Anti-sars-cov-2 fully human monoclonal antibody 6g18 and preparation method and application thereof

Abstract

The present application relates to anti-SARS-COV-2 antibodies or antigen-binding fragments thereof and applications thereof, and specifically discloses the heavy chain and light chain complementarity determining regions and the heavy chain and light chain variable region sequences of the antibody 6G18 against SARS-COV-2. The antibody of the present application is a humanized antibody, which has low side effects, high affinity and high specificity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunology, and particularly relates to anti-SARS-COV-2 (COVID-19) fully human monoclonal antibodies and a preparation method and application thereof. BACKGROUND

[0002] Among the top ten best-selling drugs in the world in 2018, eight are fully human or humanized monoclonal antibody drugs. The first is Humira, an anti-TNFa monoclonal antibody of Abbott Company for treating arthritis, which is a fully human monoclonal antibody and has been the king of drugs with sales of more than 10 billion for six consecutive years. Since the first monoclonal antibody drug was launched in 1986, monoclonal antibody drugs have gone through stages of murine monoclonal antibody drugs (such as Orthclone OKT3), chimeric monoclonal antibody drugs (Rituximab), humanized monoclonal antibody drugs (Herceptin) and fully human monoclonal antibody drugs (Humira). Due to the occurrence of anti-mouse antibody reaction (HAMA) in the human body, murine monoclonal antibody drugs and chimeric monoclonal antibody drugs have gradually been eliminated, and all the monoclonal antibody drugs currently occupying the market are humanized monoclonal antibody drugs.

[0003] Human monoclonal antibodies have high specificity and significant efficacy in the treatment of inflammation, cancer, influenza and coronavirus, etc. COVID-19 is an acute respiratory infectious disease caused by a SARS-COV-2 coronavirus, and there is still a lack of effective drugs and vaccines. The new coronavirus needs to rely on the specific molecules expressed by the virus itself to bind to the receptors on human cells when invading cells, so as to infect cells and further expand. Human antibodies that neutralize viruses are certain specific antibodies produced by human B lymphocytes, which can bind to antigens on the surface of the virus, thereby preventing the virus from adhering to target cell receptors and preventing the virus from invading cells, and can effectively prevent and treat SARS-COV-2 influenza. SUMMARY

[0004] To solve the above problems, the present application provides an antibody or antigen-binding fragment thereof against SARS-COV-2, which specifically binds to S protein of SARS-COV-2.

[0005] In one aspect, the present application provides an isolated antibody or antigen-binding fragment thereof against SARS-COV-2; which has the following any one group of three heavy chain variable region complementarity determining regions (HCDRs) and three light chain variable region complementarity determining regions (LCDRs):

[0006] Antibody 6G18

[0007] 6G18-HCDR1: GFTFSSYA SEQ ID No: 1;

[0008] 6G18-HCDR2: ISGSGGST SEQ ID No: 2;

[0009] 6G18-HCDR3: AKMVTPGYGDYSLYYFEY SEQ ID No: 3;

[0010] 6G18-LCDR1: SGSIASNY SEQ ID No: 4;

[0011] 6G18-LCDR:2: EDN SEQ ID No: 5; and

[0012] 6G18-LCDR3: QSYDSSNLIWV SEQ ID No: 6;

[0013] Another aspect of the present application provides an isolated antibody or antigen binding fragment thereof against SARS-COV-2, wherein the heavy chain variable region and the light chain variable region of the antibody 6G18 are:

[0014] 6G18 heavy chain variable region:

[0015] EVQLLESGGDLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVISGSGGST YYADSVKGRFTISRDNSKNTLNLQMSSLRAEDTAVYYCAKMVTPGYGDYSLYYFEYWGQ GTLVTVSS SEQ ID No: 7

[0016] 6G18 light chain variable region:

[0017] NFMLTQPHSVSESPGKTVTISCTRTSGSIASNYVQWYQQRPGSAPTTVMFEDNQRPSG VPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNLIWVFGGGTKLTVT SEQ ID No: 8;

[0018] In the technical solution of the present application, the antibody or antigen binding fragment thereof is a humanized antibody, more preferably a fully humanized antibody.

[0019] In the technical solution of the present application, the antibody is an IgG1 antibody.

