Human antibodies and antibody combinations that synergistically neutralize sars-cov-2 and uses thereof

Abstract

The application relates to anti-novel coronavirus human antibodies, antibody combinations and applications thereof. The antibodies are isolated from novel coronavirus pneumonia convalescents, and are 825 monoclonal antibodies, 843 monoclonal antibodies and 826 monoclonal antibodies against RBD, and 846 monoclonal antibodies against NTD. The 825 or 843 monoclonal antibodies can effectively neutralize the currently popular novel coronavirus mutant strains, such as the British strain, the South African strain, the Brazilian strain and the Indian strain. The 825 / 843 / 826 and 825 / 843 / 846 antibody combinations can effectively neutralize the currently popular novel coronavirus mutant strains, and the neutralization activity IC50 in the pseudovirus system can reach 10 ng / mL. The antibodies and the antibody combinations can be used for preparing medicines for preventing or treating novel coronavirus infection or diagnostic reagents or kits.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to human antibodies and antibody combinations that synergistically neutralize the novel coronavirus and their applications. Background Technology

[0002] The novel coronavirus pneumonia (COVID-19), currently causing a global pandemic, is caused by SARS-CoV-2, a coronavirus that can cause severe acute respiratory syndrome. It poses a serious threat to human health and the economy. To date, there have been nearly 200 million confirmed cases and over 4.2 million deaths worldwide. And we still haven't found an effective specific antiviral drug.

[0003] Monoclonal antibodies (mAbs) are undoubtedly one of the most promising classes of biological agents for therapeutic use, and have been applied to the treatment of tumors, autoimmune diseases, and infectious diseases. Rapid isolation of human monoclonal antibodies, high-throughput sequencing technology, and advancements in structural biology have made antibodies an important tool for rapidly responding to emerging infectious diseases.

[0004] Currently, human monoclonal antibodies are mainly obtained using the following strategies (see: Immunol Res. 2020 Dec; 68(6):325-339.): (1) Immunizing transgenic mice with viral antigens. These transgenic mice, due to the introduction of human antibody heavy and light chain genes, can produce antibodies derived from human antibody sequences after antigen immunization, i.e., human antibodies. However, this method requires the preparation of viral antigens for immunization, and the antibody production is in the immune environment of mice, which cannot show the production of neutralizing antibodies in natural human infection. (2) Phage display technology. This technology is used to screen from variable region fragment (scFv) libraries of immune or non-immune single-chain antibodies. Since the source is a non-immune cDNA library, the antibody has not undergone an affinity maturation process, or the antibody heavy and light chains are not originally paired, making it difficult to obtain antibodies with high affinity. (3) Cloning antibodies directly from memory B cells of recovered infected individuals. With the development of antibody technology, people can directly obtain antibodies from single B cells. Most methods employ a strategy of separating individual virus antigen-specific memory B cells from PBMCs of recovered infected individuals via flow cytometry for antibody cloning. While this method can rapidly obtain antibodies, it only yields antibodies that can bind to viral proteins, losing conformation-dependent antibodies that play important functions in the body.

[0005] SARS-CoV-2 invades host cells by fusing with the cell membrane via the spike protein, the S protein, on its surface. The S protein consists of two subunits, S1 and S2. S1 is divided into an N-terminal (NTD) and a receptor-binding domain (RBD). During viral infection, the S1-RBD first binds to the receptor on the cell membrane, triggering a conformational change in the S protein, leading to viral fusion with the cell membrane and entry into the target cell. The S1-RBD is considered the main protein that induces the production of neutralizing antibodies in the host. Therefore, in response to the COVID-19 pandemic, most researchers have used the expression of recombinant RBD proteins to sort out RBD-specific B cells to obtain neutralizing antibodies. The acquisition of such RBD antibodies has been reported, some of which have a strong ability to neutralize the virus (see: 1. Science. 2020 Aug 21; 369(6506):956-963. and 2. Nature. 2020 Aug; 584(7821):450-456.). However, this strategy may result in the loss of some antibodies that do not target S1-RBD, and may even miss the antibodies produced by the body that have important antiviral effects.

[0006] Therefore, researchers changed their strategy and used the S protein to screen for antibodies. Research teams at home and abroad have obtained neutralizing antibodies against S1-NTD from recovered patients (see: Cell. 2021 Apr 29; 184(9):2332-2347.).

