Human anti-sars-cov-2 broadly neutralizing monoclonal antibodies and uses thereof

Abstract

The application discloses a humanized anti-SARS-COV-2 virus monoclonal neutralizing antibody, the neutralizing antibody is screened by single B cell flow sorting-antibody gene amplification pairing expression technology, has a unique CDR region, can be specifically combined with SARS-COV-2 and can effectively neutralize SARS-COV-2 wild strains and Omicron BA.5, Omicron XBB.1.5, Omicron EG.5, Omicron JN.1, Omicron KP.2 and other Omicron mutant strains, the IC50 is lower than 0.1 μg / mL, has broad-spectrum virus neutralization activity, and the application also relates to application of the antibody in preparation of a new coronavirus disease treatment or prevention drug and a detection reagent, the antibody has the characteristics of full humanization, high expression and good stability, and is suitable for industrialization.

Description

TECHNICAL FIELD

[0001] The present invention discloses a polypeptide, more specifically, the present invention discloses an antibody. BACKGROUND

[0002] The novel coronavirus (SARS-CoV-2, also known as 2019-nCoV) belongs to a positive-strand RNA virus, belonging to the ß genus of the coronavirus family, which encodes four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), 16 non-structural proteins, and 5-8 auxiliary proteins. SARS-CoV-2 enters the cell by using the S protein on the surface of the virus to bind to the host cell receptor-angiotensin-converting enzyme II (ACE2). The S protein is divided into two functional units according to the protein structure function, namely S1 and S2 protein subunits. S1 can be divided into NTD (N-terminal domain) and RBD (Receptor binding site), the RBD region is about 240 amino acids long, mainly binds to the host cell receptor, and S2 plays a role in the fusion of the virus and the cell membrane. According to existing reports, neutralizing antibodies mainly act on the RBD region, and the antibody binds to the RBD to hinder the binding of the RBD to ACE2, thereby preventing the virus from infecting the cell.

[0003] Currently, there are reports of the isolation of neutralizing antibodies against the novel coronavirus both domestically and internationally. Using single-cell sorting and deep antibody genome sequencing, a number of human monoclonal antibodies targeting RBD have been isolated, including LYCoV555, REGN-COV2, TY027, CT-P59, JS016, BRII-196, BRII-198, and SCTA01. These antibodies have demonstrated strong neutralizing activity in vitro and have shown promising therapeutic effects in transgenic mice expressing ACE2, significantly reducing viral load in the lungs. However, SARS-CoV-2 is constantly mutating, and once infected with a virus with a mutated neutralizing epitope, existing neutralizing antibodies will no longer be effective. Especially since the rapid prevalence of the Omicron strain, variants such as OmicronBA.5, OmicronXBB.1.5, OmicronEG.5, OmicronJN.1, and OmicronKP.2 have been produced, among which OmicronJN.1 is the current main prevalent strain in my country, and OmicronKP.2 was listed as a "variant that needs to be monitored" by the World Health Organization on May 3, 2024. The neutralizing ability of existing monoclonal antibodies against Omicron variants has been greatly reduced, or even completely ineffective. Therefore, it is necessary to isolate more potent and broad-spectrum neutralizing antibodies as alternatives to more effectively avoid immune escape of the virus. The purpose of the present invention is to provide an anti-new coronavirus monoclonal antibody with a broad-spectrum high-neutralizing activity, and on this basis, to provide the application of the high-neutralizing activity anti-new coronavirus monoclonal antibody in the preparation of new coronavirus infection therapeutic drugs and diagnostic kits. Summary of the Invention

[0004] Based on the above invention objectives, the present invention first provides a humanized broadly neutralizing monoclonal antibody against the novel coronavirus, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region and CDR1, CDR2 and CDR3 of the light chain variable region of the antibody are as follows:

[0005] (1) Positions 26-35, 53-59 and 98-112 of SEQ ID NO.1 and positions 26-34, 52-54 and 91-103 of SEQ ID NO.3; or

[0006] (2) positions 26-33, 51-57 and 96-106 of SEQ ID NO. 5 and positions 27-32, 50-52 and 89-96 of SEQ ID NO. 7; or

[0007] (3) Positions 26-35, 53-59 and 98-111 of SEQ ID NO. 9 and positions 27-32, 50-52 and 89-95 of SEQ ID NO. 11.

