A fully human monoclonal antibody against sars-cov and sars-cov-2 and uses thereof

Abstract

The application provides a kind of anti-SARS-CoV and SARS-CoV-2 fully human monoclonal antibody and its application, belong to immunology technical field, the amino acid sequence of the variable region of heavy chain of the monoclonal antibody is as shown in SEQ ID NO:1;The amino acid sequence of the variable region of light chain of the monoclonal antibody SARS2-1 is as shown in SEQ ID NO:2;The antibody has unique CDR partition, can simultaneously recognize the S protein of the two viruses mentioned above, and has cross-neutralizing activity to the pseudo virus of the two viruses mentioned above.The antibody has the characteristics of high expression, fully human, good stability at the same time, is suitable for industrial production, has potential application value for coping with the outbreak and prevalence caused by coronavirus at present and in the future.

Description

Technical Field

[0001] This invention belongs to the field of immunology technology, and in particular relates to a fully human monoclonal antibody against SARS-CoV and SARS-CoV-2 and its application. Background Technology

[0002] Coronaviruses are a class of enveloped, single-stranded, positive-sense RNA viruses, belonging to the genus Coronavirus in the family Coronaviridae of the order Nidovirales. Among them, the β-coronavirus genus includes various pathogens that can infect humans and cause severe respiratory illnesses, such as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and the novel coronavirus (SARS-CoV-2). SARS-CoV-2 and SARS-CoV share high sequence homology, both utilizing angiotensin-converting enzyme 2 (ACE2) as the functional receptor for infecting cells. The key to viral invasion of host cells lies in the binding of the spike protein (S protein) on its surface to the ACE2 receptor on the surface of human cells. The receptor-binding domain (RBD) is an important target for neutralizing antibodies. Studies have shown that neutralizing antibodies targeting the S protein RBD can block the binding of the virus to the receptor, thereby effectively inhibiting viral infection of cells.

[0003] Fully human antibodies have become an important trend in the development of therapeutic antibody drugs due to their low immunogenicity and high safety. Currently, neutralizing monoclonal antibodies can be prepared using hybridoma technology, humanized transgenic mice, phage library screening, and single-cell PCR technology. The principle of single-cell PCR technology is that recovered viral infections or vaccinated individuals possess protective monoclonal antibodies against viruses. The gene encoding this antibody is located in a single lymphocyte in human peripheral blood. This gene can be amplified using flow cytometry sorting and single-cell PCR technology. Subsequently, through genetic engineering, candidate antibody molecules can be prepared on a large scale in vitro. This method offers advantages such as being fully human and having good natural stability, and is widely used in the development of neutralizing antibodies.

[0004] Due to the similarity between SARS-CoV and SARS-CoV-2, and the increasingly significant problem of immune escape caused by the latter's viral mutations, SARS-CoV-2, as an RNA virus, is prone to mutation during replication, leading to changes in its antigenicity. This has rendered some early-developed monoclonal antibody drugs ineffective or significantly reduced in their neutralizing ability. To address this issue, research on broad-spectrum cross-neutralizing antibodies has become an important direction. For example, CR3022 is a SARS-CoV neutralizing antibody that, through directed evolution technology, has increased its affinity for SARS-CoV-2 RBD by more than 1000 times and possesses SARS-CoV-2 neutralizing activity. Similarly, some research institutions have also developed bispecific neutralizing antibodies capable of simultaneously recognizing different coronaviruses such as SARS-CoV-2 and SARS-CoV.

[0005] Currently, there are still limited fully human monoclonal antibodies capable of simultaneously and effectively neutralizing both SARS-CoV and SARS-CoV-2, and their molecular mechanisms and application potential require further exploration. Therefore, developing fully human monoclonal antibodies with cross-neutralizing activity is of significant scientific and clinical value in addressing coronavirus mutations and potential new coronaviruses in the future. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a fully human monoclonal cross-neutralizing antibody against SARS-CoV and SARS-CoV-2 and its application; the monoclonal antibody has the potential to become a broad-spectrum anti-coronavirus drug and can be used for the prevention and treatment of coronavirus.