[0007] Using a single monoclonal antibody targeting a single epitope as an antiviral treatment is prone to viral escape, meaning that newly emerging variant viral strains can render the corresponding antibody incapable of neutralizing the virus, or even cause both combined antibodies to lose their activity (Reference: Cell Rep Med. 2021 Apr 20; 2(4):100255.). For example, a cocktail of two COVID-19 neutralizing antibody drugs from Lily (LY-CoV555 and LY-CoV016) has become ineffective against mutant viruses in South Africa and Brazil. Therefore, using multiple antibodies targeting different epitopes in combination as an antiviral treatment strategy is more effective in preventing the emergence of escape mutant viruses. To solve this problem, it is first necessary to obtain antibodies with high neutralizing activity targeting different epitopes of viral antigens, screen for antibody combinations with synergistic effects, and ensure that single antibodies or antibody combinations can effectively neutralize currently prevalent viral variants. Summary of the Invention

[0008] This invention aims to provide a human antibody and antibody combination that synergistically neutralizes the novel coronavirus and their applications. Specifically, it involves isolating a group of human monoclonal antibodies from recovered COVID-19 patients that can efficiently neutralize the novel coronavirus, including three antibodies targeting the RBD and one targeting the NTD (825, 843, 826, and 846). A single antibody (825 or 843) can effectively neutralize currently prevalent viral variants, including those from the UK, South Africa, Brazil, and India. The three antibody combinations screened (825 / 843 / 826 and 825 / 843 / 846) can effectively neutralize currently prevalent viral variants, with neutralizing activity IC50 reaching approximately 10 ng / ml in a pseudovirus system.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The first objective of this invention is to provide a human antibody against the novel coronavirus, wherein the human antibody is an 825 monoclonal antibody, an 843 monoclonal antibody, an 826 monoclonal antibody, or an 846 monoclonal antibody.

[0011] The 825 monoclonal antibody contains a heavy chain variable region and a light chain variable region. The heavy chain variable region of the 825 monoclonal antibody contains an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.1, and the light chain variable region contains an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.2.

[0012] The 843 monoclonal antibody contains a heavy chain variable region and a light chain variable region. The heavy chain variable region of the 843 monoclonal antibody contains an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.3, and the light chain variable region contains an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.4.

[0013] The 826 monoclonal antibody contains a heavy chain variable region and a light chain variable region. The heavy chain variable region of the 826 monoclonal antibody contains an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.5, and the light chain variable region contains an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.6.

[0014] The 846 monoclonal antibody contains a heavy chain variable region and a light chain variable region. The heavy chain variable region of the 846 monoclonal antibody contains an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.7, and the light chain variable region contains an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.8.

[0015] Preferably, the amino acid sequence of the heavy chain variable region of the 825 monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2;

[0016] QVQLVQSGAEVKKPGASVKVSCKASGGTFRTYAVSWVRQAPGQGLEWLGGIIPIFHIAYYAQNF QGRLTITADESTNTAYMDLSSLRSEDTAVYYCASHPVGADYSYYYYMDVWGQGTTVTVSS(SEQ ID NO.1);

[0017] QSALTQPASVSGSPGQSITISCTGTSSDIGSYDLVSWYQQHPGKAPKLMIYDVFKRPSGISNRF SGSKSGNTASLTISGLQTEDEADYYCCSYAGTSTWVFGGGTKLTVL(SEQ ID NO.2)

[0018] The amino acid sequence of the heavy chain variable region of the 843 monoclonal antibody is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4;

[0019] QMQLVQSGAEVKKPGASVKVSCKASGYAFISYHMDWVRQAPGQGLEWMGIINPSGGGASYAQKF QGRVTLTRDTSTSTVYMELSSLTSEDTAVYYCARSQGAGWDQHHFDYWGQGTLVTVSS(SEQ ID NO.3);

[0020] QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYGVHWYQQLPGTAPKLLIYGDNNRPSGVPDRF SGSSKSGTSASLAITGLQTGDEADYFCQSYDTGLSASQWVFGGGTKLTVL(SEQ ID NO.4)

[0021] The amino acid sequence of the heavy chain variable region of the 826 monoclonal antibody is shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.6.

