Anti-omicron full human monoclonal antibody 3g5 targeting rbd and application thereof

Abstract

The application provides a target RBD anti-Omicron fully human monoclonal antibody 3G5 and an application thereof, and belongs to the technical field of microbiology and immunology. The monoclonal antibody 3G5 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has three complementary determining regions, and the amino acid sequences of the three complementary determining regions are GGS I SS SS NF, I YY SG ST and ARL DLGGDIVVPPAAKPYYYMDV respectively; the light chain variable region has three complementary determining regions, and the amino acid sequences of the three complementary determining regions are SSNIGAGYN, GDI and QSYDSSLSGLYV respectively. The monoclonal antibody 3G5 provided by the application has high-efficiency and specific anti-Omicron neutralization activity, is fully human and 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] The present application relates to the technical field of microbiology and immunology, and particularly relates to an anti-Omicron full human monoclonal antibody 3G5 targeting RBD and application thereof. BACKGROUND

[0002] Coronaviruses are a class of enveloped positive-strand RNA viruses that exist widely in nature, which can infect multiple species such as birds, bats and humans, and cause diseases of the gastrointestinal tract, liver, respiratory system and nervous system, etc., and pose a significant threat to humans and domestic animals. There are seven known coronaviruses that can infect humans, four of which are low pathogenic HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, which are globally distributed and exist universally in the human population, and usually only cause respiratory infections, with mild clinical symptoms such as fever, runny nose and cough. In addition, three highly pathogenic human coronaviruses, SARS-CoV, MERS-CoV and SARS-CoV-2, have also broken out in this century, among which the coronavirus disease (COVID-19) has a very strong transmission ability and pathogenicity, and its pathogen SARS-CoV-2 (Chinese name: new coronavirus, for short: new coronavirus) has a very close evolutionary relationship with SARS-CoV, and the clinical symptoms of the two are also highly similar. In the early stage of infection, both are mainly characterized by fever, dry cough, and general malaise, and in the late stage, they are prone to develop into different degrees of respiratory failure and acute pneumonia, and the mortality rate is higher in people with low immunity and underlying diseases. The SARS-CoV-2 virus particle is spherical or oval, with a diameter of about 80-160 nm. The outermost layer of the virus particle is a lipid bilayer membrane, and there are a large number of terminal swollen spines on the membrane. The core of the virus particle is composed of non-segmented single-stranded positive-strand RNA and nucleocapsid protein (N). The viral genome is about 30 kb in size, which encodes 29 kinds of proteins, including 16 non-structural proteins (nsp1-nsp16), 4 structural proteins (S, E, M, N) and 9 auxiliary proteins (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c and ORF10). The viral particle membrane contains three different glycoproteins, namely spike protein (S), membrane protein (M) and small envelope protein (E). The S protein is the main antigenic protein of coronavirus, and its structure is a globular structure extending from the end of the membrane, which functions to mediate the binding of the virus to the host cell receptor and promote the fusion of the viral envelope and the cell membrane. The receptor binding domain (RBD) of the S protein is responsible for interacting with the receptor, and is an ideal target for neutralizing antibodies.

[0003] The coronavirus mutates during transmission, and an important node is the emergence of the Omicron mutant strain. Compared with the original strain of the new coronavirus, 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 new coronavirus vaccines and antibody drugs. At the same time, the Omicron strain is constantly mutating, and the Omicron sub-viruses with stronger escape ability have appeared, 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 new coronavirus Omicron vaccines and specific therapeutic drugs.

[0004] Therefore, it is urgent to develop a fully human monoclonal therapeutic antibody with good neutralizing effect against multiple Omicron sub-viruses. SUMMARY

[0005] In view of the above deficiencies of the prior art, the present application provides a high neutralizing activity anti-Omicron fully human monoclonal antibody 3G5 targeting RBD and its application. The monoclonal therapeutic antibody has good protective effect against the Omicron strain.

[0006] To achieve the above-mentioned purpose, the specific technical solutions of the present application are as follows:

[0007] In a first aspect, the present application provides a high neutralizing activity anti-Omicron fully human monoclonal antibody 3G5 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 GGS I SS SS NF (see SEQ ID NO: 9), I YYSGST (see SEQ ID NO: 10) and ARLDLGGDIVVPPAAKPYYYMDV (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 SSNIGAGYN (see SEQ ID NO: 12), GDI and QSYDSSLSGLYV (see SEQ ID NO: 13) respectively.

