Monoclonal antibody targeting coronavirus and application and preparation method thereof

Abstract

The invention provides a monoclonal antibody for targeting coronavirus, which is characterized in that the amino acid sequence of a heavy chain CD R of the monoclonal antibody is H-CDR1: GFTFSSYA, H-CDR2: ISSYDGSNK, H-CDR3: ARAPDCSGSCSTEYFQ H, and the amino acid sequence of the heavy chain CD R of the monoclonal antibody is H-CDR1: GFTFSSYA, H-CDR2: ISSYDGSNK, and H-CDR3: ARAPDCSGSCSTEYFQ H; the amino acid sequence of the monoclonal light chain CDR is L-CDR1: ESVDNYGLSF, L-CDR2: AAS, and L-CDR3: QQSKEVPWT (Quantitative Quantitative SKEVPWT). The monoclonal antibody provided by the invention has obvious binding and neutralizing effects on SARS-CoV, MERS-CoV and SARS-CoV-2, and can be used for treating or detecting the coronavirus infection.

Description

Field of the Invention

[0001] The present application belongs to the field of antibodies. Specifically, the present application provides monoclonal antibodies targeting coronaviruses and applications thereof. Background Art

[0002] Coronavirus (CoV) belongs to the order Nidovirales, family Coronaviridae, genus Coronavirus. It is an enveloped single positive-strand RNA virus with a genome length of 27-32kb, making it the largest known RNA virus. Currently, there are seven known coronaviruses that can cause human infection: HCoV-229E, HCoV-OC43, SARS-CoV, HKU-1, HCoV-NL63, MERS-CoV, and SARS-CoV-2.

[0003] The genetic diversity of coronaviruses has also made people more aware of the potential risk of cross-species transmission of coronaviruses. SARS-CoV, MERS-CoV and SARS-CoV-2 belong to the same genus as coronavirus β. The three have a close phylogenetic relationship and the same virus particle structure. There are four main structural proteins: spike (S) protein, envelope (E) protein, membrane (M) protein and nucleocapsid (N) protein. Among them, S protein, as an important envelope protein of coronavirus, exists on the surface of the virus membrane in the form of a trimer. It plays an important role in the process of virus infection of host cells and is an important site for the research and design of virus vaccines and related antibody therapeutic drugs. S can be divided into two subunits, S1 and S2. The receptor binding domain (RBD) on the S1 subunit mainly mediates the recognition of virus and host cell surface receptors. The receptors of different coronaviruses are different. The receptor of MERS-CoV is DPP4, while that of SARS-CoV and SARS-CoV-2 is ACE2. The S2 subunit mediates the fusion of the virus and the cell membrane through the six-stranded helical bundle formed after conformational changes, thereby enabling the virus to invade the host cell. It is generally believed that the RBD of the coronavirus is the main neutralizing epitope, while the neutralizing epitope of the S2 subunit has been less studied.

[0004] Although vaccines have been approved for use against the SARS-CoV-2 virus, due to the rapid mutation of the coronavirus, there are large differences in the protective effects of different vaccines in different regions and populations. People's health conditions and vaccine side effects also become limitations to vaccination. The vaccine plays a limited role in preventing and controlling coronavirus infections.

[0005] Monoclonal antibodies are antibodies secreted by a single B lymphocyte and only target one antigenic determinant cluster. Because of their specific and potential targeted therapeutic drug characteristics, they have been widely used in the treatment of clinical tumors and immune system diseases. Monoclonal antibodies for viral infections have been studied, and ZMapp has shown an effective therapeutic effect on Ebola virus infection in clinical practice. The serum of coronavirus infected patients in the recovery period contains a large amount of neutralizing antibodies, which have also been found to have effective clinical therapeutic effects, indicating that neutralizing antibodies can be used as an effective and safe treatment for viral infections.

