RBD-targeted anti-Omicro fully-humanized monoclonal antibody 9C4 and application thereof

Abstract

The invention provides an anti-Omicro fully-humanized monoclonal antibody 9C4 for targeting RBD (Radial Basis Decidosis) and application of the anti-Omicro fully-humanized monoclonal antibody 9C4, 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, and the amino acid sequences of three complementary determining regions of the heavy chain variable region are respectively as follows: GYAFTTYG, ISTYNGNT and ATSLGLWFGQNS; the amino acid sequences of the three complementary determining regions of the light chain variable region are respectively as follows: SGSIASNY, EDN and QTYDGSNQV. The monoclonal antibody 9C4 provided by the invention has efficient and specific anti-Omicron neutralizing activity, is completely human-derived and good in stability, and has great application potential in preparation of novel coronavirus detection products or drugs for preventing and treating novel coronavirus diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology and immunology, and in particular to an anti-Omicron fully human monoclonal antibody 9C4 targeting RBD and an application thereof. Background Art

[0002] Coronaviruses belong to the family Coronaviridae of the order Nidovirales. They are a type of enveloped positive-strand RNA virus that is widely found in nature and can infect a variety of vertebrates, including humans, rodents, cats, dogs, bats, cattle, chickens, and whales. In addition, coronaviruses can also be transmitted across species. There are currently seven known coronaviruses that can infect humans, of which four are of low pathogenicity: HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1, and three are of high pathogenicity: SARS-CoV, MERS-CoV, and SARS-CoV-2. The pathogen of the coronavirus epidemic (COVID-19) was originally named 2019-nCoV, and was later officially named SARS-CoV-2 (Chinese name: novel coronavirus, abbreviated as novel coronavirus). The virus has extremely strong transmission ability and pathogenicity, and quickly caused a pandemic worldwide.

[0003] The novel coronavirus particles are spherical or ellipsoidal, with a diameter of about 80~160 nm. The outer layer of the virus particle has a phospholipid envelope, on which there are structural proteins such as Spike trimer protein, M protein, and E protein. Inside the virus particle is a genetic material core composed of viral genome RNA and N protein. After the novel coronavirus infects the human body, it will cause respiratory disease symptoms such as fever, cough, and headache. Most patients can recover on their own, but some patients are prone to develop respiratory failure and acute pneumonia to varying degrees, especially those with low immunity and underlying diseases. The mortality rate is high. Spike protein (S protein) is a key protein that mediates the invasion of coronavirus into host cells. The receptor binding domain (RBD) of the novel coronavirus S protein is an important region for it to bind to the human ACE2 receptor protein, and it is also an ideal antigen target for neutralizing antibodies to bind.

[0004] Although various vaccines have been put into use since the outbreak of the new coronavirus, the new coronavirus has continued to mutate during its spread, and a large number of breakthrough infections have occurred (people who have been vaccinated or recovered from the new coronavirus have been infected with the new coronavirus again). Among them, the S protein of the Omicron strain carries more than 30 amino acid mutations, which has led to the Omicron strain's strong immune escape ability, which poses a serious challenge to the existing new coronavirus vaccines and antibody drugs. There are many Omicron progeny viruses, such as BA.2, BA.5, XBB.1.5, KP.1, JN.1, etc. Therefore, the development of a broad-spectrum, highly effective new coronavirus Omicron vaccine and specific therapeutic drugs has become of great public health and safety significance.

[0005] In summary, the present invention provides a fully human monoclonal therapeutic antibody with good neutralizing effect against a variety of Omicron progeny viruses. Summary of the invention

[0006] In view of the deficiencies of the above-mentioned prior art, the present invention provides a fully human monoclonal antibody 9C4 targeting RBD with high neutralizing activity against the Omicron strain of new coronavirus and its application. The monoclonal therapeutic antibody has a good protective effect against the Omicron strain.