[0008] In a preferred embodiment, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are respectively as follows:

[0009] (1) SEQ ID NO.1 and SEQ ID NO.3. The antibody having this technical solution is named “LY-2” in the present invention; or

[0010] (2) SEQ ID NO.5 and SEQ ID NO.7. The antibody having this technical solution is named “LY-3” in the present invention; or

[0011] (3) SEQ ID NO. 9 and SEQ ID NO. 11. In the present invention, an antibody having a specific technical solution of the amino acid sequence of the heavy chain variable region and the light chain variable region is named "LY-7".

[0012] In a more preferred embodiment, the amino acid sequence of the heavy chain constant region of the antibody is shown as SEQ ID NO. 13, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO. 15 (kappa chain) or SEQ ID NO. 17 (lambda chain).

[0013] Secondly, the present invention also provides a polynucleotide encoding the above-mentioned human anti-novel coronavirus broadly neutralizing monoclonal antibody. The polynucleotide encoding the heavy chain variable region of the antibody and the polynucleotide encoding the light chain variable region of the antibody are shown below:

[0014] (1) SEQ ID NO. 2 and SEQ ID NO. 4. The antibody having this technical solution is named "LY-2" in the present invention; or

[0015] (2) SEQ ID NO. 6 and SEQ ID NO. 8. The antibody having this technical solution is named “LY-3” in the present invention; or

[0016] (3) SEQ ID NO. 10 and SEQ ID NO. 12. The antibody having this technical solution is named "LY-7" in the present invention.

[0017] In a preferred embodiment, the polynucleotide sequence encoding the heavy chain constant region of the antibody is shown as SEQ ID NO. 14, and the polynucleotide sequence encoding the light chain constant region of the antibody is shown as SEQ ID NO. 16 (kappa chain) or SEQ ID NO. 18 (lambda chain).

[0018] Third, the present invention also provides an expression vector containing a polynucleotide encoding the above-mentioned human broad-spectrum, high-neutralizing activity anti-novel coronavirus monoclonal antibody, wherein the expression vector contains the above-mentioned polynucleotide encoding the heavy chain variable region of the antibody and the polynucleotide encoding the light chain variable region of the antibody. The vector can be a eukaryotic expression vector conventionally used in genetic engineering. In a specific embodiment of the present invention, the vector is IgH (heavy chain expression vector), Igκ (κ light chain expression vector), and Igλ (λ light chain expression vector) (see Tiller et al. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning, J Immunol Methods. 2008 January 1; 329(1-2): 112–124., the present invention incorporates this prior art document into the description of the present invention by reference).

[0019] Fourth, the present invention provides a host cell expressing the above-mentioned human broad-spectrum, high-neutralizing activity anti-novel coronavirus monoclonal antibody, wherein the host cell contains the above-mentioned expression vector.

[0020] The host cell may be a eukaryotic host cell conventionally used in genetic engineering. In a specific embodiment of the present invention, the host cell is a 293F cell. Other conventional eukaryotic host cells in the art, such as CHO cells, may also be used in the practice of the present invention.

[0021] Fifth, the present invention also provides an antibody composition, comprising a first antibody and a second antibody, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the first antibody are as shown in SEQ ID NO. 1 and SEQ ID NO. 3, respectively, and the amino acid sequences of the heavy chain variable region and the light chain variable region of the second antibody are as shown in:

[0022] (1) SEQ ID NO. 5 and SEQ ID NO. 7; or

[0023] (2) SEQ ID NO. 9 and SEQ ID NO. 11.

[0024] In another alternative embodiment, the composition contains a first antibody and a second antibody, the amino acid sequences of the heavy chain variable region and the light chain variable region of the first antibody are shown in SEQ ID NO. 5 and SEQ ID NO. 7 respectively, and the amino acid sequences of the heavy chain variable region and the light chain variable region of the second antibody are shown in SEQ ID NO. 9 and SEQ ID NO. 11 respectively.