[0020] In the technical solution of the present application, the antibody or antigen binding fragment thereof specifically binds to the S surface protein of SARS-COV-2.

[0021] In the technical scheme of the present application, the antibody is a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.

[0022] In the technical scheme of the present application, the antibody or antigen-binding fragment thereof specifically binds to the SARS-COV-2 surface S protein.

[0023] In another aspect of the present application, a nucleotide sequence encoding the antibody or antigen-binding fragment thereof as described above is provided.

[0024] In another aspect of the present application, a vector comprising the nucleotide sequence as described above is provided.

[0025] In another aspect of the present application, a host cell comprising the vector or vector set as described above is provided, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from a yeast cell, a mammalian cell or other cells suitable for producing the antibody or antigen-binding fragment thereof.

[0026] In another aspect of the present application, a kit comprising the antibody or antigen-binding fragment thereof as described above is provided.

[0027] In another aspect of the present application, a detection reagent comprising the antibody or antigen-binding fragment thereof as described above is provided.

[0028] In another aspect of the present application, the antibody or antigen-binding fragment thereof as described above is used as a detection reagent, and the reagent is used in enzyme-linked immunosorbent assay (ELISA), Western Blot, flow cytometry (FACS), immunohistochemistry (IHC) detection or immunoprecipitation (IPCR).

[0029] In the above immunological detection, the antibody or antigen-binding fragment thereof can be connected to a conjugate including horseradish peroxidase (HRP), alkaline phosphatase (AP), biotin, fluorescein isothiocyanate (FITC), Cy3, Cy5, magnetic beads and agarose, etc. conjugate by chemical coupling, electrostatic adsorption or hydrophobic adsorption, alone or in combination.

[0030] In the technical scheme of the present application, the detection reagent can be used for non-diagnostic or therapeutic purpose detection.

[0031] In another aspect of the present application, a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable excipient is provided.

[0032] In the technical solution of the present application, the antibody or antigen-binding fragment thereof blocks or reduces the binding of the S protein of SARS-COV-2 to the cell surface receptor of the subject, preferably the cell angiotensin-converting enzyme-related carboxypeptidase (ACE2).

[0033] In another aspect of the present application, the use of an antibody or antigen-binding fragment thereof against SARS-COV-2 in the preparation of a medicament for preventing, treating or alleviating at least one symptom or indication of SARS-COV-2 infection is provided.

[0034] In the technical solution of the present application, the medicament is an oral or injectable preparation.

[0035] In another aspect of the present application, a method for preventing, treating or alleviating at least one symptom or indication of SARS-COV-2 infection is provided, the method comprising administering the antibody or antigen-binding fragment thereof of any of the preceding or the pharmaceutical composition to a subject.

[0036] In the technical solution of the present application, the at least one symptom or indication is selected from the group consisting of lung inflammation, alveolar damage, fever, cough, dyspnea, hypoxemia, acute respiratory distress syndrome, septic shock, coagulopathy, metabolic acidosis, nasal congestion, rhinorrhea, sore throat, diarrhea, organ failure, septic shock and death.

[0037] In the technical solution of the present application, the pharmaceutical composition or the antibody or antigen-binding fragment thereof is administered in combination with a second therapeutic agent. The second therapeutic agent is selected from the group consisting of anti-inflammatory drugs (such as corticosteroids and non-steroidal anti-inflammatory drugs), antiviral drugs, different antibodies against the S protein of SARS-COV-2, vaccines for SARS-COV-2, antibiotics, dietary supplements such as antioxidants and any other palliative therapy for SARS-COV-2 infection, drugs that alleviate the above symptoms or indications.

[0038] In the technical solution of the present application, the pharmaceutical composition or the antibody or antigen-binding fragment thereof is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly or intracranially.

[0039] Advantages

[0040] (1) The anti-SARS-COV-2 antibody of the present application can specifically target and bind to the S protein of SARS-COV-2 virus, has high specificity and can effectively block the binding of the S protein on the surface of SARS-COV-2 virus to the cell surface receptor of the subject.