[0006] At present, the research on coronavirus neutralizing antibodies targets the receptor binding region RBD. These neutralizing antibodies can be derived from memory or plasma B cells, phage, yeast and ribosome libraries of recovered patients, or from immunized mice, rabbits, monkeys and alpacas. However, the understanding of non-RBD regions, especially S2, is still very limited. There are relatively few studies on antibodies that can neutralize multiple coronaviruses. Broad-spectrum neutralizing antibodies are not only helpful in treating current patients with coronavirus infection, but can also be used to treat severe infections of related coronaviruses such as SARS and MERS, which is of great significance for the prevention and control of coronavirus infection. Summary of the invention

[0007] The present invention obtains neutralizing mouse monoclonal antibodies targeting SARS-CoV, MERS-CoV and SARS-CoV-2 through screening, and confirms that they exhibit significant neutralizing effects on SARS-CoV, MERS-CoV and SARS-CoV-2 pseudoviruses. Because of their broad spectrum, they can provide new means for the prevention and treatment of coronaviruses in the future.

[0008] On the one hand, the present application provides a monoclonal antibody targeting coronavirus, wherein the amino acid sequence of the heavy chain CDR of the monoclonal antibody is H-CDR1: GFTFSSYA, H-CDR2: ISYDGSNK, H-CDR3: ARAPDCSGSCSTEYFQH; the amino acid sequence of the light chain CDR of the monoclonal antibody is L-CDR1: ESVDNYGLSF, L-CDR2: AAS, L-CDR3: QQSKEVPWT.

[0009] Furthermore, the heavy chain amino acid sequence of the monoclonal antibody is SEQ ID NO.2, and / or the light chain amino acid sequence of the monoclonal antibody is SEQ ID NO.7.

[0010] The light and heavy chain parts other than CDR and the framework part of the antibody can be designed or selected from existing parts by those skilled in the art according to requirements, such as humanization or expression production requirements.

[0011] On the other hand, the present application provides the use of the above-mentioned monoclonal antibodies in the preparation of drugs for treating diseases caused by coronavirus infection.

[0012] Furthermore, the drug is in oral or injectable form.

[0014] Examples of injections include, but are not limited to, injection solutions and powder injections; examples of oral preparations include, but are not limited to, tablets, capsules, and oral solutions. Those skilled in the art can select pharmaceutically acceptable excipients based on common knowledge in the field of pharmacy, including, but not limited to, solvents, cosolvents, stabilizers, dispersants, viscosity regulators, osmotic pressure regulators, antioxidants, sweeteners, adhesives, fillers, lubricants, coating agents, capsule shells, etc.

[0015] On the other hand, the present application provides the use of the above-mentioned monoclonal clone in the preparation of a reagent for detecting coronavirus infection.

[0016] Furthermore, the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2.

[0017] The diseases caused by the coronavirus infection include but are not limited to respiratory tract related symptoms, such as inflammation, cough, dyspnea, etc., as well as cytokine storm, nervous system damage, organ failure, etc. complicated by infection.

[0018] On the other hand, the present application provides a nucleic acid encoding the above monoclonal antibody, wherein the nucleotide sequence of the nucleic acid comprises SEQ ID NO.1 and SEQ ID NO.6.

[0019] In another aspect, the present application provides a vector comprising the above-mentioned nucleic acid.

[0020] In another aspect, the present application provides a host cell, wherein the host comprises the above-mentioned vector.

[0021] The host cells in the present application can be selected from various prokaryotic or eukaryotic host cells that can be used for antibody expression in the art, such as Escherichia coli, Bacillus subtilis, yeast, etc. For these different hosts, those skilled in the art can design different nucleotide sequences based on known codon preferences and attach suitable connections, labels and other components.

[0022] On the other hand, the present application provides a method for preparing the above-mentioned monoclonal antibody, which comprises the step of culturing the above-mentioned host cell.

[0023] Other molecular biological operation steps and protein purification steps in the method for preparing antibodies through recombinant expression can be routinely selected by those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a positive result graph for monoclonal antibody screening.

