Anti-omicron full human monoclonal antibody 9C4 targeting RBD and application thereof

Abstract

The application provides a target RBD anti-Omicron fully human monoclonal antibody 9C4 and application thereof, and belongs to the technical field of microbiology and immunology. The monoclonal antibody 9C4 comprises a heavy chain variable region and a light chain variable region, the amino acid sequences of three complementarity determining regions of the heavy chain variable region are GYAFTTYG, ISTYNGNT and ATSLGLWFGQNS respectively, and the amino acid sequences of three complementarity determining regions of the light chain variable region are SGSIASNY, EDN and QTYDGSNQV respectively. The monoclonal antibody 9C4 provided by the application has high-efficiency, specific anti-Omicron neutralization activity, is fully human, has good stability, and has great application potential in the preparation of a novel coronavirus detection product or a drug for preventing and treating a novel coronavirus disease.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and immunology, specifically to an anti-Omicron fully human monoclonal antibody 9C4 targeting RBD and its applications. Background Technology

[0002] Coronaviruses belong to the family Coronaviridae in the order Nidovirales. They are enveloped, positive-sense RNA viruses widely distributed in nature, capable of infecting a variety of vertebrates, including humans, rodents, cats, dogs, bats, cattle, chickens, and whales. Furthermore, coronaviruses can spread across species. Currently, seven coronaviruses are known to infect humans: four with low pathogenicity (HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1) and three with high pathogenicity (SARS-CoV, MERS-CoV, and SARS-CoV-2). SARS-CoV-2, in particular, possesses extremely high transmissibility and pathogenicity, rapidly causing a global pandemic.

[0003] SARS-CoV-2 is spherical or ellipsoidal in shape, with a diameter of approximately 80–160 nm. The viral particle has an outer envelope composed of phospholipids, on which structural proteins such as the Spike trimer, M protein, and E protein are present. The interior of the viral particle contains a genetic core composed of viral genomic RNA and the N protein. After infecting humans, SARS-CoV-2 causes respiratory symptoms such as fever, cough, and headache. Most patients recover spontaneously, but some are prone to developing varying degrees of respiratory failure and acute pneumonia, especially those with weakened immune systems and underlying diseases, who have a higher mortality rate. The Spike protein (S protein) is a key protein mediating the invasion of host cells by coronaviruses. The receptor-binding domain (RBD) of the SARS-CoV-2 S protein is an important region for its binding to the human ACE2 receptor protein and is also an ideal antigen target for neutralizing antibodies.

[0004] Although various vaccines for SARS-CoV-2 have been put into use, SARS-CoV-2 continues to mutate during transmission, and a large number of breakthrough infections (vaccinated and recovered people are infected with SARS-CoV-2 again) have occurred. Among them, the S protein of the Omicron strain carries more than 30 amino acid mutations, resulting in the Omicron strain acquiring extremely strong immune escape ability, which poses a serious challenge to the existing COVID-19 vaccines and antibody drugs. Omicron subviruses are more common, such as BA.2, BA.5, XBB.1.5, KP.1, JN.1, etc. Therefore, it is of great public health and safety significance to develop broad-spectrum and efficient COVID-19 Omicron vaccines and specific therapeutic drugs.

[0005] In summary, the present application provides a fully human monoclonal therapeutic antibody with good neutralizing effect against multiple Omicron subviruses. SUMMARY

[0006] In view of the above deficiencies of the prior art, the present application provides a high neutralization activity anti-COVID-19 Omicron strain fully human monoclonal antibody 9C4 targeting RBD and its application. The monoclonal therapeutic antibody has good protective effect against the Omicron strain.

[0007] To achieve the above-mentioned purpose, the specific technical solutions of the present application are as follows:

[0008] In a first aspect, the present application provides a high neutralization activity anti-Omicron fully human monoclonal antibody 9C4 targeting RBD, which comprises a heavy chain variable region (V H ) and a light chain variable region (V L ), the heavy chain variable region has three complementarity determining regions CDR1, CDR2 and CDR3 with amino acid sequences of GYAFTTYG (see SEQ ID NO: 9), ISTYNGNT (see SEQ ID NO: 10) and ATSLGLWFGQNS (see SEQ ID NO: 11) respectively; the light chain variable region has three complementarity determining regions CDR1, CDR2 and CDR3 with amino acid sequences of SGSIASNY (see SEQ ID NO: 12), EDN and QTYDGSNQV (see SEQ ID NO: 13) respectively.