[0025] Sixth, the present application provides the application of the above-mentioned humanized broad-spectrum high neutralization activity anti-SARS-CoV-2 monoclonal antibody in the preparation of SARS-CoV-2 treatment and / or prevention drugs, and the antibody can be prepared into a clinical treatment drug related to SARS-CoV-2 infection.

[0026] Finally, the present application provides the application of the above-mentioned humanized broad-spectrum high neutralization activity anti-SARS-CoV-2 monoclonal antibody in the preparation of SARS-CoV-2 detection reagents. The SARS-CoV-2 detection reagent is an immunological detection reagent based on the specific binding reaction of antigen and antibody, including but not limited to enzyme-linked immunoassay, chemiluminescence immunoassay, radioimmunoassay, and the detection sample is serum, secretion, excretion, biopsy specimen, etc. in vitro, or in situ detection.

[0027] The humanized broad-spectrum high neutralization activity anti-SARS-CoV-2 monoclonal antibody provided by the present application is obtained by screening through single B cell flow sorting-antibody gene amplification paired expression technology, has a unique CDR region, can specifically bind to SARS-CoV-2 and effectively neutralize the currently popular multiple SARS-CoV-2 mutant strains, including: SARS-CoV-2 WT, Omicron BA.5, Omicron XBB.1.5, Omicron EG.5, Omicron JN.1, and Omicron KP.2. The IC50 of the antibodies LY-3 and LY-7 to the above strains in the pseudo virus neutralization test is less than 0.07 μg / mL; the IC50 of the antibody LY-2 to the pseudo virus other than the above SARS-CoV-2 WT strain is less than 0.05 μg / mL, and the IC50 to the pseudo virus of SARS-CoV-2 WT is greater than 1.1 μg / mL. The above antibodies have significant broad-spectrum neutralization ability to multiple different SARS-CoV-2 representative strains in the world, and therefore the antibody provided by the present application can be used for preparing COVID-19 emergency prevention and / or treatment drugs, has the characteristics of full humanization, high expression, and good stability, and is suitable for industrialization. In addition, the antibody can also be used for preparing SARS-CoV-2 virus detection reagents for detecting viral antigens and for discovering effective neutralizing antigen epitopes. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1. Schematic diagram of flow cytometry sorting of novel coronavirus RBD-specific B cells;

[0029] Figure 2 . Result diagram of the neutralizing ability of antibodies against pseudoviruses. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0031] Example 1: Preparation of novel coronavirus RBD probe

[0032] 1.1 Biotinylated 2019-nCoV RBD proteins were purchased from ACROBiosystems Group with the following catalog numbers: XBB.1.5 RBD (Cat. No.: SPD-C82Q3), EG.5 RBD (Cat. No.: SPD-C82Q5).

[0033] 1.2 Fluorescent labeling of biotinylated novel coronavirus RBD protein

[0034] The XBB.1.5 RBD probe protein was fluorescently labeled with PE (phycoerythrin), and the EG.5 RBD probe protein was fluorescently labeled with APC (allophycocyanin) for use in single-cell flow cytometry sorting.

[0035] Example 2: Screening and identification of human anti-SARS-CoV-2 broadly neutralizing monoclonal antibodies

[0036] 2.1 Prepare cell lysis buffer: prepare 20 μl cell lysis buffer per well, including 0.5 μl RNase-free buffer, 5 μl 5× First Strand buffer, 1.25 μl 0.1 M DTT, 0.0625 μl Igepal, and 13.1875 μl water. Cover with sealing film and store in a refrigerator at 4°C until use.