[0022] QMQLVQSGAEVKKPGASVKVSCKTSGFSFTNYFLHWVRQAPGQGLEWMGIINPSGTITTYAQKF QDRVTMTRDTSTSTVYMELRSLRYEDTAVYYCARDQDPISWDDAFDIWGQGTMVTVSS(SEQ ID NO.5);

[0023] QSVLTQPPSVSGAPGQRVTISCTGSIATIGAGFDVHWYQQLPGTAPKLLIYGNNNRPSGVPDRF SGSKSGTSASLAITGLQAEDEADYYCQSYDSGLSVSFGTGTKVTVL(SEQ ID NO.6)

[0024] The amino acid sequence of the heavy chain variable region of the 846 monoclonal antibody is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0025] QVQLVQSGAEVKKPGASVKVSCKVSVYSLTELSMHWVRQAPGKGLEWMGGFDPENAETIYSQKF QGRVTMTEDTSTDTAYMELRSLRSEDTAVYYCAAGFAILGVISNYSYYYGMDVWGQGTTVTVSS(SEQ ID NO.7);

[0026] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVFWYQHLPGTAPKLLIYRNNQRPSGVPDRFS GSKSGNTASLTISGLQAEDEADYYCNSYTSSTTLVFGGGTKLTVL(SEQ ID NO.8)

[0027] Preferably, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the antibody 825 monoclonal antibody is shown in SEQ ID NO.9, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region of the antibody 825 monoclonal antibody is shown in SEQ ID NO.10.

[0028] CAGGTTCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCTTCTGGAGGCACCTTCAGGACCTATGCTGTCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGCTGGGAGGCATCATCCCTATCTTTCATATAGCATACTACGCACAGAA TTTCCAGGGCAGACTCACGATCACCGCGGACGAATCCACCAACACAGCCTACATGGACCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGCCATCCCGTGGGAGCAGATTATTCTTACTACTACTATATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO.9);

[0029] CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGATATTGGGAGTTATGACCTTGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGACGTCTTTAAGCG GCCCTCAGGGATTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCTGGGCTCCAGACTGAGGACGAGGCTGATTATTACTGCTGCTCATATGCAGGTACTAGCACCTGGGTGTTCGGCGGAGGGACCAAACTGACCGTCCTAG(SEQ ID NO.10)

[0030] The nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the monoclonal antibody 843 is shown in SEQ ID NO. 11, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region of the monoclonal antibody 843 is shown in SEQ ID NO. 12.

[0031] CAAATGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACGCCTTCATCAGCTATCACATGGATTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTGGCGCAAGCTACGCACAG AAGTTCCAGGGCAGAGTCACCCTGACCAGAGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGTCTGACATCTGAGGACACGGCCGTGTATTACTGTGCGAGATCGCAGGGGGCTGGATGGGACCAACATCACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG(SEQID NO.11);

[0032] CAGTTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGGTGTACACTGGTACCAGCAACTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGTGACAACAATCGGCCCT CAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGTCCAGACTGGGGACGAGGCCGATTATTTCTGCCAGTCCTATGACACCGGCCTGAGTGCTTCGCAATGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTGG(SEQ ID NO.12)

[0033] The nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the monoclonal antibody 826 is shown in SEQ ID NO. 13, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region of the monoclonal antibody 826 is shown in SEQ ID NO. 14.

[0034] CAGATGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGACATCTGGATTCAGCTTCACCAACTACTTTCTACACTGGGTGCGACAGGCCCTGGACAGGGCCTTGAGTGGATGGGAATAATCAACCCTAGTGGTACTATCACAACCTACGCACAG AAGTTCCAGGACAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGCGCAGTCTGAGATATGAGGACACGGCCGTCTATTACTGTGCGAGAGATCAAGATCCCATATCCTGGGATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAG(SEQID NO.13);

[0035] CAGTTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACTGGGAGCATCGCCACCATCGGGGCAGGTTTTGATGTCCACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGTAACAACAATCG GCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAGCGGCCTGAGTGTCTCCTTCGGAACGGGGACCAAGGTCACCGTCCTAC(SEQ ID NO.14)

[0036] The nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the antibody 846 monoclonal antibody is shown in SEQ ID NO. 15, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region of the antibody 846 monoclonal antibody is shown in SEQ ID NO. 16.