[0041] (2) Compared with the mouse-derived antibody, the gene of the full human-derived antibody of the application is completely derived from human gene and has no components of other species, and no toxic side effects such as anti-mouse antibody and the like occur in the human body, and has better biocompatibility, and is more suitable and has more potential to become a macromolecular drug for treating influenza virus.

[0042] (3) Compared with the method for preparing the human-derived monoclonal antibody against SARS-COV-2 virus provided by the prior art phage display technology, the antibody against SARS-COV-2 virus developed by the single B cell has the advantages of simple and fast operation, and the human-derived antibody produced has high affinity and specificity. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 The figure is the ELISA experimental result of Example 1. DETAILED DESCRIPTION

[0044] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application are described in detail below, but it should not be understood as limiting the scope of the present application.

[0045] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present application, the technical solutions of the present application are described in detail below in combination with specific examples, and it should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. In the examples, each raw reagent material can be commercially available, and the experimental method without specific conditions is a conventional method and conventional condition well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0046] As used herein, the term "antibody" refers to a molecule that comprises at least one antigen binding site that immunospecifically binds to a particular antigenic target of interest. Thus, the term "antibody" includes, but is not limited to, full-length antibodies and / or variants thereof, fragments thereof, peptibodies and variants thereof, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, and antibody mimetics or specified fragments or portions thereof that mimic the structure and / or function of antibodies, including single-chain antibodies and fragments thereof. Binding of an antibody to a target can elicit a variety of effects, such as, but not limited to, modulation, reduction, increase, antagonism, agonism, mitigation, slowing, blocking, inhibition, ablation, and / or interference with at least one target activity or binding, or receptor activity or binding, in vitro, in situ, and / or in vivo. Thus, antibodies of the present disclosure encompass antibody fragments that are capable of binding to a biomolecule (e.g., an antigen or receptor) or portion thereof, including, but not limited to, Fab, Fab' and F(ab')2, pFc', Fd, single-domain antibodies (sdAbs), variable fragments (Fv), single-chain variable fragments (scFv), or disulfide-linked Fv (sdFv); bifunctional antibodies or divalent bifunctional antibodies; linear antibodies; single-chain antibody molecules; multispecific antibodies formed from antibody fragments. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.

[0047] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they can be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.

[0048] As used herein, the term HCDR has the same meaning as heavy chain complementarity determining region, and LCDR has the same meaning as light chain complementarity determining region.

[0049] As used herein, a monoclonal antibody includes "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, that exhibit the desired biological activity.

[0050] As used herein, the term "SARS-COV-2", also known as "novel coronavirus", refers to the newly occurring virus causing the novel coronavirus pneumonia (COVID-19).

[0051] As used herein, S protein refers to the spike protein on the coronavirus, SARS-CoV-2 recognizes ACE2 on the surface of human cells through the spike protein on the surface of the virus and invades host cells. By blocking the S protein on the surface of the coronavirus SARS-CoV-2, the virus can effectively inhibit the adhesion of the target cell receptor, preventing the virus from invading the cell.

[0052] The term "humanized antibody" as used herein includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The humanized antibodies of the present application can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis or by in vivo somatic mutation).

[0053] The term "antigen-binding fragment" and the like as used herein includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein which specifically binds an antigen to form a complex. The term "antigen-binding fragment" of an antibody as used herein refers to one or more fragments of an antibody that have binding ability to the S protein of SARS-COV-2.

[0054] In one aspect, the present application provides an anti-SARS-COV-2 fully human monoclonal antibody or a biologically active fragment derived from the monoclonal antibody capable of specifically binding to SARS-COV-2, wherein the amino acid sequences of the heavy and light chain CDR1, CDR2 and CDR3 regions of the antibody are as follows:

[0055] Antibody 6G18

[0056] 6G18-HCDR1: GFTFSSYA SEQ ID No: 1;

[0057] 6G18-HCDR2: ISGSGGST SEQ ID No: 2;

[0058] 6G18-HCDR3: AKMVTPGYGDYSLYYFEY SEQ ID No: 3;

[0059] 6G18-LCDR1: SGSIASNY SEQ ID No: 4;

[0060] 6G18-LCDR:2: EDN SEQ ID No: 5; and

[0061] 6G18-LCDR3: QSYDSSNLIWV SEQ ID No: 6;