[0007] The present invention provides a monoclonal antibody SARS2-1 against SARS-CoV and SARS-CoV-2, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:1; and the amino acid sequence of the light chain variable region of the monoclonal antibody SARS2-1 is shown in SEQ ID NO:2.

[0008] Preferably, the amino acid sequence of the heavy chain constant region of the monoclonal antibody SARS2-1 is shown in SEQ ID NO:3; the amino acid sequence of the light chain constant region of the monoclonal antibody SARS2-1 is shown in SEQ ID NO:4.

[0009] The present invention provides a nucleic acid sequence encoding the monoclonal antibody SARS2-1, including a nucleic acid sequence encoding the heavy chain variable region as shown in SEQ ID NO:5, a nucleic acid sequence encoding the light chain variable region as shown in SEQ ID NO:6, a nucleic acid sequence encoding the heavy chain constant region as shown in SEQ ID NO:7, and a nucleic acid sequence encoding the light chain constant region as shown in SEQ ID NO:8.

[0010] The present invention provides a recombinant vector for expressing the monoclonal antibody SARS2-1, comprising an initial vector, a nucleic acid sequence encoding the heavy chain variable region of the monoclonal antibody SARS2-1, and a nucleic acid sequence encoding the light chain variable region of the monoclonal antibody SARS2-1.

[0011] Preferably, the initial vector is a eukaryotic expression vector containing a nucleic acid sequence encoding the light chain constant region of the monoclonal antibody SARS2-1 and a nucleic acid sequence encoding the heavy chain constant region of the monoclonal antibody SARS2-1.

[0012] This invention provides a recombinant cell expressing the monoclonal antibody SARS2-1, obtained by transfecting the recombinant vector into a host cell; the host cell is an Expi 293F cell or a CHO-S cell.

[0013] This invention provides the use of the monoclonal antibody SARS2-1, the nucleic acid sequence, the recombinant vector, and the recombinant cells in the preparation of drugs for the prevention and / or treatment of coronavirus infection.

[0014] This invention provides the application of the monoclonal antibody SARS2-1, the nucleic acid sequence, the recombinant vector, and the recombinant cells in the preparation of reagents for detecting coronavirus infection.

[0015] Compared with existing technologies, the present invention has the following beneficial effects: The fully human monoclonal cross-neutralizing antibody SARS2-1 provided by the present invention has a unique CDR partition, which can simultaneously recognize the S protein of the two viruses and has cross-neutralizing activity against pseudoviruses of the two viruses. The monoclonal antibody SARS2-1 binds to the SARS-CoV variants BA.1, XBB.1.16, JN.1 and the SARS-CoV antigen protein. 50 The values ​​were 15.85 ng / mL, 8.736 ng / mL, 9.804 ng / mL, and 11.68 ng / mL, respectively. In the pseudovirus neutralization experiment, the antibody showed IC50 values ​​against wild-type SARS-CoV-2 pseudovirus, JN.1, and pseudovirus SARS-CoV. 50 The values ​​were 8.305 μg / mL, 1.720 μg / mL, and 0.6233 μg / mL. The antibody effectively neutralized both the pseudovirus SARS-CoV-2 and the pseudovirus SARS-CoV.

[0016] The monoclonal antibody SARS2-1 provided by this invention has the characteristics of high expression, fully human origin, and good stability, making it suitable for industrial production and potentially valuable for responding to current and future outbreaks of coronaviruses. Attached Figure Description

[0017] Figure 1 This is a graph showing the OD values ​​of SARS-CoV and SARS-CoV-2 cross-binding antibodies screened by ELISA in Example 1;

[0018] Figure 2 This is a graph showing the SDS-PAGE results for SARS2-1 purity detection in Example 1;

[0019] Figure 3 The cross-neutralizing activity of SARS2-1 was detected by the pseudovirus in Example 2. Detailed Implementation

[0020] This invention provides a monoclonal antibody, SARS2-1, against SARS-CoV and SARS-CoV-2. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:1, and is as follows:

[0021] EVQLVESGGGLVQPGGSLRLSCSASGFTFSRYSMHWIRQTPGKGPEYVSGLSRNGDIADYADSVKGRFTISRDNAKNTLYLQMSSLRPEDTALYYCVKDVDTSMVNVFDYWGQGTLVTVSS.