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

[0008] In the first aspect, the present invention provides a fully humanized monoclonal antibody 9C4 targeting RBD with high neutralizing activity against Omicron, wherein the monoclonal antibody 9C4 comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), the amino acid sequences of the three complementary determining regions CDR1, CDR2 and CDR3 in the heavy chain variable region are GYAFTTYG (see SEQ ID NO: 9), ISTYNGNT (see SEQ ID NO: 10) and ATSLGLWFGQNS (see SEQ ID NO: 11), respectively; the amino acid sequences of the three complementary determining regions CDR1, CDR2 and CDR3 in the light chain variable region are SGSIASNY (see SEQ ID NO: 12), EDN and QTYDGSNQV (see SEQ ID NO: 13), respectively.

[0009] Furthermore, 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.

[0010] Furthermore, 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.

[0011] Furthermore, the monoclonal antibody further comprises:

[0012] An antibody with the same function obtained by replacing, deleting and / or adding one or more amino acids to the amino acid sequence of the monoclonal antibody;

[0013] Alternatively, comprising a heavy chain variable region having an amino acid sequence that is at least 80% homologous to the heavy chain variable region; and a light chain variable region having an amino acid sequence that is at least 80% homologous to the light chain variable region;

[0014] Alternatively, the invention relates to 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 The amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the above sequence; having V H and V L V H and V L Antibodies to the regions can be obtained by mutagenizing (eg, 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 gene can be converted into a scFv gene. Once the V H and V L The fragmented DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody chain gene, a Fab fragment gene, or a scFv gene.

[0017] In these operations, the DNA fragment encoding VL or VH is effectively linked to another DNA fragment encoding another protein such as an antibody constant region or a flexible linker. As used herein, the term "effectively linked" means that two DNA fragments are linked together so that the amino acid sequences encoded by the two DNA fragments remain in the reading frame.

[0018] In a second aspect, the present invention provides a nucleic acid molecule encoding the monoclonal antibody, wherein 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.

[0019] Furthermore, 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] Furthermore, 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 invention provides an expression vector comprising the nucleic acid, wherein the expression vector is capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell.

[0022] Furthermore, the vector is selected from a plasmid vector, a phage vector, a viral vector, and a mammalian expression vector.

[0023] Specifically, the present invention employs mammalian expression vectors.

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

[0025] In a fifth aspect, the present invention provides the use of the monoclonal antibody in the preparation of COVID-19 therapeutic drugs or new coronavirus detection products.

[0026] Compared with the prior art, the present invention is beneficial in that:

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

[0028] (1) Fully human: no humanization is required for clinical application.

[0029] (2) High neutralizing activity. In the SARS-COV-2 Omicron-infected safe replicon cell model, the half effective concentration (EC50) of monoclonal antibody 9C4 against Omicron BA.5 was 15.7 ng / mL; the half effective concentration (EC50) against Omicron JN.1 was 21.5 ng / mL.

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

[0031] (4) Good stability: Because the monoclonal antibody genes come from the same cell in the human body and are naturally paired, the half-life of IgG1 antibodies in the human body is known to be 21 to 28 days. In theory, publicly available monoclonal antibodies have a consistent half-life in the human body. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0033] Figure 2 Automatic nucleic acid electrophoresis instrument for detecting the pattern of monoclonal antibody variable region gene amplification;

[0034] Figure 3 This is the denaturing SDS-PAGE detection pattern after affinity chromatography purification;

[0035] Figure 4 It is a curve diagram to detect the binding activity of 9C4 and RBD protein as the concentration changes;

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

[0037] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The present invention provides a fully humanized anti-Omicron monoclonal antibody 9C4 with high neutralizing activity targeting RBD, wherein the monoclonal antibody 9C4 comprises a heavy chain variable region (V H ) and the light chain variable region (V L), the amino acid sequences of the three complementary determining regions in the heavy chain variable region are: GYAFTTYG (see SEQ ID NO: 9), ISTYNGNT (see SEQ ID NO: 10) and ATSLGLWFGQNS (see SEQ ID NO: 11); the amino acid sequences of the three complementary determining regions in the light chain variable region are: SGSIASNY (see SEQ ID NO: 12), EDN and QTYDGSNQV (see SEQ ID NO: 13).

[0039] 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.