[0025] Figure 2 This is a diagram showing the results of ELISA identification of some monoclonal antibody binding areas.

[0026] Figure 3 This is a diagram showing the results of ELISA identification of some monoclonal antibody binding areas.

[0027] Figure 4 This is a graph showing the results of identifying the luciferase activity of three pseudoviruses.

[0028] Figure 5 This is a diagram showing the inhibitory effect of monoclonal antibodies on SARS-CoV pseudovirus-infected Huh7.5 cells.

[0029] Figure 6 This is a diagram showing the inhibitory effect of monoclonal antibodies on MERS-CoV pseudovirus-infected Huh7.5 cells.

[0030] Figure 7 This is a diagram showing the inhibitory effect of monoclonal antibodies on SARS-CoV-2 pseudovirus-infected Huh7.5 cells. DETAILED DESCRIPTION

[0031] Material

[0032] Recombinant proteins: SARS-CoV and MERS-CoV full-length S, RBD, S1, and S2 proteins were purchased from Beijing Sino-Biological Technology Co., Ltd.; SARS-CoV-2 full-length S, RBD, S1, and S2 proteins were purchased from Jinan Protein Technology Co., Ltd.

[0033] Cell lines: SP2 / 0 cells, 293FT cells, Huh7.5 cells, and Vero E6 cells are all preserved by our company.

[0034] Plasmid vector: pNL4-3.luc.RE- and envelope plasmids pVRC-MERS-S, pVRC-SARS-S, and pVRC-SARS-2-S carrying the MERS-CoV, SARS-CoV, and SARS-CoV-2S genes were all constructed by our company.

[0035] Example 1 Screening and preparation of monoclonal antibodies

[0036] Mouse immunization:

[0037] 50ug of SARS-CoV recombinant S full-length protein was mixed with an equal volume of aluminum adjuvant (50ug) and then intraperitoneally immunized 6-week-old female Balb / c mice. MERS-CoV and SARS-CoV-2 recombinant S proteins were immunized once at 2 weeks and 4 weeks respectively according to the above method. At the 6th week, the mouse sera were taken to test the titers of the three S protein antibodies respectively. The mouse with the highest titer was selected and boosted with 20ug of SARS-CoV-2 recombinant S protein through the tail vein. The mouse spleen was taken 3 days later for the preparation of hybridoma cells. .

[0038] Screening of hybridoma cell lines

[0039] All spleen cells of immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and placed in HAT medium for screening and culture. When the fused cells grew to 1 / 2 of the bottom of the well, clones positive for SARS-CoV, MERS-CoV and SARS-CoV-2S were screened by indirect ELISA, and the cell lines were expanded and frozen to a monoclonal state by limiting dilution cloning.

[0040] Screening of positive clones by indirect ELISA: SARS-CoV, MERS-CoV and SARS-CoV-2S full-length recombinant proteins were coated in microplates (coating buffer carbonate buffer: 1.59g sodium carbonate, 2.93g sodium bicarbonate, fixed to 1L pure water), with a coating concentration of 1μg / mL, overnight at 4°C; 1% gelatin blocking, 150 μL per well, blocking at 37°C for 2 hours, washing the plate once with washing solution, patting dry; adding 50μL of cell culture supernatant, reacting at 37°C for 30min. Shake out the liquid in the well, wash the plate 4 times with PBST washing solution, pat dry, add 50μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:10000 with PBS), react at 37°C for 30min, wash the plate 4 times, pat dry, add 50μL / well of TMB colorimetric solution for color development at room temperature for 10min, and finally add 0.5M sulfuric acid to terminate the reaction, and measure OD with an enzyme reader. 450nm value.

[0041] Preparation of monoclonal antibody ascites

[0042] After the selected monoclonal cell lines were expanded and cultured, 0.2 ml (containing 2.5×10 6 Cells) were pretreated with Freund's incomplete adjuvant in female BALB / c mice. After about 10 days, when the abdomen of the mice was obviously swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000r / min for 10 minutes to collect the middle layer.