[0022] The heavy chain variable region of the monoclonal antibody SARS2-1 includes:

[0023] The amino acid sequence of the CDR1 region is shown in positions 26-33 of SEQ ID NO:1;

[0024] The amino acid sequence of the CDR2 region is shown in positions 51-58 of SEQ ID NO:1;

[0025] The amino acid sequence of the CDR3 region is shown in positions 97-110 of SEQ ID NO:1;

[0026] The amino acid sequence of the light chain variable region of the monoclonal antibody SARS2-1 is shown in SEQ ID NO:2, and is as follows:

[0027] DIVMTQSPPSLSASVGDRVTITCRASQTISRYLNWFQQTPGKAPKLLIFDTSTLKDGVPSRFSGSASGTDFTLTISSLQREDFATYYCQQSYTSPYTFGQGTKVEIK.

[0028] The light chain variable region of the monoclonal antibody SARS2-1 includes:

[0029] The amino acid sequence of the CDR1 region is shown in positions 27-32 of SEQ ID NO:2;

[0030] The amino acid sequence of the CDR2 region is shown in positions 50-52 of SEQ ID NO:2;

[0031] The amino acid sequence of the CDR3 region is shown in positions 89-97 of SEQ ID NO:2;

[0032] The amino acid sequence of the heavy chain constant region of the monoclonal antibody SARS2-1 is shown in SEQ ID NO:3, and is as follows:

[0033] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0034] The amino acid sequence of the light chain constant region of the monoclonal antibody SARS2-1 is shown in SEQ ID NO:4, and is as follows:

[0035] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0036] This invention provides a nucleic acid sequence encoding the monoclonal antibody SARS2-1, comprising a nucleic acid sequence encoding the heavy chain variable region as shown in SEQ ID NO:5, a nucleic acid sequence encoding the light chain variable region as shown in SEQ ID NO:6, a nucleic acid sequence encoding the heavy chain constant region as shown in SEQ ID NO:7, and a nucleic acid sequence encoding the light chain constant region as shown in SEQ ID NO:8, as detailed below:

[0037] SEQ ID NO:5:

[0038] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGGCTCTCCTGTTCAGCCTCTGGATTCACCTTTAGTAGATACTCTATGCACTGGATCCGCCAGACTCCAGGGAAGGGACCGGAATATGTGTCAGGTCTTAGTAGAAATGGGGATATCGCAGACTACG CAGACTCCGTGAAGGGCAGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTATCTTCAAATGAGCAGCCTGAGACCTGAGGACACGGCCTGTATTACTGTGTGAAAGATGGTGGATACATCTATGGTTAATGTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0039] SEQ ID NO:6:

[0040] GATATTGTGATGACTCAGTCTCCACCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGACCATTAGTAGGTATTTAAATTGGTTTCAGCAGACACCAGGGAAAGCCCCTAAGCTCCTGATCTTTGATACATCCACTT TGAAAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGCATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACGTGAAGATTTCGCAACTTATTACTGTCAACAGAGTTACACTTCCCCGTATACTTTTGGCCAGGGGACCAAGGTGGAGATCAAA

[0041] SEQ ID NO:7:

[0042] GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAGGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCAGGTAAA

[0043] SEQ ID NO:8:

[0044] CGTACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGA GAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGTACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT.

[0045] This invention provides a recombinant vector for expressing the monoclonal antibody SARS2-1, comprising an initial vector, a nucleic acid sequence encoding the heavy chain variable region of the monoclonal antibody SARS2-1, and a nucleic acid sequence encoding the light chain variable region of the monoclonal antibody SARS2-1. In this invention, the initial vector is a eukaryotic expression vector containing a nucleic acid sequence encoding both the light chain constant region and the heavy chain constant region of the monoclonal antibody SARS2-1. Preferably, the nucleic acid sequences of the heavy chain variable region and the light chain variable region are integrated into the initial vector via homologous recombination. In this invention, the homologous arm sequences and the initial vector are described in patent CN114480501A, "A Human Antibody Expression Plasmid and Its Construction Method".