[0062] In some embodiments, the heavy chain variable region amino acid sequence of the antibody 6G18 is as set forth in SEQ ID NO: 7, or an amino acid sequence having equivalent functionality that is formed by replacing, deleting or adding one or several amino acids from this sequence;

[0063] EVQLLESGGDLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVISGSGGST YYADSVKGRFTISRDNSKNTLNLQMSSLRAEDTAVYYCAKMVTPGYGDYSLYYFEYWGQ GTLVTVSS SEQ ID No: 7

[0064] and

[0065] The light chain variable region amino acid sequence of the antibody is as set forth in SEQ ID NO: 8, or an amino acid sequence having equivalent functionality that is formed by replacing, deleting or adding one or several amino acids from this sequence NFMLTQPHSVSESPGKTVTISCTRTSGSIASNYVQWYQQRPGSAPTTVMFEDNQRPSGVPD RFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNLIWVFGGGTKLTVT SEQ ID No: 8.

[0066] It has been verified by ELISA experiments that the anti-SARS-COV-2 fully human monoclonal antibody described in the present application can target and bind to the S protein of SARS-COV-2 virus. The antibody described in the present application is a fully human monoclonal antibody. Compared with a mouse-derived antibody, a fully human antibody is completely derived from human genes and does not contain components of other species. It does not cause toxic side effects such as anti-mouse antibody in the human body, has better biocompatibility, and is more suitable and has more potential to become a macromolecular drug for treating influenza virus.

[0067] In another aspect, the present application provides a gene encoding the anti-SARS-COV-2 fully human monoclonal antibody described in the present application. In some embodiments, the gene comprises a nucleotide sequence encoding an amino acid as set forth above.

[0068] In some specific embodiments, the nucleotide sequence is as set forth below (the following sequence is only exemplary, and those skilled in the art can design other nucleotide sequences that can be translated into the desired amino acid sequence according to the specific amino acid sequence):

[0069] The nucleotide sequence encoding the heavy chain variable region of the antibody 6G18 is:

[0070] GaggtgcagctgttggagtctgggggagacttggtacagccgggggggtccctgagactctcctgtgcagcctccggattcacgtttagtagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagttattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgaatctgcaaatgagcagcctgagagccgaggacacggccgtatattactgtgcgaaaatggtgacccccgggtacggtgactactccttgtactactttgaatactggggccagggaaccctggtcaccgtctcctcaSEQ ID No: 9

[0071] The nucleotide sequence encoding the variable region of the heavy chain of antibody 6G18 is:

[0072] AattttatgctgactcagccccactctgtgtcggagtctccggggaagacggtaaccatctcctgcacccgcaccagtggcagcattgccagcaactatgtgcagtggtaccagcagcgcccgggcagtgcccccaccactgtgatgtttgaggataaccaaagaccctctggggtccctgatcggttctctggctccatcgacagctcctccaactctgcctccctcaccatctctggactgaagactgaggacgaggctgattactactgtcagtcttatgatagcagcaatcttatttgggtgttcggcggagggaccaagctgaccgtcacaSEQ ID No: 10.

[0073] The sequences of the variable region of the heavy chain and the variable region of the light chain of the antibody of the present application are shown in the underlined regions below, wherein the CDR regions are shown in bold.

[0074] In another aspect, the present application provides a vector containing the gene as described above.

[0075] In still another aspect, the present application provides a cell containing the gene as described above or the vector as described above.

[0076] In another aspect, the present application provides a method for producing the anti-SARS-COV-2 fully human monoclonal antibody or a biologically active fragment derived from the monoclonal antibody capable of specifically binding to SARS-COV-2, which comprises culturing a genetically engineered cell containing the above-mentioned gene or the above-mentioned vector encoding the heavy and light chains of the anti-SARS-COV-2 fully human monoclonal antibody or directly culturing the above-mentioned cell, collecting and purifying to obtain the anti-SARS-COV-2 fully human monoclonal antibody.