[0037] 2.2 Sample preparation:

[0038] (1) PBMCs cell recovery of convalescent patients with novel coronavirus infection: After taking the frozen cell tube out of liquid nitrogen, it is quickly placed in a 37°C water bath, and when it is melted to have an ice core, it is taken out and opened in a biological safety cabinet. Slowly drop into RPMI-1640 (10% FBS) culture medium containing Benzonase (5 mL of culture medium for 1 cell). Centrifuge at 1500 rpm for 10 minutes, discard the supernatant, suspend the cells with the residual liquid, add 10 mL of the above-mentioned culture medium, mix well, take 50 μl for cell counting, centrifuge at 1500 rpm for 10 minutes; adjust the cell concentration: discard the supernatant, suspend the cells with the residual liquid, adjust the cell concentration with the above-mentioned culture medium, add 2.5 x 10 6 cells / well to a 96-well U-shaped plate;

[0039] (2) Prepare a 2 mM EDTA / PBS solution, denoted as E-PBS hereinafter;

[0040] (3) Place the 96-well cell plate in the centrifuge, centrifuge at 2000 rpm for 3 minutes at 4°C, discard the supernatant (operate in a biological safety cabinet, paper towel high-pressure table);

[0041] (4) Add 50 μl of Vivid working solution to each well (prepare Vivid (UV) working solution: 1:1000 dilution with PBS, mix well), mix well, avoid light, and incubate on ice for 20 minutes;

[0042] (5) Add 150 μl of E-PBS to each well, centrifuge at 2000 rpm for 3 minutes at 4°C;

[0043] (6) Discard the supernatant, add 50 μl of extracellular antibody mixture (2 μl CD3-Pacific Blue, 2 μl CD8-Pacific Blue, 1.5 μl CD14-Pacific Blue, 1 μl CD19-BV510, 2 μl CD20-ECD, 2.5 μl CD27-APCCy7, 5 μl IgG-FITC, 2.5 μl IgM-PercpCy5.5, 2.5 μl PD-1-PECy7, 1 μl CXCR5-APC-R700, 5 μl CXCR3-PECy5, 1 μl CD45RA-BV650, 1 μl CD4-BV605, 5 μl XBB-RBD-PE, 5 μl EG.5-RBD-APC, 11 μl E-PBS), mix well, avoid light, and incubate on ice for 60 minutes;

[0044] (7) Add 150 μL E-PBS to each well, centrifuge at 2000 rpm for 3 minutes at 4°C, discard the supernatant;

[0045] (8) Add 200 μL of E-PBS to each well, centrifuge at 2000 rpm for 3 minutes at 4°C, and discard the supernatant;

[0046] (9) Add 200 μL of E-PBS to each well, mix well, filter the cell suspension of the same sample into the same flow-through tube, and store at 4°C in the dark for sorting;

[0047] (10) Single-stained tube control: set up 1 unstained tube control (200 μl E-PBS with 1 drop of compensation microspheres) and 15 single-stained tubes (1 drop of compensation microspheres was added to each tube, and 2 μl CD3-Pacific Blue, 2 μl CD8-Pacific Blue, 1.5 μl CD14-Pacific Blue, 1 μl CD19-BV510, 2 μl CD20-ECD, 2.5 μl CD27-APCCy7, 5 μl IgG-FITC, 2.5 μl IgM-PercpCy5.5, 2.5 μl PD-1-PECy7, 1 μl CXCR5-APC-R700, 5 μl CXCR3-PECy5, 1 μl CD45RA-BV650, 1 μl CD4-BV605, 50 μl UV working solution were added respectively), mix well, incubate on ice in the dark for 20 minutes, and then add 150 μl to each well. Centrifuge at 2000 rpm for 3 minutes at 4°C in E-PBS, discard the supernatant, and resuspend in 200 μl E-PBS.

[0048] 2.3 Single B cell flow cytometry sorting: selection of CD3 - CD8 - CD14 - CD19 + CD20 + CD27 + IgG + IgM - RBD + The cells were sorted, and a total of 254 cells were sorted. First, single lymphocytes were identified, and then CD3 - Live cells of CD8-CD14- were used to exclude T cells and macrophages, and CD19 + CD20 + B cells, and then identify CD27 + Memory B cells, then circle IgG + IgM -B cells, and finally circle the memory B cells that bind to the probe RBD. These B cells are sorted into 96-well plates containing the following lysis system (Table 1) at 1 per well. After sorting, immediately seal the 96-well plates with sealing film, freeze on dry ice, and transfer to a -80°C refrigerator overnight, and then perform PCR the next day. Results: The results of flow sorting are shown in Fig. Figure 1

[0049] Table 1. B cell lysis system

[0050]

[0051] 2.4 Amplification of human antibody variable region genes using RT-PCR technology

[0052] The RT-PCR reaction system is configured as shown in Table 2, and 6 μl is added per well to perform the RT-PCR reaction.