[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 replacing, deleting and / or adding one or more amino acids to the amino acid sequence of the monoclonal antibody; or, a heavy chain variable region having an amino acid sequence with at least 80% homology to the heavy chain variable region; and a light chain variable region having an amino acid sequence with at least 80% homology to the light chain variable region; or, 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 The amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the above sequence; having V H and V L V H and V L Antibodies to the regions can be obtained by mutagenizing (eg, 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 gene can be converted into a scFv gene. Once the V H and V L The fragmented DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region gene into a full-length antibody chain gene, a Fab fragment gene, or a scFv gene.

[0044] In these operations, the encoding V L or V H The DNA fragment of the present invention is operably linked to another DNA fragment encoding another protein such as an antibody constant region or a flexible linker. As used herein, the term "operably linked" means that two DNA fragments are linked together so that the amino acid sequences encoded by the two DNA fragments remain in the reading frame.

[0045] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0046] Example 1

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

[0048] 1. Prepare a 96-well plate: add 20 μL RNase-free water + 20 U RNase inhibitor to each well, cover with a sealing film, and place at 4 °C for later use;

[0049] 2. Prepare samples:

[0050] (1) Cell resuscitation: Take out the frozen PBMC cells isolated from the peripheral blood of patients who have recovered from the novel coronavirus infection from -80℃ and quickly place them in warm water at 37℃. After the cells thaw, centrifuge them at 800 rpm for 5 min and discard the supernatant. Resuspend them in 2-3mL PBS in a flow tube, balance them and centrifuge them at 800 rpm for 5 min, discard the supernatant. Resuspend them in 2-3mL PBS, centrifuge them and discard the supernatant. Finally, add 100 μL PBS to resuspend them and take 2 μL to dilute them 10 times for cell counting.

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

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

[0053] (4) Cells for sorting: 1 tube, first determine the number of cells, 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 of PBS to each tube, centrifuge at 800 g for 5 min at 4 °C, discard the supernatant, and repeat the wash twice. Add streptavidin-APC dye and adjust the system to 100 μL. Incubate the sample at 4 °C in the dark for 0.5 h.

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

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

[0058] 3. Flow cytometry: Select CD19 + , CD3 - , CD4 - , CD8 - , IgD - , CD38 + , CD20 - , S1 + The results of flow cytometry sorting are shown in Figure 1 , first select lymphocytes, then select non-adherent single cells, then select live cells, then select CD19 + 、CD3 - 、CD4 - 、CD8 - B cells, and then select IgD - The mature B cells finally select S1 + The mature B cells are our target cells.

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

[0060] (1) Reverse transcription PCR reference manual (QIAGEN, 210212), the procedure is briefly described as follows: 94 cells were sorted by flow cytometry. All of the following specific primers for each subtype of heavy chain (H), kappa light chain (κ), and Lambda light chain (λ) were added to each reaction system at the same time (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: Reverse transcription PCR primers

[0066]

[0067] The PCR reaction system contained: 6 μL of 5× buffer, 1.2 μL of dNTP, 1.2 μL of reverse transcriptase (Takara Biotechnology Co., Ltd., RR036A), primers as above, single cell as template, and water to 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, 40 cycles of 95 °C for 40 s, 55 °C for 30 s, and 72 °C for 1 min, and finally extension at 72 °C for 10 min.

[0069] (2) Nested PCR

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

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

[0072]

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

[0074]

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

[0076]

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

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

[0079] The PCR reaction conditions were as follows: pre-denaturation at 94 °C for 4 min, followed by 40 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 45 min, and a final extension at 72 °C for 10 min.

[0080] (3) Detection by automatic nucleic acid electrophoresis instrument

[0081] Some 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 in the figure.