[0046] This invention provides a recombinant cell expressing the monoclonal antibody SARS2-1, obtained by transfecting the recombinant vector into a host cell; the host cell is Expi 293F cell or CHO-S cell. This invention does not specify a particular method for constructing the recombinant cell; conventional recombinant cell construction methods in the art can be used.

[0047] This invention provides the application of the monoclonal antibody SARS2-1, the nucleic acid sequence, the recombinant vector, and the recombinant cells in the preparation of medicaments for the prevention and / or treatment of coronavirus infection. In this invention, the medicaments include antibody drugs, antibody-drug conjugates, vaccines, and pharmaceutical compositions.

[0048] This invention provides the application of the monoclonal antibody SARS2-1, the nucleic acid sequence, the recombinant vector, and the recombinant cells in the preparation of reagents for detecting coronavirus infection.

[0049] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] Screening and preparation of human monoclonal antibodies

[0052] 1. Blood sample collection and PBMC separation

[0053] After obtaining informed consent, 20 mL of blood samples were collected from recovered SARS-CoV-2 patients 14 days after infection. PBMCs were separated using Ficoll density gradient centrifugation and the lymphocytes were resuspended in PBS buffer for later use.

[0054] 2. Flow cytometry sorting of antigen-specific memory B cells

[0055] Lymphocytes were counted and stained with fluorescent dyes (PE-Anti Human IgG, Alexa Fluor 700-Anti Human CD19, PerCP-Anti Human CD3, PE Cy7-Anti Human CD27) and biotinylated antigen (XBB.1.16, purchased from ACRObiosystems, catalog number: SPN-C524s), and incubated at 4°C in the dark for 1 h. After washing twice with FPBS, cells were stained with streptavidin conjugated with Alexa Fluor 488 and incubated at 4°C in the dark for 30 min. After washing twice with FPBS, cells were resuspended in FPBS and antigen-specific individual memory B cells were sorted using a cell sorter (SONY, MA900). The sorting strategy was as follows: CD3- / CD19+ / IgG+ / CD27+ / XBB.1.16+. Individual memory B cells were directly sorted into 96-well plates, each containing 20 U of RNase inhibitor and 20 μL of RNase-free water, and stored at -80°C.

[0056] 3. Amplification of antibody variable region genes

[0057] (1) Reverse transcription PCR

[0058] Add reverse transcription PCR reagent to a 96-well PCR plate containing a single B cell. The reagent consists of: 6 μL 5× buffer, 1.2 μL dNTPs, 1.2 μL reverse transcriptase, and 0.2 μM specific primers for each subtype of the heavy chain (H), κ light chain, and λ light chain (see Table 3, paragraph 0032, of Chinese Patent CN115850465A for primers). Add water to a final volume of 30 μL. The PCR reaction conditions are: reverse transcription at 50℃ for 30 min, pre-denaturation at 95℃ for 15 min, followed by 40 cycles of 95℃ for 40 s, 55℃ for 30 s, and 72℃ for 1 min, with a final extension at 72℃ for 10 min. The amplified cDNA is stored at -20℃.

[0059] (2) Nested PCR

[0060] Using the reverse transcription product as a template, nested PCR was performed to amplify the H, κ, and λ chains of the antibody (primers are listed in Table 4, paragraph 0035, of Chinese Patent CN115850465A). The PCR reaction system contained: 2.5 μL of 10× buffer, 0.5 μL of 10 mM dNTPs, 0.25 μL of DNA polymerase, 0.2 μM primers for each of the H, κ, and λ chains, 1 μL of the reverse transcription product as template, and water to a final volume of 25 μL. The PCR reaction conditions were: 94℃ pre-denaturation for 4 min; followed by 40 cycles of 94℃ for 30 s, 57℃ for 30 s, and 72℃ for 45 s; and a final extension at 72℃ for 10 min. Positive clones were identified by nucleic acid electrophoresis, and the amplification products of positive clones were sequenced.