[0008] Further, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 3G5 is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the monoclonal antibody 3G5 is shown in SEQ ID NO: 3.

[0009] Further, the amino acid sequence of the heavy chain constant region of the monoclonal antibody 3G5 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain constant region of the monoclonal antibody 3G5 is shown in SEQ ID NO: 7.

[0010] Further, the monoclonal antibody further comprises:

[0011] 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;

[0012] 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;

[0013] or, comprises an antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] Further, the polynucleotide sequences encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody 3G5 are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0019] Further, the polynucleotide sequences encoding the heavy chain constant region and the light chain constant region of the monoclonal antibody 3G5 are shown in SEQ ID NO: 6 and SEQ ID NO: 8, respectively.

[0020] In a third aspect, the present application provides an expression vector comprising the nucleic acid, which can express the nucleic acid in a prokaryotic or eukaryotic host cell.

[0021] Further, the vector is selected from one of a plasmid vector, a bacteriophage vector, a viral vector, and a mammalian expression vector.

[0022] Specifically, the present application employs a mammalian expression vector.

[0023] In a fourth aspect, the present application provides an engineered bacterium or eukaryotic host cell comprising the expression vector.

[0024] 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.

[0025] Compared with the prior art, the present application has the following advantages:

[0026] The targeting RBD high neutralization activity anti-Omicron fully human monoclonal antibody 3G5 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 3G5 has wide application prospects in the preparation of COVID-19 therapeutic drugs. Specifically, the targeting RBD high neutralization activity anti-Omicron fully human monoclonal antibody 3G5 provided by the present application has the following advantages:

[0027] (1) Fully human, no humanization modification is needed in clinical application.

[0028] (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 3G5 on Omicron BA.5 is 26.8 ng / mL; the half effective concentration (EC50) of the monoclonal antibody 3G5 on Omicron JN.1 is 11.8 ng / mL.

[0029] (3) clear mechanism of action: monoclonal antibody 3G5 binds to RBD with high specificity, showing that the monoclonal antibody is targeted to the receptor binding region, and plays an antiviral effect by specifically blocking the binding of the virus to the receptor.

[0030] (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

[0031] Figure 1 is a flow cytometry single cell sorting chart;

[0032] Figure 2 is a detection map of automatic nucleic acid electrophoresis of monoclonal antibody variable region gene amplification;

[0033] Figure 3 is a denaturing SDS-PAGE detection map after affinity chromatography purification;

[0034] Figure 4 is a curve graph of the binding activity of 3G5 and RBD protein changing with concentration;

[0035] Figure 5 is an EC50 determination curve graph of the antibody on the safe replicon cell model; wherein, Figure 5 A is the half effective concentration of monoclonal antibody 3G5 on Omicron BA.5; Figure 5 B is the half effective concentration of monoclonal antibody 3G5 on Omicron JN.1. DETAILED DESCRIPTION

[0036] 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.

[0037] The present application provides a high neutralizing activity anti-Omicron monoclonal antibody 3G5 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: GGSISSSSNF (see SEQ ID NO: 9), IYYSGST (see SEQ ID NO: 10), and ARLDLGGDIVVPPAAKPYYYMDV (see SEQ ID NO: 11); and the light chain variable region has the amino acid sequences of the three complementarity determining regions as follows: SSNIGAGYN (see SEQ ID NO: 12), GDI, and QSYDSSLSGLYV (see SEQ ID NO: 13).

[0038] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3G5 is shown in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region of the monoclonal antibody 3G5 is shown in SEQ ID NO: 3.

[0039] The amino acid sequence of the heavy chain constant region of the monoclonal antibody 3G5 is shown in SEQ ID NO: 5; and the amino acid sequence of the light chain constant region of the monoclonal antibody 3G5 is shown in SEQ ID NO: 7.

[0040] 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.

[0041] 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.

[0042] In some embodiments, the variable region genes can be converted into scFv genes, once the V H and V LDNA fragments of the fragments, i.e., the variable region genes, 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.

[0043] In these manipulations, the DNA fragment encoding V L or V H 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.

[0044] Unless otherwise specifically indicated, all materials, reagents, instruments and equipment used in the present application are commercially available or are prepared by known methods.

[0045] Example 1

[0046] Screening and preparation of human anti-Omicron monoclonal antibodies

[0047] 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;

[0048] 2. Prepare samples:

[0049] (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, centrifuge at 800 rpm for 5 min after adjusting the volume, and discard the supernatant. Resuspend in 2-3 mL PBS and centrifuge, and discard the supernatant. Finally, resuspend in 100 μL PBS, take 2 μL for dilution by 10 times, and perform cell counting.