[0043] Purification of monoclonal antibodies (Protein G)

[0044] The ascites was centrifuged at 12000r / min for 5 minutes, the supernatant was diluted 10 times with binding buffer (20mM PBS, 150mM NaCl, pH7.4), filtered through a 0.22um filter, and the filtered sample was pumped into the Protein G purification column equilibrated with binding buffer at a low speed through a peristaltic pump, connected to an AKTA purifier, washed with binding buffer for 5-10 column volumes until the UV absorption peak was washed flat, and then eluted with elution buffer (0.1M glycine, pH 2.7), and the elution peak was collected. The collected sample was adjusted to neutral with 1MTris-HCl, pH9, and placed in a dialysis bag (MW: 8000-14000), and dialyzed in a 0.01M PBS pH7.4 solution at 2-8°C for 14 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000r / min for 5 minutes. The supernatant was the purified monoclonal antibody. The purified monoclonal antibody was subjected to SDS-PAGE electrophoresis, and its concentration was determined by a micro-ultraviolet spectrophotometer, and then stored in aliquots.

[0045] A total of 7 monoclonal antibodies that were positive for SARS-CoV, MERS-CoV, and SARS-CoV-2S were screened, and they all showed high binding affinity to the three recombinant proteins (e.g. Figure 1 shown).

[0046] Example 2 ELISA identification of monoclonal antibody binding domain

[0047] SARS-CoV, MERS-CoV and SARS-CoV-2 RBD, S1, S2 recombinant proteins were coated in microplates (coating buffer carbonate buffer: 1.59g sodium carbonate, 2.93g sodium bicarbonate, fixed to 1L pure water), the coating concentration was 1μg / mL, overnight at 4°C, and the blocking conditions were as above. The monoclonal antibody was diluted to 1μg / mL (using FluA monoclonal antibody as a control), 50ul was added to each well, and reacted at 37°C for 30min. The liquid in the well was shaken out, the plate was washed 4 times with PBST washing solution, and 50μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:10000 with PBS) was added after patting dry, reacted at 37°C for 30min, and the plate was washed 4 times again. After patting dry, 50μL / well of TMB color development solution was added for color development at room temperature for 10min, and finally 0.5M sulfuric acid was added to terminate the reaction, and OD was measured with an enzyme reader. 450nm value.

[0048] SARS-CoV, MERS-CoV and SARS-CoV-2 RBD, S1, S2 recombinant proteins were coated respectively to identify and screen the binding regions of the monoclonal antibodies. It was found that 2D9 and 11G2 both reacted with S1 but not with RBD, which may be bound to the region other than RBD on S1. The remaining 5 monoclonal antibodies all bound to the S2 region (such as Figure 2 shown).

[0049] Example 3 Pseudovirus Neutralization Experiment

[0050] Fake virus packaging

[0051] 1) Preparation of 293FT cells: One day before transfection, subculture 293FT cells in a T25 cell culture flask, change the medium the next morning, and transfect when 70% of the cells have grown into sheets.

[0052] 2) Preparation of plasmid mixture: Add 300μl OPTI-MEM to an enzyme-free 1.5ml EP tube, add 1.2μg membrane protein plasmids pVRC-MERS-S, pVRC-SARS-S, pVRC-SARS-2-S and 4.8μg backbone plasmid pNL4-3.luc.RE mixture respectively, gently shake to mix, then add 12μl HP transfection reagent, gently flick the tube wall and let stand at room temperature for 15 minutes, add the mixture of plasmid and transfection reagent to 293FT cells and culture in a CO2 incubator.

[0053] 3) Replacement of pseudovirus medium: 10 hours after transfection, discard the culture medium and add 8 ml of 5% FBS-DMEM medium to continue culturing. 72 hours after transfection, harvest the supernatant containing pseudovirus, centrifuge at 2500 r / min for 5 minutes to remove cell debris, obtain the supernatant, and store at -80°C.