[0061] 4. Preliminary screening of binding antibodies using linear expression cassettes

[0062] Antibodies were rapidly screened by constructing a linear expression cassette from the antibody variable region gene. The designed linear expression cassette contained all the elements for monoclonal antibody expression in mammalian cells. The linear expression cassette, from the 5' end, sequentially contained the CMV promoter sequence (Genbank accession number: X03922.1), the coding sequence of the antibody leader peptide, the antibody variable region (absorbed from single cells, nucleotide sequence SEQ ID NO: 5~6), the antibody constant region (synthesized by bioengineering, the DNA coding sequence of the heavy chain constant region is shown in SEQ ID NO: 7, and the DNA coding sequence of the Kappa type light chain constant region is shown in SEQ ID NO: 8), and the poly-A tail (Genbank accession number: X03896.1) linked together. (For the specific construction method, see paragraph 0039 of Chinese Patent CN115947838A).

[0063] The linear form of DNA was transfected into cells for antibody expression. Antibody binding activity was measured in the cell supernatant containing the antibody: One day prior to the experiment, 2 µg / mL of XBB.1.16 S protein was coated onto 96-well ELISA plates (100 µL per well) overnight at 4°C. On the day of the experiment, the plates were washed 5 times, and 100 µL of blocking buffer was added to each well, blocking at 37°C for 1 hour. After washing 5 times, the cell supernatant was added to microplates and incubated at 37°C for 1 hour. After washing 5 times, HPR-labeled goat anti-human IgG secondary antibody was added to the microplates and incubated at 37°C for 1 hour. After washing 5 times, 100 µL of TMB single-component chromogenic solution was added to each well, and the plates were incubated at 37°C in the dark for 3 minutes. Then, 50 µL of stop solution was added to each well to terminate the reaction. The absorbance at 450 nm was measured using a microplate reader with 630 nm as a reference. Wells without the test sample were used as negative controls, and wells with OD450-630 greater than 2.1 times that of the negative control were considered positive.

[0064] 5. Constructing antibody expression vectors

[0065] The variable region gene of the selected antibody-binding gene was amplified by PCR, and homologous arms were added at the 5' and 3' ends. The variable region was then ligated into a eukaryotic expression vector containing the light and heavy chain constant region gene via homologous recombination (the homologous arm sequence and vector are shown in paragraph 0030 of Chinese Patent CN 114480501A, "A Human Antibody Expression Plasmid and Its Construction Method"). The homologous recombination product was transformed into TOP10 competent cells and cultured overnight on plates containing ampicillin. Single clones were picked and sequenced, and clones whose sequences matched the nested product sequencing results were selected for plasmid extraction.

[0066] 6. Antibody Expression and Purification

[0067] Take 15 μg each of the antibody light and heavy chain expression plasmids and transfect them into 30 mL of Expi293F cells according to the instructions of the transfection kit (ExpiFectamine™ 293Transfection Kit, ThermoFisher, catalog number: A14525). After culturing at 125 rpm and 5% CO2 for 120 h, the culture product was centrifuged at 3000 g for 10 min to collect the expression supernatant. After filtration through a 0.22 μm syringe filter, the supernatant was purified by rProtein A affinity. The collected antibody was concentrated and replaced with PBS, and the antibody concentration was determined by spectrophotometry.

[0068] 7. Results

[0069] The results are as follows Figure 1As shown, ELISA detection revealed that antibody SARS2-1 exhibits cross-binding activity against SARS-CoV and SARS-CoV-2 antigen proteins from the amplified paired antibodies. It also showed binding ECMO activity against SARS-CoV variants BA.1, XBB.1.16, JN.1, and SARS-CoV antigen proteins (all purchased from ACRO Biosystems, catalog numbers: SPN-C52Hz, SPN-C524s, SPN-C5221, and SPN-S5221, respectively). 50 The values ​​were 15.85 ng / mL, 8.736 ng / mL, 9.804 ng / mL, and 11.68 ng / mL, respectively. Expression plasmids were further constructed, expressed, and purified. The purity of the target antibody protein was confirmed by SDS-PAGE, as shown below. Figure 2 As shown, the light and heavy chains of the SARS2-1 antibody after unwinding can be clearly observed.