[0009] Further, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 9C4 is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the monoclonal antibody 9C4 is shown in SEQ ID NO: 3.

[0010] Further, the amino acid sequence of the heavy chain constant region of the monoclonal antibody 9C4 is shown as SEQ ID NO: 5, and the amino acid sequence of the light chain constant region of the monoclonal antibody 9C4 is shown as SEQ ID NO: 7.

[0011] Further, the monoclonal antibody further comprises:

[0012] the amino acid sequence of the monoclonal antibody is substituted, deleted and / or added with one or more amino acids to obtain an antibody with the same function;

[0013] or, comprises a heavy chain variable region having an amino acid sequence with at least 80% homology with the heavy chain variable region; and a light chain variable region having an amino acid sequence with at least 80% homology with the light chain variable region;

[0014] or, comprises an antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

[0015] In some embodiments, V H and / or V L may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the above sequences; antibodies having V H and V L regions with a high degree of (i.e., 80% or more) homology to the V H and V L regions of the above sequences can be obtained by mutagenizing (e.g., 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 the functional assays described herein.

[0016] In some embodiments, the variable region genes can be converted to scFv genes, and once DNA fragments encoding V H and V L fragments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, e.g., to convert the variable region genes to full-length antibody chain genes, Fab fragment genes, or scFv genes.

[0017] In these manipulations, the DNA fragment encoding the VL or VH is operatively linked to another DNA segment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked" as used herein means that the two DNA segments are connected in such a way that the amino acid sequences encoded by the two DNA segments remain in-frame.

[0018] In a second aspect, the present application provides a nucleic acid molecule encoding the monoclonal antibody, which comprises a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region.

[0019] Further, the polynucleotide sequences encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody 9C4 are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0020] Further, the polynucleotide sequences encoding the heavy chain constant region and the light chain constant region of the monoclonal antibody 9C4 are shown in SEQ ID NO: 6 and SEQ ID NO: 8, respectively.

[0021] In a third aspect, the present application provides an expression vector comprising the nucleic acid, which is capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell.

[0022] Further, the vector is selected from one of a plasmid vector, a bacteriophage vector, a viral vector, and a mammalian expression vector.

[0023] Specifically, the present application employs a mammalian expression vector.

[0024] In a fourth aspect, the present application provides an engineered bacterium or eukaryotic host cell comprising the expression vector.

[0025] In a fifth aspect, the present application provides use of the monoclonal antibody in the preparation of a COVID-19 therapeutic drug or a SARS-CoV-2 detection product.

[0026] Compared with the prior art, the present application has the following advantages:

[0027] The targeting RBD high neutralization activity anti-Omicron fully human monoclonal antibody 9C4 provided by the present application exhibits good neutralization protection effect on SARS-COV-2 infected cells, and the results of the present application show that the monoclonal antibody 9C4 has a wide application prospect in the preparation of COVID-19 therapeutic drugs. Specifically, the targeting RBD high neutralization activity anti-Omicron fully human monoclonal antibody 9C4 provided by the present application has the following advantages:

[0028] (1) Fully human, no humanization modification is needed in clinical application.

[0029] (2) High neutralization activity, in a safe type replicon cell model of SARS-COV-2 Omicron infection, the half effective concentration (EC50) of the monoclonal antibody 9C4 on Omicron BA.5 is 15.7 ng / mL; the half effective concentration (EC50) of the monoclonal antibody 9C4 on Omicron JN.1 is 21.5 ng / mL.

[0030] (3) clear mechanism of action: monoclonal antibody 9C4 binds to RBD with high specificity, showing that the monoclonal antibody targets the receptor binding region, and plays an antiviral effect by specifically blocking the binding of the virus to the receptor.