[0053] Table 2. RT-PCR reaction system

[0054]

[0055] The RT-PCR reaction program is set as follows: 42°C for 10 min, 25°C for 10 min, 50°C for 50 min, 94°C for 5 min, and 25 μl of water is added per well at the end of the reaction, and it is stored at 4°C.

[0056] Two rounds of PCR reactions are performed, and the reaction technical parameters are as follows.

[0057] Table 3. First round of PCR reaction system

[0058]

[0059] Table 4. Primer sequences for first round of PCR amplification

[0060]

[0061] Table 5. Amplification target fragments of the first round of PCR amplification primers

[0062]

[0063] Table 6. First round of PCR program

[0064]

[0065] After the first round of PCR reaction, the second round of PCR reaction is performed, and the second round of PCR reaction technical parameters are as follows.

[0066] ​Table 7. Second round PCR reaction system

[0067]

[0068] Table 8. Second round PCR primer sequences

[0069]

[0070] Table 9. Target fragments amplified by the second round of PCR primers

[0071]

[0072] Table 10. Second round PCR program

[0073]

[0074] (2) Electrophoresis, sequencing, and pedigree analysis

[0075] The second-round PCR products were detected by electrophoresis, and the heavy and light chain products were directly sequenced. The sequencing results were analyzed using the Antibody Family Gene Database (http: / / www.imgt.org / IMGT_vquest / vquest), and antibody variable region sequences with enzyme cleavage sites (heavy chain: 5′-Age I, 3′-Sal I; kappa chain: 5′-Age I, 3′-BsiW I; lambda chain: 5′-Age I, 3′-Xho I) were designed and synthesized. A total of 78 pairs of heavy and light chain paired clones were obtained.

[0076] 2.5 Monoclonal Antibody Expression Vector Construction and Plasmid Transformation

[0077] The synthesized gene was recovered after enzyme digestion with the corresponding enzyme and gel electrophoresis, and the variable region gene was connected with the corresponding vector IgH (heavy chain expression vector), IgK (Kappa light chain expression vector), IgL (lambda light chain expression vector) (for details, see Tiller et al. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning, J Immunol Methods. 2008 January 1; 329(1-2): 112-124. The prior art document is incorporated into the specification of the present application by reference) using T4 DNA ligase on a 16°C ligation instrument overnight. Then plasmid transformation was performed, 10 μL of the ligation product was added to 50 μL of DH5α competent cells, shaken and mixed, and then placed in an ice bath for 30 minutes, and then heated at 42°C for 45 seconds. After placing the centrifuge tube in an ice bath for 2 minutes, 1 mL of antibiotic-free LB medium was added, and the culture was incubated at 37°C and 200 rpm for 1 hour, and then centrifuged at 4000 rpm for 4 minutes. The residual bacterial solution was spread on the corresponding antibiotic-resistant LB plate. Incubate at 37°C for 14-16 hours. Single colonies were picked and inoculated into the corresponding antibiotic-resistant LB liquid medium, and then incubated at 37°C and 200 rpm for 14-16 hours before plasmid extraction (Plasmid Midi Kit from Omega).

[0078] 2.6 Antibody expression and purification

[0079] The concentration of 293F cells was adjusted to 1.2×10 6 6 / mL, and incubated for 2 hours. Solution A: 2.5 mL of opti-MEM was added with 50 μg of antibody heavy chain DNA and 50 μg of antibody light chain DNA, and solution B: 2.5 mL of opti-MEM was added with 0.5 mL of PEI transfection reagent, and left to stand for 5 minutes. Solutions A and B were mixed and left to stand for 20 minutes, and then added dropwise to 100 mL of 293F cell suspension while shaking, and then incubated at 8% CO2, 37°C, and 200 rpm for 5 days.