[0037] CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAAGTTTCCGTATACAGCCTCACTGAATTATCCATGCACTGGGTGCGACAGGCTCCTGGAAAAGGGCTTGAGTGGATGGGAGGTTTTGATCCTGAAAATGCTGAAACAATCTACTCACAGAAGTTCCA GGGCAGGGTCACCATGACCGAGGACACATCTACAGACACAGCCTACATGGAACTGCGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCCGCAGGGTTTGCGATTCTGGGAGTTATTAGTAATTACTCCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO.15);

[0038] CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCCTTGTTCTGGAAGCAGCTCCAACATTGGAAGTAATTATGTATTCTGGTACCAGCACCTCCCAGGAACGGCCCCCAAGCTCCTCATCTATAGGAATAATCAGCGG CCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGACTCCAGGCTGAGGACGAGGCTGATTATTACTGCAACTCATATACAAGCAGCACCACTCTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO.16)

[0039] Preferably, the human antibody combination contains at least one of the antibodies described above.

[0040] Preferably, the antibody combination includes at least one of antibody combination 1, antibody combination 2, antibody combination 3, and antibody combination 4, wherein,

[0041] Antibody combination 1 consists of monoclonal antibody 825, monoclonal antibody 843, monoclonal antibody 826 and monoclonal antibody 846;

[0042] Antibody combination 2 consists of monoclonal antibody 825, monoclonal antibody 843 and monoclonal antibody 826;

[0043] Antibody combination 3 consists of monoclonal antibody 825, monoclonal antibody 843, and monoclonal antibody 846.

[0044] Antibody combination 4 consists of monoclonal antibody 825 and monoclonal antibody 843.

[0045] Another object of the present invention is to provide the use of the above-described human antibody or combination of the above-described human antibodies in the preparation of drugs for novel coronavirus and its variants.

[0046] Preferably, the novel coronavirus strain includes, but is not limited to, variants of the British strain, the South African strain, the Brazilian strain, and the Indian strain.

[0047] Another object of the present invention is to provide the use of the human antibody as described above in the preparation of a medicament, diagnostic reagent or kit for the prevention or treatment of novel coronavirus SARS-CoV-2 infection.

[0048] Another object of the present invention is to provide the use of the human antibody combination as described above in the preparation of a medicament, diagnostic reagent or kit for the prevention or treatment of novel coronavirus SARS-CoV-2 infection.

[0049] Preferably, the drug further includes pharmaceutical excipients.

[0050] Currently, there is a lack of effective prevention and treatment strategies for COVID-19. This invention utilizes a patented technology combining B cell culture and antibody cloning to selectively identify antibodies with highly efficient neutralizing activity targeting different epitopes of the viral antigen, including three antibodies targeting the RBD and one targeting the NTD (825, 843, 826, and 846). A single antibody (825 or 843) can effectively neutralize currently prevalent viral variants, including those from the UK, South Africa, Brazil, and India. The selected antibody combinations (825 / 843 / 826 and 825 / 843 / 846) can effectively neutralize currently prevalent viral variants, with neutralizing activity IC50 reaching approximately 10 ng / ml in a pseudovirus system. Drugs or formulations prepared from these antibody combinations can be used for passive prevention of SARS-CoV-2 infection in high-risk groups and for the treatment of COVID-19.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] (1) The present invention adopts a screening strategy of B cell culture without deviation combined with antibody cloning, which can isolate conformation-dependent functional antibodies that exist in vivo but are difficult to imitate in vitro. This antibody isolation strategy does not require antigen labeling of memory B cells, and is therefore not limited by labeled antigens. It can simultaneously screen antibodies that bind to different target proteins.

[0053] (2) Using the technology of the present invention, four neutralizing antibodies against different regions of the spike protein on the surface of SARS-CoV-2 were obtained. The pairing of the heavy chain and light chain of the antibody is naturally generated and has high affinity, making it more suitable for practical applications.

[0054] (3) The two sets of three antibody combinations screened by this invention can effectively neutralize the currently popular viral variants, including the British strain, the South African strain, the Brazilian strain and the Indian strain. Their neutralizing activity IC50 in the pseudovirus system can reach about 10 ng / ml.

[0055] (4) The human antibodies and antibody combinations that synergistically neutralize the novel coronavirus provided by the present invention can be used in the preparation of drugs, diagnostic reagents or kits for the prevention or treatment of SARS-CoV-2 infection. Attached Figure Description

[0056] Figure 1 Sequence feature map of the newly obtained antibody;

[0057] Figure 2 The diagram shows the binding results of antibodies to the viral S protein and the truncated S protein.

[0058] Figure 3 The SDS-PAGE gel image of the obtained new antibody protein was reduced.