[0070] Example 2

[0071] Identification of pseudovirus neutralizing activity of monoclonal antibody SARS2-1

[0072] (1) Antibody dilution: The SARS2-1 antibody to be tested was serially diluted three-fold in DMEM cell culture medium containing 10% FBS in a 96-well cell culture plate. The initial concentration of the antibody to be tested in each well was 100 μg / mL. A total of 8 dilution concentrations were set, and 3 replicates were set for each dilution gradient. (2) Pseudovirus dilution: The pseudovirus was diluted to the appropriate titer in DMEM cell culture medium containing 10% FBS. 50 μL of pseudovirus dilution solution was added to each well. Wells without pseudovirus were used as survival controls, and wells with pseudovirus but without antibody were used as death controls. The antibody and pseudovirus were co-incubated at 37℃ for 1 h. (3) Cell infection: ACE2-293T cells were diluted to 2.5 × 10⁻⁶ cells in DMEM cell culture medium containing 10% FBS. 5Cells / mL, seeded into the above 96-well cell culture plate, add 100 μL of cell suspension to each well, and culture in a cell culture incubator at 37℃ for 48 h. (4) Detection of Luciferase reading and data processing: discard 100 μL of cell culture supernatant, add 100 μL of chromogenic substrate, and incubate in the dark for 2 min. Transfer 150 μL to a 96-well white microplate, and read the Luciferase signal value using the Tecan Spark multi-functional microplate detector. Calculate the neutralization rate of the antibody to be tested using [1-(Luc sample wells-Luc survival control) / (Luc death control-Luc survival control)] × 100%, and perform statistical analysis on the data using GraphPadPrism 8. First, convert the X value to logarithm, X=Log (X), and then use dose-dependent three-parameter nonlinear regression analysis: log (inhibitor) vs. response (three parameters) to calculate the antibody IC50. 50 Value. Result as follows Figure 3 As shown.

[0073] Depend on Figure 3 It can be seen that the antibody SARS2-1 provided by this invention has an IC50 value against wild-type, JN.1, and pseudovirus SARS-CoV. 50 The values ​​were 8.305 μg / mL, 1.720 μg / mL, and 0.6233 μg / mL. The antibody exhibited neutralizing activity against both SARS-CoV and SARS-CoV-2 pseudoviruses.

[0074] As can be seen from the above embodiments, the antibody SARS2-1 provided by the present invention can simultaneously recognize the S proteins of the two viruses and has cross-neutralizing activity against the pseudoviruses of the two viruses. The antibody SARS2-1 has the characteristics of high expression, fully human origin, and good stability, making it suitable for industrial production. It has potential application value in responding to current and future outbreaks caused by coronaviruses.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody SARS2-1 against SARS-CoV and SARS-CoV-2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the monoclonal antibody SARS2-1 is shown as SEQ ID NO:

2.

2. The monoclonal antibody SARS2-1 according to claim 1, characterized in that, The amino acid sequence of the heavy chain constant region of the monoclonal antibody SARS2-1 is shown as SEQ ID NO: 3; the amino acid sequence of the light chain constant region of the monoclonal antibody SARS2-1 is shown as SEQ ID NO:

4.

3. A recombinant vector expressing the monoclonal antibody SARS2-1 according to claim 1 or 2, characterized in that, The initial vector, a nucleic acid sequence encoding the heavy chain variable region of the monoclonal antibody SARS2-1, and a nucleic acid sequence encoding the light chain variable region of the monoclonal antibody SARS2-1.

4. The recombinant vector of claim 3, wherein, The initial vector is a eukaryotic expression vector comprising a nucleic acid sequence encoding the light chain constant region and a nucleic acid sequence encoding the heavy chain constant region of the monoclonal antibody SARS2-1.

5. A recombinant cell expressing the monoclonal antibody SARS2-1 according to claim 1 or 2, characterized in that, The recombinant cell is obtained by introducing the recombinant vector of claim 3 or 4 into a host cell; the host cell is an Expi 293F cell or a CHO-S cell.

6. Use of the monoclonal antibody SARS2-1 of claim 1 or 2, the recombinant vector of claim 3 or 4, or the recombinant cell of claim 5 in the preparation of a drug for preventing and / or treating SARS-CoV, SARS-CoV-2 coronavirus infection.

7. Use of the monoclonal antibody SARS2-1 of claim 1 or 2, the recombinant vector of claim 3 or 4, or the recombinant cell of claim 5 in the preparation of a reagent for detecting SARS-CoV, SARS-CoV-2 coronavirus infection.

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