[0050] (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.

[0051] (3) Naked cell control: 1 tube, 1×10 6 cells, and supplement with PBS to 50 μL.

[0052] (4) Cells for sorting: 1 tube, first determine the number of cells, final concentration of 100 μL system (1~8×10 6 cells), while adding FVS-780, CD3-BV510, CD4-BV510, CD8-BV510, CD19-PE, IgD-BB700, CD20-BV421, CD38-FITC and Biotin-S1 fluorescent dye.

[0053] (5) Incubate the sample at 4 ℃ in the dark for 0.5 h.

[0054] (6) Add 1 mL PBS to each tube, centrifuge at 800 g at 4 ℃ for 5 min, discard the supernatant, repeat the washing twice. Add streptavidin-APC dye, adjust the system to 100 μL, incubate at 4 ℃ in the dark for 0.5 h.

[0055] (7) Add 1 mL PBS to each tube, centrifuge at 800 g at 4 ℃ for 5 min, discard the supernatant, repeat the washing twice.

[0056] (8) After resuspension with 400 μL PBS, remove cell clumps with a 40 μm cell strainer, and store at 4 ℃ in the dark for sorting.

[0057] 3, Flow sorting: select cells of 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 of CD19 + , CD3 － , CD4 － , CD8 － , then select mature B cells of IgD － , and finally select mature B cells of S1 + , which are our target cells.

[0058] 4, Amplify full human monoclonal antibody variable region genes by single cell-PCR technology

[0059] (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).

[0060] Primers:

[0061] H: 5′L-VH 1, L-VH 3, L-VH 4 / 6, 5′L-VH 5, Hu IgG-const-anti, 3′CμCH1

[0062] κ: 5′L Vκ1 / 2, 5′L Vκ3, 5′L Vκ4, 3′Cκ543–566

[0063] 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

[0064] Table 1: Primers for Reverse Transcription PCR

[0065]

[0066] 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.

[0067] 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.

[0068] (2) Nested PCR

[0069] 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.

[0070] Table 2: Primers for amplifying the variable region of the heavy chain

[0071]

[0072] Table 3: Primers for amplifying the variable region of the Kappa light chain

[0073]

[0074] Table 4: Primers for amplifying the variable region of lambda light chain

[0075]

[0076] 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.

[0077] 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.

[0078] 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.

[0079] (3) Automatic nucleic acid electrophoresis instrument for detection

[0080] Some results of automatic nucleic acid electrophoresis detection are shown in Figure 2 , only the bands of 3G5 heavy chain and light chain variable region are shown.

[0081] 5. Synthesis of polynucleotide sequence of variable region sequence

[0082] 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.

[0083] 6. Construction of plasmid expression vector

[0084] The Suzhou Jinyuzhi Biotechnology Co., Ltd. was commissioned to synthesize the antibody heavy chain constant region DNA sequence and the antibody light chain constant region DNA sequence according to the antibody heavy chain constant region DNA sequence and the antibody light chain constant region DNA sequence, add a restriction enzyme cutting site (5'-GAGCTCGGTACC-3', see SEQ ID NO: 66) in front of the heavy chain and light chain constant region DNA sequence, and add a restriction enzyme cutting site (5'-GCTAGC-3') at the end of the sequence. The antibody heavy chain constant region DNA sequence and the antibody light chain constant region DNA sequence with the restriction enzyme cutting site were synthesized (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., item number P0165) and the antibody heavy chain constant region DNA sequence were subjected to enzyme cutting using the restriction endonuclease Nhe I and Sac I, and the cut plasmid and the antibody constant region DNA fragment were connected 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 subjected to enzyme cutting using the restriction endonuclease Nhe I and Sac I, and the cut plasmid and the antibody constant region DNA fragment were connected using T4 DNA ligase to obtain a plasmid vector containing the antibody light chain constant region.

[0085] The constructed plasmid vector containing the antibody heavy chain constant region and the antibody heavy chain variable region synthesized in step 5 were subjected to enzyme cutting using the restriction endonuclease EcoR I and Kpn I, and the cut products were connected to obtain a eukaryotic expression vector that can be used to express the 3G5 antibody heavy chain. The constructed plasmid vector containing the antibody light chain constant region and the antibody light chain variable region synthesized in step 5 were subjected to enzyme cutting using the restriction endonuclease EcoR I and Kpn I, and the cut products were connected using T4 DNA ligase to obtain a eukaryotic expression vector that can be used to express the 3G5 antibody light chain. The two vectors described above can be used to express the antibody by transfecting eukaryotic cells.