[0054] The average RLU detection value of SARS-CoV, MERS-CoV and SARS-CoV-2 pseudovirus particles packaged in different batches was above 300,000, indicating that the pseudovirus was packaged successfully and could meet the detection requirements (see Figure 3 ).

[0055] Pseudovirus TCID50 detection

[0056] 1) Preparation of Huh7.5 cells: One day before pseudovirus harvesting, Huh7.5 cells were seeded in a 96-well cell culture plate and 10% FBS-DMEM medium was added. When the cell density reached 50%-70%, the 96-well plate was washed with 100 μl serum-free medium and repeated.

[0057] 2) Sample addition: Dilute the pseudovirus supernatant 5-fold with serum-free medium, add the diluted virus solution to a 96-well Huh7.5 cell culture plate, set up 6 replicate wells for each dilution, and set up 8 normal cell control wells, and culture in a CO2 incubator.

[0058] 3) Medium replacement: After 24 hours, discard the liquid in the 96-well plate, add 2% FBS-DMEM maintenance medium, 100 μl / well, and continue culturing for 48 hours.

[0059] 4) Detection: Use the BioLux Gaussia luciferase detection kit to read the fluorescence reading RLU.

[0060] Pseudovirus neutralization assay

[0061] 1) Preparation of Huh7.5 cells: One day before pseudovirus harvesting, Huh7.5 cells were passaged in 96-well cell culture plates and 10% FBS-DMEM medium was added, with 100 μl / well of the cell and medium mixture. When the cell density reached 50%-70%, the 96-well plates were washed with 100 μl serum-free medium, and the washing was repeated.

[0062] 2) Sample addition: dilute the monoclonal antibody to 1 μg / ml, take 100 μl and mix with equal volumes of 200TCID50 SARS-CoV, MERS-CoV and SARS-CoV-2 pseudoviruses, incubate at 37°C, inoculate the monoclonal antibody-pseudovirus mixture into Huh7.5 96-well cell culture plates after 1 hour, set up negative and positive serum controls, and culture at 37°C and 5% CO2.

[0063] 3) Medium replacement: After 24 hours, discard the liquid in the 96-well plate, add 100 μl / well of 2% FBS-DMEM medium, and continue culturing in a 37° C., 5% CO 2 incubator for 48 hours.

[0064] 4) Detection: Use BioLux Gaussia luciferase detection kit to read the fluorescence reading RLU. Neutralization percentage (%) = (pseudovirus control RLU-detection monoclonal antibody RLU) / (pseudovirus control RLU-empty cell control RLU)×100%.

[0065] The monoclonal antibodies were diluted 10 times from 10 μg / ml and incubated with 200 TCID50 pseudoviruses to infect the coronavirus-sensitive cell line Huh7.5 cells. A monoclonal antibody against influenza A virus was used as an experimental irrelevant antibody control. The pseudovirus neutralization efficiency of the monoclonal antibody was calculated by comparing the luciferase activity of the monoclonal antibody group and the control cell group. The results showed that 6E2 had significant neutralizing activity against SARS-CoV, MERS-CoV and SARS-CoV-2 pseudoviruses, and its monoclonal antibody concentration was dose-dependent with the virus neutralization potency. The IC50 value for SARS-CoV and SARS-CoV-2 pseudoviruses reached about 0.13 μg / ml, and the IC50 for MERS-CoV pseudovirus was also within 1 μg / ml (see Figure 5-7 ).

[0066] The monoclonal antibody 6E2 involved in the present invention can specifically bind to the S2 subunit of SARS-CoV, MERS-CoV and SARS-CoV-2, and has good affinity. At the same time, it can significantly inhibit the infection of SARS-CoV, MERS-CoV and SARS-CoV-2 pseudoviruses to sensitive cell lines. It is a broad-spectrum neutralizing antibody targeting the S2 subunit and has application value as a drug for the prevention and treatment of coronavirus.