[0077] There is a method for preparing an anti-SARS-COV-2 virus human monoclonal antibody using phage display technology in the prior art, although this method has the advantages of low production cost and no need for immunization and cell fusion, etc., but its disadvantages are also obvious. The antibodies obtained from non-immune antibody library often have insufficient affinity, are limited by exogenous gene transformation rate, and the library capacity of the antibody library is insufficient to cover the antibody diversity of animals. The present application separates B cells secreting antibodies from the blood of patients, then extracts RNA and synthesizes cDNA, clones the genes secreting the target antibodies therefrom, and finally recombines and expresses the fully human monoclonal antibody. This technology is simple and fast in operation, and the human antibodies produced have high affinity and specificity. In addition, the improved technology for separating the monoclonal antibody of the present application from memory B cells having a neutralizing virus function or a tumor killing function can further greatly reduce the cumbersome operation and cost.

[0078] In another aspect, the present application provides a pharmaceutical composition comprising the anti-SARS-COV-2 fully human monoclonal antibody or a biologically active fragment derived from the monoclonal antibody capable of specifically binding to SARS-COV-2 of the present application.

[0079] In another aspect, the present application provides the use of the anti-SARS-COV-2 fully human monoclonal antibody or a biologically active fragment derived from the monoclonal antibody capable of specifically binding to SARS-COV-2 or the pharmaceutical composition of the present application in the preparation of a medicament for treating a disease caused by SARS-COV-2 virus.

[0080] In another aspect, the present application provides a kit for detecting the level of SARS-COV-2 virus, which contains the anti-SARS-COV-2 fully human monoclonal antibody or a biologically active fragment derived from the monoclonal antibody capable of specifically binding to SARS-COV-2 of the present application. In some embodiments, the kit further contains a second antibody and an enzyme or a fluorescent or radioactive label for detection, as well as a buffer. The second antibody is, for example, an anti-antibody against the monoclonal antibody of the present application.

[0081] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific examples, it should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. In the examples, each original reagent material can be commercially available, and the experimental method without specific conditions is a conventional method and conventional condition familiar to the art, or according to the conditions recommended by the instrument manufacturer.

[0082] Example 1

[0083] (1) Construction of NTH-3T3 cell line stably expressing CD40L (3T3-CD40L)

[0084] 3T3-CD40L feeder cells were established using lentivirus. The lentivirus expression vector pLVX-CD40L was constructed, transfected into 293T cells, and the virus supernatant was collected on the fourth day of transfection. Activated NIH-3T3 cells were infected with lentivirus after being cultured for 3 generations, and then were continuously cultured and passaged for 3 times. Cells with FITC fluorescence intensity near MFI were sorted by flow cytometry, and then were re-added to culture bottles for culture and detection in a 37℃, 5% CO2 incubator. The 3T3 cells expressing CD40L and the 3T3 cells transfected with empty vector pLVX (with ZxGreen) were respectively stained with APC-labeled anti-CD40L, and then were analyzed by flow cytometry. The results showed that all 3T3-CD40L feeder cells expressed CD40L. When the cells grew to 80% to 90%, the cells were collected by digestion, and the concentration was 1×10 7 cells per milliliter. The cells were placed in an irradiator for 5000 rad irradiation, and then were resuspended in freezing solution at a concentration of 3.5×10 7 cells per milliliter. One milliliter of the resuspended solution was aliquoted in a freezing tube, and then was frozen in liquid nitrogen (can be stored for 2 years).

[0085] (2) Sorting and activation of memory B cells

[0086] The PBMCs of a recovered patient who had been infected with SARS-COV-2 virus were separated and frozen by lymph separation solution, and the concentration was 10 to 50×10 6 cells per tube, which were frozen in a liquid nitrogen tank. A PBMC flow cytometry staining solution was prepared, and the components are shown in Table 1

[0087] Table 1 PBMC flow cytometry staining solution

[0088] Antibody Volume (μL) CD19-PE-Cy7 0.5 IgM-PE 1.0 IgA-APC 2.5 IgD-FITC 2.5 PBS-1% (wt / vol) BSA 43.5

[0089] The PBMCs were thawed, and then were added with the above-mentioned PBMC flow cytometry staining solution and sorted on a flow cytometer. CD19 + IgM – IgA – IgD– Memory B cells, cell purity should be more than 90%, if less than 90%, repeat the sorting process. Mixed culture medium for activated B cells, as shown in Table 2 below:

[0090] Table 2

[0091] Component Volume Complete IMDM medium 336 mL IL-2 (10,000 U mL -1 )]]> 3.5 mL IL-21 (100 μg mL -1 )]]> 175 μL 3T3-CD40L obtained in Step (1) 10 mL

[0092] Memory B cells are added to the mixed culture medium, mixed and diluted in a 384-well plate, 1 cell per well, 50 μl in volume, and placed in a 37°C, 5% CO2 incubator for static culture. After 13 days, the supernatant is taken for ELISA to obtain human monoclonal antibodies.