[0080] The antibody was purified using a Protein A affinity column (GE health product). The antibody concentration was determined using a NanoDrop2000 ultramicro spectrophotometer (Thermo product), and the sample was stored at 4°C before detection. Of the 16 selected light / heavy chain paired antibodies, 14 antibodies were expressed (antibody amount >1 mg, antibody concentration >1000 μg / mL).

[0081] Example 3. Assay of neutralizing activity of antibodies on SARS-CoV-2 pseudovirus

[0082] 3.1 Packaging of pseudovirus: The full-length gene of S protein of SARS-CoV-2 (GenBank: MN908947) was artificially synthesized and inserted into pcDNA3.1 expression plasmid to construct pcDNA-SARS-CoV-2-S. The mutation sites of pseudovirus with mutations are shown in Table 11, which are introduced on the S gene.

[0083] Table 11. Mutation sites of pseudovirus

[0084]

[0085] 3 x 10 6 (3 million) 293T / 17 cells were inoculated in a T75 cell culture flask and incubated at 37°C, 5% CO2 for 20-24 hours. Transfection was performed using Fugene 6 Transfection Reagent (Promega, Cat#E2691): 30 μg of plasmid pcDNA-SARS-CoV-2-S was transfected into 293T cells in a T75 culture flask, and 1.05 x 10 6 TCID50 of G*ΔG-VSV virus was used to infect 293T, and the medium was changed after 8 hours. After 24 hours of transfection, the culture supernatant was collected and filtered to obtain the pseudovirus of SARS-CoV-2 S protein, which was stored at -80°C.

[0086] 3.2 Neutralization experiment:

[0087] In a 96-well plate, 100 μL of gradient-diluted antibody diluent was added to each well, followed by dilution of the pseudovirus to 1.3 x 10 4 TCID50 / mL, and 50 μL was added to each well in columns 3-11, so that the amount of pseudovirus added was 650 TCID50 / well. The above 96-well plate was incubated in a cell incubator (37°C, 5% CO2) for 1 hour. When the incubation time was half an hour, the prepared Vero cells were taken out, the culture medium was aspirated, the cells were washed with PBS buffer, the PBS was discarded, and the cells were trypsin-EDTA digested and centrifuged, then resuspended in complete culture medium, and the cells were counted. The cell suspension was diluted to 2 x 10 5 TCID50 / mL. After 1 hour of incubation, 100 μL of cells were added to each well of the 96-well plate, so that the Vero cells in each well were 2 x 10 4Gently rock the 96-well plate back and forth and side to side to evenly distribute the cells throughout the wells. Place the 96-well plate in a cell culture incubator and incubate at 37°C, 5% CO2 for 28 hours. Remove the 96-well plate from the cell culture incubator and use a multichannel pipette to aspirate 150 μL of supernatant from each well. Then add 100 μL of luciferase assay reagent and incubate at room temperature in the dark for 2 minutes. After the reaction is complete, shake on a plate shaker to mix thoroughly and read the luminescence value on a multi-function plate reader. Calculate the neutralization inhibition rate: Based on the neutralization inhibition rate results, calculate the IC50 of the antibody.

[0088]

[0089] From the tested antibodies, we screened out 3 antibodies with high neutralizing ability against the new coronavirus WT, OmicronBA.5, OmicronXBB.1.5, OmicronEG.5, OmicronJN.1, and OmicronKP.2, namely LY-2, LY-3, and LY-7. The detailed neutralization results of the monoclonal antibodies are shown in Figure 2 Antibodies LY-3 and LY-7 had IC50 values ​​for neutralization of pseudoviruses of the aforementioned strains of less than 0.07 μg / mL, and less than 0.01 μg / mL for neutralization of OmicronKP.2 pseudovirus, demonstrating strong neutralizing activity against multiple variants of the novel coronavirus. Antibody LY-2 had IC50 values ​​for neutralization of pseudoviruses other than the aforementioned SARS-CoV-2 WT strain of less than 0.05 μg / mL, and its IC50 for neutralization of pseudoviruses of SARS-CoV-2 WT was greater than 1.1 μg / mL. These antibodies exhibit significant broad-spectrum neutralizing activity against multiple representative strains of the novel coronavirus worldwide.