[0059] Figure 4 Diagram showing the binding of the combined antibody to the RBD protein;

[0060] Figure 5 This is a diagram showing the binding of the combined antibody to the S1 protein.

[0061] Figure 6 Antibodies compete with each other to bind to RBD or S1 proteins;

[0062] Figure 7 The results of the IC50 assay for antibody neutralization against various mutant pseudoviruses;

[0063] Figure 8 The figure shows the results of the antibody neutralization experiment against the Lambda pseudovirus. Detailed Implementation

[0064] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments.

[0065] The flow cytometer is a Beckman Coulter MoFlo Astrios EQ ultra-high-speed flow cytometer cell sorting system, which can be purchased from Shanghai Zequan Instrument Equipment Co., Ltd.

[0066] The cDNA synthesis kit used was the SuperScript III First Strand Synthesis System, which is available from Invitrogen, USA, product number #18080051.

[0067] The fluorescently labeled antibody is a group of fluorescent antibodies, including IgD-FITC, CD19-ECD, CD27-PC7, CD38-APC A750, IgM-PB and CD45-KO fluorescent antibodies, purchased from Beckman Coulter.

[0068] The cell growth factors CpG, IL21, IL2, and PBMCs from healthy individuals who have undergone radiation exposure can be purchased from Guangzhou Haojin Biotechnology Co., Ltd.

[0069] The Phusion high-fidelity DNA polymerase is Phusion High-Fidelity PCR Master Mix with GC Buffer, available from NEB Ltd., product number #M0532s.

[0070] Example 1: Identification and sorting of memory B cells

[0071] 1.1 Isolation of peripheral blood mononuclear cells: EDTA-anticoagulated peripheral venous blood was collected from convalescent COVID-19 patients. Peripheral blood mononuclear cells were isolated using density gradient centrifugation and aliquoted into 5×10⁶ cells / ml containers. 6 / tube, placed in liquid nitrogen for cryopreservation.

[0072] 1.2 Fluorescent Antibody Staining: Peripheral blood mononuclear cells were thawed in a 37°C water bath, washed three times with PBS buffer, and then stained with antibodies. The cells were incubated at room temperature in the dark for 15 min, washed again with PBS buffer, and then resuspended in 400 μL of PBS buffer before being fed into a flow cytometer. The flow cytometer used was a Beckman Coulter MoFlo Astrios EQ ultra-high-speed flow cytometry cell sorting system. Fluorescent antibody staining was performed in nine analysis tubes. Tubes 1-7 contained the corresponding single fluorescently labeled antibody, tube 9 contained a mixture of seven fluorescently labeled antibodies, and tube 8 was a blank tube containing only cells. The sample tubes were stained in the same manner as tube 9. The fluorescent labels included IgD-FITC, CD19-ECD, CD27-PC7, CD38-APC A750, IgM-PB, and CD45-KO fluorescent antibodies.

[0073] Table 1. Information on fluorescently labeled antibody combinations in each tube for memory B cell sorting.

[0074]

[0075]

[0076] 1.3 Sorting of Memory B Cells: Sample tubes were analyzed. Live CD45-positive leukocytes were circled based on 7-AAD and CD45 levels, while B cells were circled based on CD19 levels. IgD was defined within the IgM and IgD-negative B cell population. - IgM - CD27 + CD38 low The population consisted of memory B cells, which were sorted into 96-well cell culture plates containing cell culture medium at a density of 100 cells per well.

[0077] Example 2: Culture of memory B cells and screening of antibodies from culture supernatant

[0078] Cell growth factors CpG, IL21, IL2, radiation-irradiated healthy human PBMCs, and B95.8 cell culture supernatant containing EBV were added to 96-well cell culture plates containing memory B cells and cultured for 7-10 days. The presence of antibodies against SARS-CoV-2S protein in the B cell culture supernatant was screened using a capture ELISA.

[0079] Example 3: Cloning of Antibodies

[0080] 3.1 cDNA synthesis: For B cells that were screened and found to contain antibodies against SARS-CoV-2S protein in the supernatant, RNA was extracted and then reverse transcribed into cDNA.

[0081] 3.2 Nested PCR amplification of the heavy and light chain variable regions of the antibody: For PCR primer sequences and specific PCR amplification procedures, please refer to the reference: J Immunol Methods. 2008 Jan 1; 329(1-2):112-24. The reaction was performed using Phusion high-fidelity DNA polymerase (Phusion High-Fidelity PCR Master Mix with GC Buffer, NEB, #M0532s).