[0093] (3) Human monoclonal antibody binding to SARS-COV-2 virus surface antigen S protein experiment

[0094] The surface antigen S protein is purchased from Yiqiao Shenzhou Company and has immunogenicity, and the anti-S protein antibody can be SARS-COV-2 influenza virus. The supernatant human monoclonal antibody obtained above is subjected to ELISA experiment, specifically:

[0095] (1) 100 ng / 100 μL of SARS-COV-2 virus HA protein is coated in a 96-well enzyme-labeled plate, 100 μl per well;

[0096] (2) Place in a 4°C refrigerator overnight;

[0097] (3) Wash three times with PBST solution, add 5% skimmed milk powder solution 200 μL per well, 37 ℃ Incubate for 1 hour;

[0098] (4) Wash three times with PBST solution, add 100 μl of normal human serum without virus infection (negative control) or supernatant, each with three repeats;

[0099] (5) Wash three times with PBST solution after incubation at 37°C for 1 hour;

[0100] (6) Dilute the HRP-labeled anti-human IgG antibody (abcam) by 1:5000, add to the enzyme-labeled plate, 100 μL per well;

[0101] (7) Wash three times with PBST solution after incubation at 37°C for 1 hour;

[0102] (8) Add 100 μL TMB substrate solution (Thermo Scientific) per well, 37°C for 5 minutes;

[0103] (9) Add 100 μL of stop solution 2M sulfuric acid per well, immediately detect the absorbance value at 450 nm wavelength in the enzyme-labeled instrument. The results are as followsFigure 1 As shown, ELISA experiments show that the human monoclonal antibody obtained by the application can target and bind to the S protein of SARS-COV-2 virus.

[0104] Example 2 Cloning of humanized monoclonal antibody gene

[0105] The B cells obtained in Example 1 capable of secreting antibodies that bind to SARS-COV-2 virus were lysed, and the lysate was subjected to reverse transcription of RNA to obtain a PCR template cDNA of the human antibody gene. Primers for cloning the antibody gene were designed and synthesized, and the genes of the heavy chain and light chain of the antibody were cloned using the cDNA as a template and were sent to the company for sequencing. Specifically:

[0106] (1) The lysed B cell solution was transferred to a 96-well plate (Eppendorf, 030133366).

[0107] (2) Reverse transcription system: 150 ng random primers (invitrogen, 48190-011), 0.5 μL 10 mM dNTP (Invitrogen, 18427-088), 1 μL 0.1 M DTT (Invitrogen, 18080-044), 0.5% v / v Igepal CA-630 (Sigma, I3021-50ML), 4 U RNAsin (Promega), 6 U Prime RNAse Inhibitor (Eppendorf) and 50 U III reverse transcriptase (Invitrogen, 18080-044), and DEPC water was added to 14 μL / well.

[0108] (3) Reverse transcription reaction program: 42°C, 10 min; 25°C, 10 min; 50°C, 60 min; 94°C, 5 min.

[0109] (4) The cDNA was stored at -20°C.

[0110] (5) Design and synthesis of primers:

[0111] (6) The heavy chain and light chain of the antibody gene were amplified by PCR using the KOD-Plus-Neo (TOYOBO, KOD401) kit, 40 μL system: 3.5 μL cDNA, 20 nM mixed primers, 4 μL buffer, 4 μL 2 mM dNTPs, 2.4 μL MgSO4, 1 μL KOD.

[0112] (7) Reaction program: 94°C, 2 min; 45 cycles: 98°C, 10 s; 58°C, 30 s; 68°C, 28 s.

[0113] (8) The amplification products were subjected to agarose gel.