[0090] Example 4: Antigen-antibody affinity kinetics determination

[0091] The affinity of monoclonal antibodies was determined using BLI technology using the Octet Red 96 system (Fortebio, USA) and a streptavidin biosensor. The biotinylated SARS-CoV-2 RBD proteins used were purchased from ACROBiosystems Group under the following catalog numbers: WT RBD (Cat. No.: SPD-C82E9), XBB.1.5 RBD (Cat. No.: SPD-C82Q3), and EG.5 RBD (Cat. No.: SPD-C82Q5). The sequence of the homemade JN.1 RBD (319-537) is shown in Table 11. The biotinylated SARS-CoV-2 RBD was diluted to a concentration of 2 μg / mL in PBST (PBS containing 0.02% Tween 20 and 0.1% bovine serum albumin) and then immobilized on a streptavidin biosensor (Sartorius AG, Germany) for 60 seconds. After a 60-second wash step with PBST, the biosensor probe was immersed in wells containing serially diluted antibodies (200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM) and allowed to bind for 120 seconds, followed by a 300-second dissociation step. KD values ​​were calculated using a 1:1 binding model in Data Analysis Software 12.2. The affinity constants (KD) for the three monoclonal antibodies to the SARS-CoV-2 RBD are shown in Table 12. Most KD values ​​were <0.001 nM (0.001 nM is the lower limit of affinity determined by BLI technology), and all KD values ​​were <1.6 nM, indicating that these three antibodies have extremely strong affinity for the SARS-CoV-2 RBD.

[0092] Table 12. Antibody affinity

[0093]

[0094] The present invention screened three novel coronavirus monoclonal antibodies, sequenced the gene amplification products of their heavy and light chains, and obtained the gene coding sequences and amino acid sequences of the heavy and light chains of the antibodies as follows:

[0095] The nucleotide sequence of the heavy chain variable region of the LY-2 antibody is shown in SEQ ID NO.2, the amino acid sequence is shown in SEQ ID NO.1, and the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in amino acids 26-35, 53-59, and 98-112 of SEQ ID NO.1; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.4, the amino acid sequence is shown in SEQ ID NO.3, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown in amino acids 26-34, 52-54, and 91-103 of SEQ ID NO.3. The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.13, and the nucleic acid sequence of the heavy chain constant region is shown in SEQ ID NO.14. The light chain of the LY-2 antibody is a lambda chain, the amino acid sequence is shown in SEQ ID NO.17, and the nucleic acid sequence of the light chain constant region is shown in SEQ ID NO.18.

[0096] The nucleotide sequence of the heavy chain variable region of the LY-3 antibody is shown in SEQ ID NO.6, the amino acid sequence is shown in SEQ ID NO.5, and the amino acid sequences of the CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in amino acids 26-33, 51-57, and 96-106 of SEQ ID NO.5. The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.8, the amino acid sequence is shown in SEQ ID NO.7, and the amino acid sequences of the CDR1, CDR2, and CDR3 of the light chain variable region are shown in amino acids 27-32, 50-52, and 89-96 of SEQ ID NO.7. The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.13, and the nucleic acid sequence of the heavy chain constant region is shown in SEQ ID NO.14. The light chain of the LY-3 antibody is a kappa chain, the amino acid sequence is shown in SEQ ID NO.15, and the nucleic acid sequence of the light chain constant region is shown in SEQ ID NO.16.

[0097] The nucleotide sequence of the heavy chain variable region of LY-7 is shown in SEQ ID NO.10, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.9, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are shown as amino acids 26-35, 53-59, and 98-111 of SEQ ID NO.9, respectively. The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.12, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.11, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown as amino acids 27-32, 50-52, and 89-95 of SEQ ID NO.11, respectively. The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.13, and the nucleic acid sequence of the heavy chain constant region is shown in SEQ ID NO.14. The light chain of the LY-7 antibody is a kappa chain, and the amino acid sequence is shown in SEQ ID NO.15, and the nucleic acid sequence of the light chain constant region is shown in SEQ ID NO.16.

