Asn Gly Val Glu 20 <210> 10 <211> 7 <212> PRT <213> Artificial sequence <221> SARS-CoV-2 RBM epitope (core sequence) <400> 10 Tyr Gln Ala Gly Ser Thr Pro 1 5 <210> 11 <211> 324 <212> DNA <213> Artificial sequence <221> Heavy chain variable region <400> 11 cagtctggac ctgagctgaa gaaacctgga gagacagtca agatctcctg caaggcttct 60 gggtatacct tcacaaaata tagaatgcac tgggtgaagc aggctccagg aaagggttta 120 aagtggatgg gctggataaa caccaacact ggagagtcaa catatgctga tgatttcaag 180 ggacggtttg ccttctcttt ggaaacctct gccagcactg cctatttgca gatcaacaac 240 ctcaaaaatg aggacacggc tacatatttc tgtgcgtcct ctatggacta ctggggccaa 300 gggaccacgg tcaccgtctc ctca 324 <210> 12 <211> 321 <212> DNA <213> Artificial sequence <221> Light chain variable region <400> 12 gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60 atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120 caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180 ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggagg aggaggatt tgcaacctat tactgtcagc acattaggg gcttacacgt tcggaggggg gaccaagctg g <210> 13 <211> 19 <212> PRT <213> The snowstorm <221> aa 467-4 <400> 13 Cys Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn 1 5 10 15 Gly Val Glue <210> 14 <211> 17 <212> PRT <213> The snowstorm <221> aa 469-4 <400> 14 Cys Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val 1 5 10 15 Glu <210> 15 <211> 15 <212> PRT <213> The snowstorm <221> aa 471-4 <400> 15 Cys Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu 1 5 10 15 <210> 16 <211> 13 <212> PRT <213> The snowstorm <221> aa 473-4 <400> 16 Cys Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu 1 5 10 <210> 17 <211> 12 <212> PRT <213> Artificial sequence <221> aa 474-484 <400> 17 Cys Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu 1 5 10 <210> 18 <211> 11 <212> PRT <213> Artificial sequence <221> aa 475-484 <400> 18 Cys Ala Gly Ser Thr Pro Cys Asn Gly Val Glu 1 5 10 <210> 19 <211> 19 <212> PRT <213> Artificial sequence <221> aa 465-482 <400> 19 Cys Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro 1 5 10 15 Cys Asn Gly <210> 20 <211> 17 <212> PRT <213> Artificial sequence <221> aa 465-480 <400> 20 Cys Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro 1 5 10 15 Cys <210> twenty one <211> 16 <212> PRT <213> Artificial sequence <221> aa 465-479 <400> twenty one Cys Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro 1 5 10 15 <210> twenty two <211> 15 <212> PRT <213> Artificial sequence <221> aa 465-478 <400> twenty two Cys Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 1 5 10 15 <210> twenty three <211> 13 <212> PRT <213> Artificial sequence <221> aa 465-476 <400> twenty three Cys Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly 1 5 10 <210> twenty four <211> 11 <212> PRT <213> Artificial sequence <221> aa 465-474 <400> twenty four Cys Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln 1 5 10 <210> 25 <211> twenty two <212> DNA <213> Artificial sequence <221> Heavy chain variable region upstream primer <400> 25 aggtsmarct gcagsagtcw gg 22 <210> 26 <211> 32 <212> DNA <213> Artificial sequence <221> Heavy chain variable region downstream primer <400> 26 tgaggagacg gtgaccgtgg tcccttggcc cc 32 <210> 27 <211> 39 <212> DNA <213> Artificial sequence <221> Light chain variable region upstream primer <400> 27 actagtcgac atggagwcag acacactsct gytatgggt 39 <210> 28 <211> 30 <212> DNA <213> Artificial sequence <221> Light chain variable region downstream primer <400> 28 cccaagctta ctggatggtg ggaagatgga 30

Claims

1. A monoclonal antibody targeting the SARS-CoV-2 receptor binding motif, characterized in that: It comprises VHCDR1, VHCDR2 and VHCDR3 whose amino acid sequences are shown in SEQ ID NOs: 1-3, and VLCDR1, VLCDR2 and VLCDR3 whose amino acid sequences are shown in SEQ ID NOs: 4-6.

2. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.

8.

3. The monoclonal antibody according to claim 1, characterized in that The heavy chain constant region of the monoclonal antibody is of IgG1 type, and the light chain constant region is of Kappa type.

4. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody specifically binds to the antigenic epitope of SARS-CoV-2 RBM, which partially overlaps with the ACE2 recognition site, and its amino acid sequence is shown in SEQ ID NO.9 or SEQ ID NO.

10.

5. A nucleic acid molecule, characterized in that The method comprises a gene sequence encoding the monoclonal antibody according to claim 1 or 2.

6. An expression cassette, recombinant vector, recombinant cell or recombinant bacterium containing the nucleic acid molecule according to claim 5.

7. Use of the monoclonal antibody according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, or the expression cassette, recombinant vector, recombinant cell or recombinant bacteria according to claim 6, which is any of the following: Use in the preparation of drugs for preventing and / or treating diseases caused by SARS-CoV-2 infection; Application in the preparation of SARS-CoV-2 immunoassay reagents or kits.

8. A pharmaceutical composition, characterized in that The composition comprises the monoclonal antibody according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier, diluent or excipient.

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