[0114] (9) The sequencing results of the antibody gene heavy chain variable region PCR product are as follows, wherein:

[0115] The nucleotide sequence of the heavy chain variable region of antibody 6G18 is as shown in SEQ ID No: 9:

[0116] Gaggtgcagctgttggagtctgggggagacttggtacagccgggggggtccctgagactctcctgtgcagcctccggattcacgtttagtagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagttattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgaatctgcaaatgagcagcctgagagccgaggacacggccgtatattactgtgcgaaaatggtgacccccgggtacggtgactactccttgtactactttgaatactggggccagggaaccctggtcaccgtctcctca SEQ ID No:9;

[0117] The amino acid sequence of the heavy chain variable region of antibody 6G18 is as shown in SEQ ID No: 7:

[0118] EVQLLESGGDLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVISGSGGST YYADSVKGRFTISRDNSKNTLNLQMSSLRAEDTAVYYCAKMVTPGYGDYSLYYFEYWGQ GTLVTVSS SEQ ID No:7;

[0119] The nucleotide sequence of the light chain variable region of antibody 6G18 is as shown in SEQ ID No: 10,

[0120] AattttatgctgactcagccccactctgtgtcggagtctccggggaagacggtaaccatctcctgcacccgcaccagtggcagcattgccagcaactatgtgcagtggtaccagcagcgcccgggcagtgcccccaccactgtgatgtttgaggataaccaaagaccctctggggtccctgatcggttctctggctccatcgacagctcctccaactctgcctccctcaccatctctggactgaagactgaggacgaggctgattactactgtcagtcttatgatagcagcaatcttatttgggtgttcggcggagggaccaagctgaccgtcacaSEQ ID No: 10;

[0121] The amino acid sequence of the heavy chain variable region of antibody 6G18 is shown in SEQ ID No: 7

[0122] NFMLTQPHSVSESPGKTVTISCTRTSGSIASNYVQWYQQRPGSAPTTVMFEDNQRPSG VPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNLIWVFGGGTKLTVT SEQ ID No: 8.

[0123] Correspondingly, the CDR region sequences of each antibody are as follows:

[0124] Antibody 6G18

[0125] 6G18-HCDR1 : GFTFSSYA SEQ ID No: 1 ;

[0126] 6G18-HCDR2: ISGSGGST SEQ ID No: 2;

[0127] 6G18-HCDR3: AKMVTPGYGDYSLYYFEY SEQ ID No: 3;

[0128] 6G18-LCDR1 : SGSIASNY SEQ ID No: 4;

[0129] 6G18-LCDR:2: EDN SEQ ID No: 5; and

[0130] 6G18-LCDR3: QSYDSSNLIWV SEQ ID No: 6;

[0131] The above results show that the supernatant contains antibodies capable of binding to SARS-COV-2 virus.

[0132] Finally, it should be pointed out that the above examples are only used to illustrate the implementation process and characteristics of the present application, and are not intended to limit the technical solutions of the present application. Although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that the present application can still be modified or equivalently replaced without departing from the spirit and scope of the present application. Any modification or partial replacement that does not depart from the spirit and scope of the present application should be covered within the protection scope of the present application. SEQUENCE LISTING <110> Shenzhen Advanced Technology Research Institute <120> Anti-SARS-COV-2 fully human monoclonal antibody 6G18 and its preparation method and application <130> CP121011135C <160> 10 <170> PatentIn version 3.3 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <400> 1 Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial sequence <400> 2 Ile Ser Gly Ser Gly Gly Ser Thr 1 5 <210> 3 <211> 18 <212> PRT <213> Artificial sequence <400> 3 Ala Lys Met Val Thr Pro Gly Tyr Gly Asp Tyr Ser Leu Tyr Tyr Phe 1 5 10 15 Glu Tyr <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <400> 4 Ser Gly Ser lie Ala Ser Asn Tyr 1 5 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <400> 5 Glu Asp Asn 1 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <400> 6 Gln Ser Tyr Asp Ser Ser Asn Leu lie Trp Val 1 5 10 <210> 7 <211> 125 <212> PRT <213> Artificial Sequence <400> 7 Glu Val Gin Leu Leu Glu Ser Gly Gly Asp Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val lie Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Asn 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Met Val Thr Pro Gly Tyr Gly Asp Tyr Ser Leu Tyr Tyr Phe 100 105 110 Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 8 <211> 112 <212> PRT <213> Artificial Sequence <400> 8 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Thr Ser Gly Ser Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Thr Val 35 40 45 Met Phe Glu Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gin Ser Tyr Asp Ser 85 90 95 Ser Asn Leu He Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Thr 100 105 110 <210> 9 <211> 375 <212> DNA <213> Artificial Sequence <400> 9 gaggtgcagc tgttggagtc tgggggagac ttggtacagc cgggggggtc cctgagactc 60 tcctgtgcag cctccggatt cacgtttagt agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagtt attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgaat 240 ctgcaaatga gcagcctgag agccgaggac acggccgtat attactgtgc gaaaatggtg 300 acccccgggt acggtgacta ctccttgtac tactttgaat actggggcca gggaaccctg 360 gtcaccgtct cctca 375 <210> 10 <211> 336 <212> DNA <213> Artificial Sequence <400> 10 aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60 tcctgcaccc gcaccagtgg cagcattgcc agcaactatg tgcagtggta ccagcagcgc 120 ccgggcagtg cccccaccac tgtgatgttt gaggataacc aaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgattactac tgtcagtctt atgatagcag caatcttatt 300 tgggtgttcg gcggagggac caagctgacc gtcaca 336