[0031] (4) good stability: because the monoclonal antibody gene comes from the same cell in the human body, it is naturally paired, and the half-life of IgG1 antibody in the human body is known to be 21-28 days. Theoretically, the disclosed monoclonal antibody has a consistent half-life in the human body. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is a flow cytometry single cell sorting chart;

[0033] Figure 2 is a detection map of the amplification of the variable region gene of the monoclonal antibody by automatic nucleic acid electrophoresis;

[0034] Figure 3 is a denaturing SDS-PAGE detection map after affinity chromatography purification;

[0035] Figure 4 is a curve graph showing the binding activity of 9C4 and RBD protein changes with concentration;

[0036] Figure 5 is an EC50 determination curve of the antibody on the safe replicon cell model; wherein, Figure 5 A is the half effective concentration of monoclonal antibody 9C4 on Omicron BA.5; Figure 5 B is the half effective concentration of monoclonal antibody 9C4 on Omicron JN.1. DETAILED DESCRIPTION

[0037] The technical solutions of the present application will be described below. Obviously, the described embodiments are only a part of the embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0038] The present application provides a high neutralizing activity anti-Omicron monoclonal antibody 9C4 targeting RBD, which comprises a heavy chain variable region (V H ) and a light chain variable region (V L), the heavy chain variable region has the amino acid sequences of the three complementarity determining regions as follows: GYAFTTYG (see SEQ ID NO: 9), ISTYNGNT (see SEQ ID NO: 10), and ATSLGLWFGQNS (see SEQ ID NO: 11), respectively; and the light chain variable region has the amino acid sequences of the three complementarity determining regions as follows: SGSIASNY (see SEQ ID NO: 12), EDN, and QTYDGSNQV (see SEQ ID NO: 13), respectively.

[0039] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 9C4 is shown in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region of the monoclonal antibody 9C4 is shown in SEQ ID NO: 3.

[0040] The amino acid sequence of the heavy chain constant region of the monoclonal antibody 9C4 is shown in SEQ ID NO: 5; and the amino acid sequence of the light chain constant region of the monoclonal antibody 9C4 is shown in SEQ ID NO: 7.

[0041] The monoclonal antibody also includes an antibody having the same function obtained by substituting, deleting, and / or adding one or more amino acids in the amino acid sequence of the monoclonal antibody; or, includes a heavy chain variable region having an amino acid sequence with at least 80% homology with the heavy chain variable region; and a light chain variable region having an amino acid sequence with at least 80% homology with the light chain variable region; or, includes an antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

[0042] In some embodiments, V H and / or V L may have an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the above sequences; an antibody having V H and V L regions that are highly (i.e., 80% or more) homologous to the V H and V L regions of the above sequences can be obtained by mutagenizing (e.g., 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 the functional assays described herein.

[0043] In some embodiments, the variable region genes can be converted to scFv genes, and once DNA fragments encoding V H and V L fragments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, e.g., to convert the variable region genes to full-length antibody chain genes, Fab fragment genes, or scFv genes.

[0044] In these manipulations, the DNA segment encoding V L or V H is operably linked to another DNA segment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used herein means that the two DNA segments are joined in such a way that the amino acid sequences encoded by the two DNA segments remain in-frame.

[0045] Unless otherwise specifically indicated, all of the various raw materials, reagents, instruments and equipment, etc. used in the present application can be purchased on the market or can be prepared by existing methods.

[0046] Example 1

[0047] Screening and preparation of human anti-Omicron monoclonal antibodies

[0048] 1. Prepare 96-well plates: 20 μL RNase-free water + 20 U RNase inhibitor per well, cover with sealing film, and place at 4 ℃ for standby;

[0049] 2. Prepare samples:

[0050] (1) Resuscitate cells: Take the PBMC cells isolated from the peripheral blood of a novel coronavirus infection recovered person from -80 ℃ and quickly place them in warm water at 37 ℃. After the cells are thawed, centrifuge at 800 rpm for 5 min, and discard the supernatant. Resuspend in 2-3 mL PBS in a flow tube, and after equalizing, centrifuge at 800 rpm for 5 min, and discard the supernatant. Resuspend in 2-3 mL PBS, centrifuge, and discard the supernatant. Finally, resuspend in 100 μL PBS, and take 2 μL for dilution 10 times for cell counting.

[0051] (2) Single staining tube: 7 tubes (FVS-780, CD3-BV510, CD4-BV510, CD8-BV510, CD19-PE, IgD-BB700, CD20-BV421, CD38-FITC), 1×10 6 cells per tube, and add the dyes to the flow tubes containing the cells according to the recommended antibody concentration in the instructions, and supplement each reaction volume with PBS to 50 μL.

[0052] (3) Naked cell control: 1 tube, 1×10 6 cells, and supplement with PBS to 50 μL.

[0053] (4) Cells for sorting: 1 tube, first determine the number of cells, and the final concentration is 100 μL system (1~8×10 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.

[0054] (5) Incubate the sample at 4°C in the dark for 0.5 h.