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

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

[0084] 6. Construction of plasmid expression vector

[0085] Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to add restriction enzyme sites (5'-GAGCTCGGTACC-3', see SEQ ID NO: 66) before the heavy chain and light chain constant region DNA sequences and add restriction enzyme sites (5'-GCTAGC-3') at the end of the sequence according to the antibody heavy chain constant region DNA sequence and light chain constant region DNA sequence, and synthesize antibody heavy chain constant region DNA sequence and light chain constant region DNA sequence with restriction enzyme sites (the heavy chain constant region sequence is shown by SEQ ID NO: 5, the DNA coding sequence is shown by SEQ ID NO: 6, the Kappa type light chain constant region sequence is shown by SEQ ID NO: 7, and the DNA coding sequence is shown by SEQ ID NO: 8). The pCAGGS empty plasmid (which can be purchased from Shanghai Yuanmu Biotechnology Co., Ltd., product number P0165) and the antibody heavy chain constant region DNA sequence were digested with restriction endonucleases Nhe I and Sac I, and the digested plasmid and antibody constant region DNA fragments were connected with 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 endonucleases Nhe I and Sac I, and the digested plasmid and antibody constant region DNA fragments were connected with T4 DNA ligase to obtain a plasmid vector containing the antibody light chain constant region.

[0086] The constructed plasmid vector containing the constant region of the antibody heavy chain and the antibody heavy chain variable region synthesized in step 5 were digested with restriction endonucleases EcoR I and Kpn I, and the digestion products were connected 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 constant region of the antibody light chain and the antibody light chain variable region synthesized in step 5 were digested with restriction endonucleases EcoR I and Kpn I, and the digestion products were connected with T4 DNA ligase to obtain a eukaryotic expression vector that can be used to express the light chain of the 9C4 antibody. The above two vectors can be used to express antibodies by transfecting eukaryotic cells.

[0087] The amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region of monoclonal antibody 9C4 are shown in the amino acid sequences of positions 26-33, 51-58 and 97-108 of SEQ ID NO: 1, respectively; the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the light chain variable region are shown in the amino acid sequences of positions 26-33, 51-53 and 92-100 of SEQ ID NO: 3, respectively. The details are shown in Table 5.

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

[0089]

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

[0091] Using the Expi293 expression system, 15 ug of heavy chain and 15 ug of light chain were mixed and transfected into Expi 293F cells. The operation was carried out according to the instructions of the transfection reagent (Shanghai Liji Biotechnology Co., Ltd., EZ Trans Plus, AC04L011). The culture medium was harvested after 5-6 days. After centrifugation, the supernatant was about 50 mL. A pre-loaded Protein A affinity column with a volume of 5 mL was used. Before loading, it was balanced with 20mM PBS. After the conductivity showed a baseline, the sample was injected. After the loading was completed, the column was washed with 20mM PBS until the baseline was stable. The target protein was eluted with 0.1M pH 3.0 glycine buffer. After 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 the column was washed with 20% ethanol. The results of SDS-PAGE detection of monoclonal antibodies after affinity chromatography purification are shown in Figure 3 .Depend on Figure 3 It can be seen that the monoclonal antibody 9C4 was successfully purified and obtained.

[0092] Example 2

[0093] Analysis of the binding activity of fully human anti-Omicron monoclonal antibody 9C4 to RBD

[0094] 1. Coating: dilute the RBD antigen with coating solution to a concentration of 3 μg / mL, coat the ELISA plate with 100 μL per well, and coat overnight at 4°C.

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

[0096] 3. Sample incubation: Add 300 μL PBST washing solution to each well, wash 3 times × 3 min / time; tap the liquid in the well, add purified monoclonal antibody diluted with PBS, 9 ug / mL in the first well, 3-fold serial dilution 100 μL / well, incubate at 37 ℃ for 2 h.

[0097] 4. Secondary antibody incubation: Wash 5 times and perform the same operation as above; add HRP goat anti-human Fc secondary antibody (1:20,000 dilution), 100 μL / well, and incubate at 37 °C for 1 h.

[0098] 5. Color development: Wash 5 times, operate as above; add 100 μL TMB single-component color development solution to each well, color at 37 ℃ for 15 minutes, add 50 μL stop solution to each well to terminate the reaction, and use an enzyme reader to detect the absorbance at 450 nm. Use Graph Pad nonlinear regression and four-parameter fitting to draw the standard curve, and calculate the EC50 concentration of the monoclonal antibody based on the standard curve and dilution multiple.