Claims

1. An isolated antibody or antigen-binding fragment thereof against SARS-COV-2, having three heavy chain complementarity determining regions and three light chain complementarity determining regions as shown below: 6G18-HCDR1: GFTFSSYA SEQ ID No: 1; 6G18-HCDR2: ISGSGGST SEQ ID No: 2; 6G18-HCDR3: AKMVTPGYGDYSLYYFEY SEQ ID No: 3; 6G18-LCDR1: SGSIASNY SEQ ID No: 4; 6G18-LCDR:2: EDN SEQ ID No: 5; and 6G18-LCDR3: QSYDSSNLIWV SEQ ID No:

6.

2. The isolated antibody or antigen-binding fragment thereof against SARS-COV-2 according to claim 1, the sequences of the heavy chain variable region and the light chain variable region of the antibody are respectively: 6G18 heavy chain variable region: EVQLLESGGDLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVISGSGGSTYYADSVKGRFTISRDNSKNT LNLQMSSLRAEDTAVYYCAKMVTPGYGDYSLYYFEYWGQGTLVTVSS SEQ ID No: 7, 6G18 light chain variable region: NFMLTQPHSVSESPGKTVTISCTRTSGSIASNYVQWYQQRPGSAPTTVMFEDNQRPSGVPDRFSGSIDSSSNSASLTIS GLKTEDEADYYCQSYDSSNLIWVFGGGTKLTVT SEQ ID No:

8.

3. The isolated antibody or antigen-binding fragment thereof against SARS-COV-2 according to claim 2, which is a humanized antibody or antigen-binding fragment thereof.

4. An isolated nucleic acid molecule, characterized by:

4. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-3.

5. A vector, characterized by:

5. A vector comprising the nucleic acid molecule according to claim 4.

6. A host cell, characterized by:

6. A host cell comprising the vector according to claim 5.

7. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-3.

8. A detection reagent comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-3.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-3 as a detection reagent for enzyme-linked immunosorbent assay, immunoblotting, flow cytometry, immunohistochemical detection or immunoprecipitation.

10. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1-3 and a pharmaceutically acceptable excipient.

11. Use of the antibody or antigen-binding fragment thereof against SARS-COV-2 of any one of claims 1-3, or the pharmaceutical composition of claim 10, in the manufacture of a medicament for preventing, treating, or ameliorating at least one symptom or indication of SARS-COV-2 infection.

12. The use of claim 11, wherein the at least one symptom or indication is selected from the group consisting of COVID-19, alveolar damage, fever, cough, dyspnea, hypoxemia, acute respiratory distress syndrome, septic shock, coagulopathy, metabolic acidosis, nasal congestion, rhinorrhea, sore throat, diarrhea, organ failure, septic shock, and death.

13. The use of claim 11, wherein the at least one symptom or indication is lung inflammation.

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