[0055] (6) Add 1 mL PBS to each tube, centrifuge at 800 g for 5 min at 4°C, discard the supernatant and repeat the washing twice. Add streptavidin-APC dye, adjust the system to 100 μL, and incubate the sample at 4°C in the dark for 0.5 h.

[0056] (7) Add 1 mL PBS to each tube, centrifuge at 800 g for 5 min at 4°C, discard the supernatant and repeat the washing twice.

[0057] (8) Resuspend with 400 μL PBS, remove cell clumps with a 40 μm cell strainer, and store at 4°C in the dark for sorting.

[0058] 3. Flow sorting: Select cells with CD19 + , CD3 - , CD4 - , CD8 - , IgD - , CD38 + , CD20 - , S1 + for sorting. The results of flow sorting are shown in Figure 1 , first select lymphocytes, then select single cells that are not adhered, then select live cells, then select B cells with CD19 + , CD3 － , CD4 － , CD8 － , then select mature B cells with IgD － , and finally select mature B cells with S1 + , which are our target cells.

[0059] 4. Amplify the variable region genes of fully human monoclonal antibodies using single-cell PCR technology

[0060] (1) The reverse transcription PCR procedure was described in the instruction manual (QIAGEN, 210212). The procedure was briefly described as follows: 94 cells were sorted by flow cytometry. The following specific primers for each subtype of heavy chain (H), kappa light chain (κ), and Lamda light chain (λ) were added to each reaction system simultaneously (primer sequences are shown in Table 1).

[0061] Primers:

[0062] H: 5′L-VH 1, L-VH 3, L-VH 4 / 6, 5′L-VH 5, Hu IgG-const-anti, 3′CμCH1

[0063] κ: 5′L Vκ1 / 2, 5′L Vκ3, 5′L Vκ4, 3′Cκ543–566

[0064] λ: 5′L Vλ1, 5′L Vλ2, 5′L Vλ3, 5′L Vλ4 / 5, 5′L Vλ6, 5′L Vλ7, 5′L Vλ8, 3′Cλ

[0065] Table 1: Primers for Reverse Transcription PCR

[0066]

[0067] The PCR reaction system contains: 6 μL of 5× buffer, 1.2 μL of dNTPs, 1.2 μL of reverse transcriptase (Takara Biotech Ltd., RR036A), primers as above, single cell template, and water to a final volume of 30 μL.

[0068] 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, 95 °C for 40 s, 55 °C for 30 s, 72 °C for 1 min, for 40 cycles, and finally extension at 72 °C for 10 min.

[0069] (2) Nested PCR

[0070] Use 1 μL of the reverse transcription product as a template to perform PCR to amplify the variable regions of H, κ, and λ; the primers for amplifying the variable regions of the heavy chain, Kappa light chain, and λ light chain are shown in Table 2-4.

[0071] Table 2: Primers for amplifying the variable region of the heavy chain

[0072]

[0073] Table 3: Primers for amplifying the variable region of the Kappa light chain

[0074]

[0075] Table 4: Primers for amplifying the variable region of lambda light chain

[0076]

[0077] Note: In Tables 2-4, the underlined part is for fusion with the upstream fragment, and the underlined bold part is for fusion with the downstream fragment.

[0078] The PCR reaction system contains: DNA polymerase mixture (Kangwei Century Biotechnology Co., Ltd., CW2849) 12.5 μL, primers as above, template is reverse transcription product 1 μL, water to 25 μL.

[0079] The PCR reaction conditions are: 94 ℃ pre-denaturation for 4 min, then 94 ℃ for 30 s, 57 ℃ for 30 s, 72 ℃ for 45 min, 40 cycles, and finally 72 ℃ extension for 10 min.

[0080] (3) Automatic nucleic acid electrophoresis instrument for detection

[0081] Part of the results of automatic nucleic acid electrophoresis detection are shown in Figure 2 , only the 9C4 heavy chain and light chain variable region bands are shown.

[0082] 5. Synthesis of polynucleotide sequence of variable region sequence

[0083] The Suzhou Jinyuizhi Biotechnology Co., Ltd. was commissioned to synthesize the variable region DNA sequence of the antibody according to the above polynucleotide sequence. A signal peptide sequence and a restriction enzyme cutting site (5'- GAATTCGCCACCATGGAGACAGACACCCTGCTCCTGTGGGTGCTGCTG-3', see SEQ ID NO: 65) were added before the variable region of the heavy chain and the variable region of the light chain of the antibody, and a restriction enzyme cutting site (5'- GGTACC-3') was added at the tail.