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

[0100] Example 3

[0101] Neutralization activity analysis of SARS-CoV-2 safe replicon cell model

[0102] 1. Rescue of SARS-CoV-2 safe replicons: rescue different safe replicons according to the spike protein (S protein) of the Omicron mutant strain. The SARS-CoV-2 N gene was stably transfected into Caco-2 cells to construct Caco-2-N cells that stably expressed the N protein. The SARS-CoV-2 genomic RNA containing different S genes (the N gene was replaced with the Luciferase reporter gene) was transfected into Caco-2-N cells by electroporation (Gene Pulser Xcell™, BIO-RAD). After 72 hours, the supernatant containing the SARS-CoV-2 safe replicons was collected, centrifuged at 3000 rpm for 10 minutes, aliquoted, and frozen at 80 °C. The TCID50 of the safe replicons was used to infect Caco-2-N cells using a series of dilutions, and its TCID50 was calculated. The SARS-CoV-2 safe replicon can be continuously passaged and amplified in Caco-2-N cells. It has the characteristics of easy operation, amplification, high safety, relatively low biosafety requirements (can be operated in a biosafety level II laboratory), and can effectively simulate the coronavirus replication cycle.

[0103] 2. Antibody dilution: SARS-CoV-2 safe replicon (3×10 5 TCID50 / well) and diluted serum were incubated in a 96-well white plate at room temperature for 30 min, and then incubated with trypsin-treated Caco-2-N cells at 2×10 4 / hole density mix.

[0104] 3. Detection and analysis of luciferase readings: After 16 h of culture, the culture medium of the infected cells was removed, and the cells were lysed with 1× Bright-Glo luciferase assay reagent (Promega), and chemiluminescence detection was performed using a SpectraMax iD3 multi-well luminometer (Molecular devices). GraphPad Prism 7 software was used to calculate the 50% neutralization dilution titer (NT50), and nonlinear regression curve fitting was performed.

[0105] Results Figure 5 (The horizontal axis represents the antibody concentration, and the vertical axis represents the neutralization effect % relative to the negative control group). Figure 5 It can be seen that in the safe replicon cell model of SARS-COV-2 Omicron infection, the half effective concentration (EC50) of monoclonal antibody 9C4 for Omicron BA.5 was 15.7 ng / mL; the half effective concentration (EC50) for Omicron JN.1 was 21.5 ng / mL.

[0106] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A fully humanized monoclonal antibody targeting RBD, characterized in that: The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as SEQ ID NOs: 9-11; the amino acid sequences of the complementary determining regions CDR1 and CDR3 of the light chain variable region are respectively shown as SEQ ID NOs: 12-13, and the amino acid sequence of CDR2 is EDN.

2. The fully humanized anti-Omicron 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 in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

3.

3. The fully humanized anti-Omicron 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 in SEQ ID NO:5, and the amino acid sequence of the light chain constant region of the monoclonal antibody is shown in SEQ ID NO:

7.

4. The fully humanized anti-Omicron monoclonal antibody targeting RBD according to claim 1, characterized in that: The monoclonal antibody also includes: An antibody with the same function obtained by replacing, deleting and / or adding one or more amino acids to the amino acid sequence of the monoclonal antibody; Alternatively, comprising a heavy chain variable region having an amino acid sequence that is at least 80% homologous to the heavy chain variable region; and a light chain variable region having an amino acid sequence that is at least 80% homologous to the light chain variable region; Alternatively, the invention relates to an antibody obtained by connecting a tag 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 molecules include 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 according to claim 5, characterized in that The polynucleotide sequences encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

7. The nucleic acid molecule according to claim 5, characterized in that The polynucleotide sequences encoding the heavy chain constant region and the light chain constant region of the monoclonal antibody are shown in SEQ ID NO: 6 and SEQ ID NO: 8, respectively.

8. An expression vector comprising the nucleic acid molecule according to claim 5, characterized in that 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 according to claim 8.

10. Use of the monoclonal antibody according to any one of claims 1 to 4 in the preparation of COVID-19 therapeutic drugs or new coronavirus detection products.

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