[0067] Monoclonal antibody 6E2 sequencing results:

[0068] 6E2 Heavy Chain:

[0069] Nucleotide sequence

[0070] Caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgca

[0071] gcctctggattcaccttcagtagctatgctatgcactgggtccgccaggctccaggcaaggggctggag

[0072] tgggtggcagttatatcatatgatggaagcaataaatactacgcagactccgtgaagggccgattcacc

[0073] atctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagatgaggacacggct

[0074] gtgtattactgtgcgagagctcctgattgtagtggtggtagctgctccactgaatacttccagcactggggccagggcaccctggtcaccgtctcctca (SEQ ID NO. 1);

[0075] Amino acid sequence

[0076] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRDEDTAVYYCARAPDCSGGSCSTEYFQHWGQGTLVTVSS(SEQ ID NO.2);

[0077] CDR amino acid sequences

[0078] CDR1: GFTFSSYA (SEQ ID NO.3);

[0079] CDR2: ISYDGSNK (SEQ ID NO.4);

[0080] CDR3: ARAPDCSGGSCSTEYFQH (SEQ ID NO.5);

[0081] 6E2 light chain:

[0082] Nucleotide sequence:

[0083] Gatgttttgatgacccaaactccagcttctttggctgtgtctcaagggcagagggccaccatctcctgc

[0084] agagccagcgaaagtgttgataattatggccttagttttatgaactggttccaacagaaaccaggacag

[0085] ccacccaaactcctcatctatgctgcatccaaccaaggatccggggtccctgccaggtttagtggcagt

[0086] gggtctgggacagacttcagcctcaacatccatcctatggaggaggatgatactgcaatgtatttctgtcagcaaagtaaggaggttccgtggacgttcggtggaggcaccaagtggaaatcaaac(SEQ ID NO.6)

[0087] Amino acid sequence:

[0088] DVLMTQTPASLAVSQGQRATISCRASESVDNYGLSFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKWKSN(SEQ ID NO.7)

[0089] CDR amino acid sequence:

[0090] CDR1: ESVDNYGLSF (SEQ ID NO.8)

[0091] CDR2: AAS (SEQ ID NO. 9, due to the restriction of at least 4 amino acids in WIPO Sequence, sequence 9 in the sequence list is counted as AASN)

[0092] CDR3: QQSKEVPWT (SEQ ID NO. 10).

Claims

1. A monoclonal antibody targeting coronavirus, characterized in that: The amino acid sequence of the heavy chain CDR of the monoclonal antibody is H-CDR1: GFTFSSYA, H-CDR2: ISYDGSNK, H-CDR3: ARAPDCSGGSCSTEYFQH; the amino acid sequence of the light chain CDR of the monoclonal antibody is L-CDR1: ESVDNYGLSF, L-CDR2: AAS, L-CDR3: QQSKEVPWT.

2. The monoclonal antibody according to claim 1, wherein the amino acid sequence of the heavy chain of the monoclonal antibody is SEQ ID NO. 2, and / or the amino acid sequence of the light chain of the monoclonal antibody is SEQ ID NO.

7.

3. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a medicament for treating diseases caused by coronavirus infection.

4. The use according to claim 3, wherein the drug is in oral or injectable form.

5. Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a reagent for detecting coronavirus infection.

6. The use according to any one of claims 3 to 5, wherein the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2.

7. A nucleic acid encoding the monoclonal antibody according to claim 1 or 2, characterized in that: The nucleotide sequence of the nucleic acid comprises SEQ ID NO.1 and SEQ ID NO.

6.

8. A carrier, characterized in that The vector comprises the nucleic acid according to claim 7.

9. A host cell, characterized in that The host comprises the vector according to claim 8.

10. A method for preparing the monoclonal antibody according to claim 1 or 2, characterized in that: The method comprises the step of culturing the host cell according to claim 9.

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