[0084] 6. Construction of plasmid expression vector

[0085] The Suzhou Jinyuzhi Biotechnology Co., Ltd. was commissioned to add restriction enzyme sites (5'-GAGCTCGGTACC-3', see SEQ ID NO: 66) to the front of the heavy chain and light chain constant region DNA sequences and add restriction enzyme sites (5'-GCTAGC-3') to the end of the sequences, and synthesize the antibody heavy chain constant region DNA sequence and the light chain constant region DNA sequence with the restriction enzyme sites (the heavy chain constant region sequence is shown in SEQ ID NO: 5, the DNA coding sequence is shown in SEQ ID NO: 6, the Kappa type light chain constant region sequence is shown in SEQ ID NO: 7, and the DNA coding sequence is shown in SEQ ID NO: 8). The pCAGGS empty plasmid (available from Shanghai Yuanmu Biotechnology Co., Ltd., catalog number P0165) and the antibody heavy chain constant region DNA sequence were digested with restriction endonuclease Nhe I and Sac I, and the digested plasmid and antibody constant region DNA fragment were ligated using T4 DNA ligase to obtain 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 with restriction endonuclease Nhe I and Sac I, and the digested plasmid and antibody constant region DNA fragment were ligated using T4 DNA ligase to obtain a plasmid vector containing the antibody light chain constant region.

[0086] 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 endonuclease EcoR I and Kpn I, and the digested products were ligated to obtain a eukaryotic expression vector that can be used to express the heavy chain of the 9C4 antibody. 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 endonuclease EcoR I and Kpn I, and the digested products were ligated using T4 DNA ligase to obtain a eukaryotic expression vector that can be used to express the light chain of the 9C4 antibody. The two vectors described above can be used to express the antibody by transfecting eukaryotic cells.

[0087] The amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of the monoclonal antibody 9C4 are shown in SEQ ID NO: 1 at positions 26-33, 51-58, and 97-108, 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 at positions 26-33, 51-53, and 92-100, respectively. The specific sequences are shown in Table 5.

[0088] Table 5: Amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region and the light chain variable region of the monoclonal antibody 9C4

[0089]

[0090] 7. Transient expression and affinity chromatography purification of monoclonal antibodies

[0091] 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 transfection reagent instructions (Shanghai Liji Biotechnology Co., Ltd., EZ Trans Plus, AC04L011). After 5-6 days, the culture medium was harvested, and approximately 50 mL of supernatant was collected after centrifugation. A 5 mL pre-packed Protein A affinity chromatography column was used. Before loading, the column was equilibrated with 20 mM PBS. After the conductivity reached baseline, the column was injected. After loading, the column was washed with 20 mM PBS until the baseline stabilized. The target protein was eluted with 0.1 M pH 3.0 glycine buffer. Once the OD280 was close to baseline, collection was stopped. The column was washed with at least 3 column volumes of 20 mM PBS until the baseline stabilized, and then washed with 20% ethanol. The SDS-PAGE results of the affinity-purified monoclonal antibody are shown below. Figure 3 .Depend on Figure 3 It can be seen that the monoclonal antibody 9C4 was successfully purified.

[0092] Example 2

[0093] Binding activity analysis of fully human anti-Omicron monoclonal antibody 9C4 with RBD

[0094] 1. Coating: Dilute RBD antigen with coating buffer to a concentration of 3 μg / mL, coat ELISA plates with 100 μL per well, and incubate overnight at 4 ℃.

[0095] 2. Blocking: Add 300 μL of PBST washing buffer to each well and wash 3 times for 3 min each time; blot the liquid out of the well, add 2% BSA, 250 μL / well, and block at 37 ℃ for 1 h.

[0096] 3. Sample incubation: Add 300 μL of PBST washing buffer to each well and wash 3 times for 3 min each time; blot the liquid out of the well, add purified monoclonal antibody diluted with PBS, 9 ug / mL for the first well, and serially dilute 3 times to 100 μL / well, and incubate at 37 ℃ for 2 h.

[0097] 4. Secondary antibody incubation: Wash 5 times, as above; add HRP goat anti-human Fc secondary antibody (1:20000 dilution), 100 μL / well, and incubate at 37 ℃ for 1 h.