Claims

1. A humanized anti-novel coronavirus monoclonal antibody, characterized in that: The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region and CDR1, CDR2 and CDR3 of the light chain variable region of the antibody are shown below: (1) Positions 26-35, 53-59 and 98-112 of SEQ ID NO.1 and positions 26-34, 52-54 and 91-103 of SEQ ID NO.3; or (2) positions 26-33, 51-57 and 96-106 of SEQ ID NO. 5 and positions 27-32, 50-52 and 89-96 of SEQ ID NO. 7; or (3) Positions 26-35, 53-59 and 98-111 of SEQ ID NO. 9 and positions 27-32, 50-52 and 89-95 of SEQ ID NO.

11.

2. The human anti-novel coronavirus monoclonal antibody according to claim 1, characterized in that The amino acid sequences of the heavy chain variable region and light chain variable region of the human anti-novel coronavirus monoclonal antibody are as follows: (1) SEQ ID NO. 1 and SEQ ID NO. 3; or (2) SEQ ID NO. 5 and SEQ ID NO. 7; or (3) SEQ ID NO. 9 and SEQ ID NO.

11.

3. The human anti-novel coronavirus monoclonal antibody according to claim 2, characterized in that The amino acid sequence of the heavy chain constant region of the human anti-novel coronavirus monoclonal antibody is shown in SEQ ID NO.13, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.15 or SEQ ID NO.

17.

4. A polynucleotide encoding the human anti-novel coronavirus monoclonal antibody according to claim 2 or 3, characterized in that: The polynucleotide sequences encoding the heavy chain variable region and the polynucleotide sequences encoding the light chain variable region of the human anti-novel coronavirus monoclonal antibody are as follows: (1) SEQ ID NO. 2 and SEQ ID NO. 4; or (2) SEQ ID NO. 6 and SEQ ID NO. 8; or (3) SEQ ID NO. 10 and SEQ ID NO.

12.

5. The polynucleotide encoding the human anti-novel coronavirus monoclonal antibody according to claim 4, characterized in that The polynucleotide sequence encoding the heavy chain constant region of the human anti-novel coronavirus monoclonal antibody is shown in SEQ ID NO.14, and the polynucleotide sequence encoding the light chain constant region is shown in SEQ ID NO.16 or SEQ ID NO.

18.

6. An expression vector containing a polynucleotide encoding the human anti-novel coronavirus monoclonal antibody according to claim 2 or 3, characterized in that: The expression vector contains the polynucleotide encoding the heavy chain variable region of the human anti-novel coronavirus monoclonal antibody as described in claim 4 and the polynucleotide encoding the light chain variable region of the human anti-novel coronavirus monoclonal antibody.

7. A host cell containing the expression vector according to claim 6.

8. The host cell according to claim 7, characterized in that The host cell is 293F cell.

9. An antibody composition, characterized in that The composition comprises a first antibody and a second antibody, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the first antibody are shown as SEQ ID NO.1 and SEQ ID NO.3, respectively, and the amino acid sequences of the heavy chain variable region and the light chain variable region of the second antibody are shown as follows: (1) SEQ ID NO. 5 and SEQ ID NO. 7; or (2) SEQ ID NO. 9 and SEQ ID NO.

11.

10. An antibody composition, characterized in that The composition contains a first antibody and a second antibody, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the first antibody are shown as SEQ ID NO.5 and SEQ ID NO.7, respectively, and the amino acid sequences of the heavy chain variable region and the light chain variable region of the second antibody are shown as SEQ ID NO.9 and SEQ ID NO.11, respectively.

11. Use of the human anti-novel coronavirus monoclonal antibody according to any one of claims 1 to 3 in the preparation of a drug for the treatment and / or prevention of novel coronavirus disease.

12. Use of the human anti-novel coronavirus monoclonal antibody according to any one of claims 1 to 3 in the preparation of a novel coronavirus detection reagent.

Images