[0082] 3.3 The PCR products of the heavy and light chain variable regions of the antibodies were digested with enzymes (VH, AgeI / SalI, VK, AgeI / Xhol, VL, AgeI / BsiWI) and cloned into an antibody expression vector containing the human IgG1 constant region. The sequence information of monoclonal antibodies 825, 843, 826, and 846, obtained by IMGT / V-Quest gene sequence analysis, is shown in Table 1 below.

[0083] Table 2. Sequence information of monoclonal antibodies 825, 843, 826, and 846

[0084]

[0085] Example 4: Characterization of Antibodies

[0086] 4.1 Antibody production: The heavy chain and light chain vectors of the constructed antibody with defined sequences were co-transfected into 293T cells, and the culture supernatant containing the antibody was harvested 5 days later.

[0087] 4.2 Antibody purification: The culture supernatant containing the antibody was purified using a protein A affinity column, and the protein content was determined.

[0088] 4.3 Antibody binding reaction and neutralization reaction with S protein:

[0089] The S protein in the supernatant was captured by anti-tag antibodies, and an ELISA method was established. The binding of the antibody to the S protein was analyzed by the capture ELISA method.

[0090] Biological membrane optical interferometry (BLI) was used to analyze the binding of antibodies or combination antibodies to RBD or S1; specifically, a Protein A sensor was used to sequentially capture anti-tagged antibodies and tagged RBD or S1 proteins to detect the binding of antibodies to RBD or S1.

[0091] The BLI (tandem method) technique is used to detect the competitive binding of antibodies to RBD or S1 in pairs. Specifically, the Protein A sensor is used to sequentially capture the anti-tagged antibody, the tagged RBD or S1 protein, the primary antibody, and the competing antibody. Competition is determined by a decrease or absence of detection signal.

[0092] All the labels used were D7; the biomolecular interaction analyzer used was purchased from ForteBio, model Octet K2.

[0093] Experimental results: Figure 1 The obtained monoclonal antibodies 825, 843, 826, and 846 and their sequence characteristics are shown in the image. Figure 2The diagram shows the binding of monoclonal antibodies 825, 843, 826, and 846 to the viral S protein and the truncated S protein. Experimental results show that monoclonal antibodies 825, 843, and 826 are RBD antibodies, while monoclonal antibody 846 is an NTD antibody. Monoclonal antibody 846 does not bind to RBD. Specifically, monoclonal antibody 825 binds to the S and S1 proteins with EC50 values ​​of 0.012 μg / mL and 0.039 μg / mL, respectively; monoclonal antibody 843 binds to the S and S1 proteins with EC50 values ​​of 0.002 μg / mL and 0.006 μg / mL, respectively; monoclonal antibody 826 binds to the S and S1 proteins with EC50 values ​​of 0.008 μg / mL and 0.030 μg / mL, respectively; and monoclonal antibody 846 binds to the S and S1 proteins with EC50 values ​​of 0.136 μg / mL and 0.049 μg / mL, respectively.

[0094] like Figure 3 As shown, the reduced SDS-PAGE gel image reveals the heavy and light chain sizes of monoclonal antibodies 825, 843, 826, and 846.

[0095] like Figure 4 and 5 As shown, the antibody combination of 825 / 843 / 826 or the three antibodies 825 / 843 / 846 can bind to the RBD or S1 protein simultaneously.

[0096] like Figure 6 The results showed that in the antibody competition experiments for binding to RBD or S1 protein, antibodies 825, 843 and 826 bound to different epitopes on RBD.

[0097] 4.4 Antibody activity in neutralizing SARS-CoV-2 pseudovirus

[0098] Establishment of pseudovirus neutralization system: Pseudoviruses were packaged by co-transfecting Expi293F with an expression plasmid containing the SARS-CoV-2 spike protein S gene and pPAX2 / pLV-EGFP (1:1).

[0099] Micro-neutralization assay with pseudovirus: 50 μL of sample was aspirated, and 800 TCID50 pseudovirus was added. After incubation for 1 h, 50,000 HEK293T cells were added to each well. The cells were cultured for another 48 h before plate testing. Neutralizing activity was defined as a decrease in fluorescence value, i.e., an inhibition rate greater than 50%.