[0098] 5. Color development: Wash 5 times, operation as above; add 100 μL TMB single-component color developing liquid to each well, and after color development at 37 °C for 15 min, add 50 μL stop solution to each well to stop the reaction, and use an enzyme label instrument to detect the absorbance value at 450 nm. Use Graph Pad nonlinear regression, four-parameter fitting to draw a standard curve, and calculate the EC50 concentration of the monoclonal antibody according to the standard curve and dilution factor.

[0099] 6. Results: See Figure 4 . Figure 4 In the present embodiment, the curve shows the detection results of the monoclonal antibody 9C4 and RBD, and the results show that the specific binding of the monoclonal antibody 9C4 and RBD presents a dose-response relationship, indicating that the monoclonal antibody is specific to RBD.

[0100] Example 3

[0101] Analysis of neutralization activity on SARS-CoV-2 safe replicon cell model

[0102] 1. Rescue of SARS-CoV-2 safe replicon: Different safe replicons were rescued according to the Omicron mutant spike protein (S protein). The SARS-CoV-2 N gene was stably transfected into Caco-2 cells to construct Caco-2-N cells stably expressing N protein. The coronavirus genome RNA containing different S genes (N gene replaced by Luciferase reporter gene) was transfected into Caco-2-N cells by an electric transfection instrument (Gene Pulser Xcell™, BIO-RAD). After 72 hours, the supernatant containing SARS-CoV-2 safe replicon was collected, centrifuged at 3000 rpm for 10 minutes, aliquoted, and stored at -80 °C. The TCID50 of the safe replicon was calculated by infecting Caco-2-N cells with a series of dilutions. The SARS-CoV-2 safe replicon can be continuously passaged and amplified in Caco-2-N cells, has the characteristics of easy operation, amplification, high safety, relatively low biological safety requirement level (can be operated in a biological safety level II laboratory), and can effectively simulate the replication cycle of coronavirus.

[0103] 2. Dilute the antibody: SARS-CoV-2 safe replicon (3×10 5 TCID50 / well) was incubated with diluted serum at room temperature for 30 min, and then mixed with trypsin-treated Caco-2-N cells at a density of 2×10 4 / well.

[0104] 3. Luciferase readout and analysis: After 16 h of incubation, the culture medium was removed from the infected cells, which were lysed with 1x Bright-Glo luciferase assay reagent (Promega), and chemiluminescence detection was performed using a SpectraMax iD3 multi-well luminometer (Molecular devices). The 50% neutralization titer (NT50) was calculated using GraphPad Prism 7 software, and the curve was fitted by nonlinear regression.

[0105] Results are shown in Figure 5 (Figure 2). (The abscissa represents the antibody concentration, and the ordinate represents the neutralization effect % relative to the negative control group). It can be seen that Figure 5 the half maximal effective concentration (EC50) of monoclonal antibody 9C4 on SARS-COV-2 Omicron BA.5 is 15.7 ng / mL; the half maximal effective concentration (EC50) of monoclonal antibody 9C4 on SARS-COV-2 Omicron JN.1 is 21.5 ng / mL.

[0106] The above specific embodiments describe the implementation of the present application in detail, but the present application is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concepts of the present application, various simple modifications and changes can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A target RBD anti-Omicron fully human monoclonal antibody, 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 is shown as SEQ ID NO:

3.

2. The anti-Omicron full human monoclonal antibody targeting RBD according to claim 1, characterized in that, The amino acid sequence of the heavy chain constant region of the monoclonal antibody is shown as SEQ ID NO: 5, and the amino acid sequence of the light chain constant region of the monoclonal antibody is shown as SEQ ID NO:

7.

3. The anti-Omicron full human monoclonal antibody targeting RBD according to claim 1, wherein, The monoclonal antibody further comprises an antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

4. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 3, characterized in that, 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; the polynucleotide sequences encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown as SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

5. The nucleic acid molecule of claim 4, wherein, The polynucleotide sequences encoding the heavy chain constant region and the light chain constant region of the monoclonal antibody are shown as SEQ ID NO: 6 and SEQ ID NO: 8, respectively.

6. An expression vector comprising the nucleic acid molecule of claim 4. The expression vector is capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell.

7. An engineered bacterium or eukaryotic host cell comprising the expression vector of claim 6.

8. Use of the monoclonal antibody of any one of claims 1-3 in the preparation of a COVID-19 therapeutic drug or a novel coronavirus detection product.

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