[0086] The amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of the monoclonal antibody 3G5 are shown in SEQ ID NO: 1, positions 26-35, 53-59, and 98-120, 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, positions 26-34, 52-54, and 91-102, respectively. The details are shown in Table 5.

[0087] 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 3G5

[0088]

[0089] 7. Transient expression of mAb and affinity chromatography purification

[0090] Using Expi293 expression system, 15 ug heavy chain and 15 ug light chain were mixed and transfected into Expi 293F cells, following the instructions of transfection reagent (Shanghai Liji Biological Technology Co., Ltd., EZ Trans Plus, AC04L011). After 5-6 days, the culture solution was harvested, and about 50 mL of supernatant was obtained after centrifugation. A pre-packed Protein A affinity chromatography column with a volume of 5 mL was used. Before loading, the column was equilibrated with 20 mM PBS. When the conductivity showed to the baseline, the sample was loaded. After loading, the column was washed with 20 mM PBS until the baseline was stable. The target protein was eluted with 0.1 M glycine buffer at pH 3.0. When the OD280 was close to the baseline, the collection was stopped. The column was washed with at least 3 column volumes of 20 mM PBS until the baseline was stable, and then washed with 20% ethanol. The results of SDS-PAGE detection of mAb after affinity chromatography purification are shown in Figure 3 . It can be seen that the monoclonal antibody 3G5 is successfully purified. Figure 3

[0091] Example 2

[0092] Binding activity analysis of fully human anti-Omicron monoclonal antibody 3G5 and RBD

[0093] 1. Coating: Dilute the RBD antigen with coating solution to a concentration of 3 ug / mL, coat the enzyme-labeled plate with 100 uL per well, and incubate at 4°C overnight.

[0094] 2. Blocking: Add 300 uL of PBST washing solution to each well and wash 3 times for 3 min each time. Tap off the liquid in the well, add 2% BSA, 250 uL per well, and incubate at 37°C for 1 h.

[0095] 3. Sample incubation: Add 300 uL of PBST washing solution to each well and wash 3 times for 3 min each time. Tap off the liquid in the well, add the purified mAb diluted with PBS, 9 ug / mL in the first well, 100 uL per well with 3-fold serial dilution, and incubate at 37°C for 2 h.

[0096] 4. Secondary antibody incubation: Wash 5 times as above. Add HRP goat anti-human FC secondary antibody (1:20000 dilution), 100 uL per well, and incubate at 37°C for 1 h.

[0097] ​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.

[0098] 6. Results: See Figure 4 . Figure 4 In the present embodiment, the curve shows the detection results of the monoclonal antibody 3G5 and RBD, and the results show that the specific binding of the monoclonal antibody 3G5 and RBD presents a dose-response relationship, indicating that the monoclonal antibody is specific to RBD.

[0099] Example 3

[0100] Analysis of neutralization activity on SARS-CoV-2 safe replicon cell model

[0101] 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 genomic 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.

[0102] 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.

[0103] 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.

[0104] Results are shown in Figure 5 (Figure 3). (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 3G5 on SARS-COV-2 Omicron BA.5 is 26.8 ng / mL; the half maximal effective concentration (EC50) of monoclonal antibody 3G5 on SARS-COV-2 Omicron JN.1 is 11.8 ng / mL.

[0105] 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 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as SEQ ID NO: 9-11; the amino acid sequences of the complementarity determining regions CDR1 and CDR3 of the light chain variable region are respectively shown as SEQ ID NO: 12-13, and the amino acid sequence of CDR2 is GDI.

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 variable region of the monoclonal antibody is shown as SEQ ID NO: 1; and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO:

3.

3. The anti-Omicron full human monoclonal antibody targeting RBD according to claim 1, wherein, 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.

4. 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 label to the N-terminus and / or C-terminus of the monoclonal antibody.

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

6. The nucleic acid molecule of claim 5, wherein, The polynucleotide sequences encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO: 2 and SEQ ID NO:

4.

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

8.

8. An expression vector comprising the nucleic acid molecule of claim 5. The expression vector is capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell.

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

10. Use of the monoclonal antibody of any one of claims 1-4 in the preparation of a COVID-19 therapeutic drug or a novel coronavirus detection product.

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