[0100] Experimental results: such as Figure 7 As shown, based on the IC50 results of antibody neutralization experiments against various mutated pseudoviruses, antibodies 825, 843, 846, and 826, as well as antibody combinations 825 / 843, 825 / 843 / 826, and 825 / 843 / 846, can effectively neutralize various mutated pseudoviruses of COVID-19; for example... Figure 8The image shown is a graph illustrating the results of an antibody neutralization experiment against Lambda pseudovirus. Figure 8 A and Figure 8 As shown in B, the IC50 values ​​of antibodies 825, 843, 826, and 846 for neutralizing the SARS-CoV-2 variant Lambda strain were 0.004 μg / mL, 0.011 μg / mL, 0.4538 μg / mL, and >10 μg / mL, respectively. The IC50 values ​​of antibody combinations 825 / 843 / 826 and 825 / 843 / 846 were both <0.0004 μg / mL. Therefore, antibodies 825, 843, 846, and 826, as well as their combinations 825 / 843 / 826 and 825 / 843 / 846, can all effectively neutralize the Lambda strain.

[0101] Therefore, the antibodies 825, 843, 846, and 826 provided by this invention, as well as the antibody combinations 825 / 843 / 826 and 825 / 843 / 846, can effectively neutralize the activity of the SARS-CoV-2 pseudovirus. Moreover, with the same amount of antibodies, the antibody combinations 825 / 843 / 826 and 825 / 843 / 846 achieve a better neutralizing effect than using antibodies 825, 843, 826, and 846 alone.

[0102] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A human antibody against the novel coronavirus, characterized in that, The human antibody is 825 monoclonal antibody, 843 monoclonal antibody, 826 monoclonal antibody or 846 monoclonal antibody; The 825 monoclonal antibody contains a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region of the 825 monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2. The 843 monoclonal antibody contains a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region of the 843 monoclonal antibody is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4. The 826 monoclonal antibody contains a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region of the 826 monoclonal antibody is shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

6. The 846 monoclonal antibody contains a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region of the 846 monoclonal antibody is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

2. The human antibody according to claim 1, characterized in that, The nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the monoclonal antibody 825 is shown in SEQ ID NO.9, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region of the monoclonal antibody 825 is shown in SEQ ID NO.

10. The nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the monoclonal antibody 843 is shown in SEQ ID NO.11, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region of the monoclonal antibody 843 is shown in SEQ ID NO.

12. The nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the monoclonal antibody 826 is shown in SEQ ID NO.13, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region of the monoclonal antibody 826 is shown in SEQ ID NO.

14. The nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the antibody 846 monoclonal antibody is shown in SEQ ID NO.15, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region of the antibody 846 monoclonal antibody is shown in SEQ ID NO.

16.

3. A combination of human antibodies that synergistically neutralize the novel coronavirus, characterized in that, The human antibody combination contains at least one of the antibodies according to any one of claims 1-2.

4. The human antibody combination according to claim 3, characterized in that, The antibody combination includes at least one of antibody combination 1, antibody combination 2, antibody combination 3, and antibody combination 4, wherein, Antibody combination 1 consists of monoclonal antibody 825, monoclonal antibody 843, monoclonal antibody 826 and monoclonal antibody 846; Antibody combination 2 consists of monoclonal antibody 825, monoclonal antibody 843 and monoclonal antibody 826; Antibody combination 3 consists of monoclonal antibody 825, monoclonal antibody 843, and monoclonal antibody 846. Antibody combination 4 consists of monoclonal antibody 825 and monoclonal antibody 843.

5. The use of a human antibody as described in any one of claims 1-2 or a combination of human antibodies as described in any one of claims 3-4 in the preparation of drugs against novel coronaviruses and their variants, characterized in that, The novel coronavirus strains and their variants include PsV-D614G, PsV-alpha, PsV-beta, PsV-gamma, PsV-gamma, PsV-kappa, and PsV-lambda.

6. The use of a human antibody as described in any one of claims 1-2 in the preparation of a medicament, diagnostic reagent, or kit for the prevention or treatment of novel coronavirus SARS-CoV-2 infection.

7. The use of a human antibody combination as described in any one of claims 3-4 in the preparation of a medicament, diagnostic reagent, or kit for the prevention or treatment of novel coronavirus SARS-CoV-2 infection.

8. The application according to any one of claims 5-7, characterized in that, The drug also includes pharmaceutical excipients.

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