Fully Human Monoclonal Antibody Against SARS-CoV-2, Its Preparation and Application

Abstract

This invention relates to anti-SARS-CoV-2 antibodies or their antigen-binding fragments and their applications, specifically disclosing the complementarity-determining regions and variable regions of the 6Y13 heavy and light chains of anti-SARS-CoV-2 antibodies. The antibodies of this invention are humanized antibodies with low side effects and high affinity and specificity.

Description

Technical Field

[0001] This invention belongs to the field of immunology, specifically relating to a fully human monoclonal antibody against SARS-CoV-2 (COVID-19) and its preparation and application. Background Technology

[0002] Of the top ten best-selling drugs globally in 2018, eight were fully human or humanized monoclonal antibody drugs. The number one drug was Humira, an anti-TNFα monoclonal antibody developed by AbbVie for the treatment of arthritis. This fully human monoclonal antibody has been the top-selling drug for six consecutive years, exceeding $10 billion in sales. Since the first monoclonal antibody drug was launched in 1986, monoclonal antibody drugs have gone through stages including murine monoclonal antibody drugs (such as Ortholone OKT3), chimeric monoclonal antibody drugs (Rituximab), humanized monoclonal antibody drugs (Herceptin), and fully human monoclonal antibody drugs (Humira). Due to the emergence of anti-mouse antibody response (HAMA) in humans, murine and chimeric monoclonal antibody drugs have gradually been phased out, and currently, all monoclonal antibody drugs on the market are humanized monoclonal antibody drugs.

[0003] Human monoclonal antibodies have shown significant efficacy with high specificity in treating inflammation, cancer, influenza, and especially coronaviruses. COVID-19 is an acute respiratory infectious disease caused by the SARS-CoV-2 coronavirus, and effective drugs and vaccines are still lacking. When the novel coronavirus invades cells, it relies on specific molecules expressed by the virus binding to receptors on human cells to infect cells and further amplify. Human antibodies that neutralize the virus are specific antibodies produced by human B lymphocytes that can bind to antigens on the surface of the virus, thereby preventing the virus from adhering to target cell receptors and preventing viral invasion. This provides highly effective prevention and treatment of SARS-CoV-2 influenza. Summary of the Invention

[0004] To address the above problems, the present invention provides an antibody against SARS-CoV-2 or its antigen-binding fragment, which specifically binds to the S protein of SARS-CoV-2.

[0005] This invention provides, in one aspect, an isolated antibody against SARS-CoV-2 or its antigen-binding fragment thereof; which has complementarity-determining regions (HCDRs) of three heavy chain variable regions and complementarity-determining regions (LCDRs) of three light chain variable regions, comprising any one of the following groups:

[0006] Antibody 6Y13

[0007] 6Y13-HCDR1: GGSINTYY SEQ ID No: 1;

[0008] 6Y13-HCDR2: ISYSRST SEQ ID No: 2;

[0009] 6Y13-HCDR3: ARRDPQYNLFDH SEQ ID No: 3;

[0010] 6Y13-LCDR1:SGSIASNY SEQ ID No: 4;

[0011] 6Y13-LCDR:2: EDN SEQ ID No: 5; and

[0012] 6Y13-LCDR3: QSYDSSNLIWV SEQ ID No: 6;

[0013] Another aspect of the present invention provides an isolated anti-SARS-CoV-2 antibody or its antigen-binding fragment, wherein the heavy chain variable region and light chain variable region of antibody 6Y13 are:

[0014] 6Y13 heavy chain variable region:

[0015] QVQLQESGPGLVKPSETLSLTCTVSGGSINTYYWSWIRQPPGKGLEYIGHISYSRSTTSN PSLKSRVTISVHTSKNQFSLKLSSVTAADTAVYYCARRDPQYNLFDHWGQGTLVTVSSSEQ ID No: 7

[0016] 6Y13 light chain variable region:

[0017] NFMLTQPHSVSESPGKTVTISCTRTGSSIASNYVQWYQQRPGSAPTTVMFEDNQRPSG VPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNLIWVFGGGTKLTVLV SEQ ID No: 8;

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

[0019] In the technical solution of this invention, the antibody is an IgG1 antibody.

[0020] In the technical solution of the present invention, the antibody or its antigen-binding fragment specifically binds to the S protein on the surface of SARS-CoV-2.

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

[0022] In the technical solution of the present invention, the antibody or its antigen-binding fragment specifically binds to the S protein on the surface of SARS-CoV-2.

[0023] In another aspect, the present invention provides a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof as described above.

[0024] In another aspect, the present invention provides a carrier comprising the aforementioned nucleotide sequence.

[0025] In another aspect, the present invention provides a host cell comprising the aforementioned vector or vector group, preferably, the host cell being prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or their antigen-binding fragments.

[0026] In another aspect, the present invention provides a kit comprising an antibody or an antigen-binding fragment thereof as described above.

[0027] In another aspect, the present invention provides a detection reagent comprising an antibody or an antigen-binding fragment thereof as described above.

[0028] In another aspect, the present invention provides the use of the above-mentioned antibody or its antigen-binding fragment as a detection reagent, said reagent being used for: enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry (FACS), immunohistochemistry (IHC) detection, or immunoPCR.

[0029] In the aforementioned immunological assays, antibodies or their antigen-binding fragments can be used alone or linked by chemical bonds, electrostatic adsorption, or hydrophilic / hydrophobic adsorption. The conjugates include horseradish peroxidase (HRP), alkaline phosphatase (AP), biotin, fluorescein isothiocyanate (FITC), Cy3, Cy5, magnetic beads, and agarose, among other conjugates.

[0030] In the technical solution of the present invention, the detection reagent can be used for non-diagnostic or therapeutic detection purposes.

[0031] In another aspect, the present invention provides a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof as described above and pharmaceutically acceptable excipients.

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

[0033] In another aspect, the present invention provides the use of an antibody against SARS-CoV-2 or an antigen-binding fragment thereof in the preparation of a medicament for the prevention, treatment or relief of at least one symptom or indication of SARS-CoV-2 infection.

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

[0035] In another aspect, the present invention provides a method for preventing, treating or alleviating at least one symptom or indication of SARS-CoV-2 infection, the method comprising administering an antibody or antigen-binding fragment thereof or a pharmaceutical composition thereof to a subject.

[0036] In the technical solution of the present invention, 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, coagulation dysfunction, metabolic acidosis, nasal congestion, runny nose, sore throat, diarrhea, organ failure, septic shock, and death.

[0037] In the technical solution of the present invention, the pharmaceutical composition or the antibody or its antigen-binding fragment 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 nonsteroidal anti-inflammatory drugs), antiviral drugs, different antibodies against the S protein of SARS-CoV-2, vaccines against SARS-CoV-2, antibiotics, dietary supplements such as antioxidants, and any other palliative therapies for treating SARS-CoV-2 infection, and drugs for relieving the above symptoms or indications.

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

[0039] Beneficial effects

[0040] (1) The anti-SARS-COV-2 antibody described in this invention can target and bind to the S protein of SARS-COV-2 virus. It has high specificity and can effectively block the binding of the S protein on the surface of SARS-COV-2 virus to the receptor on the surface of the subject's cells.

[0041] (2) Compared with mouse-derived antibodies, the genes of the fully human antibody of this invention are entirely derived from human genes and do not contain components from other species. It does not produce toxic side effects such as anti-mouse anti-antibody in the human body, has better biocompatibility, and is more suitable and has greater potential to become a macromolecular drug for treating influenza virus.

[0042] (3) Compared with the existing phage display technology for preparing human monoclonal antibodies against SARS-CoV-2, the method of developing antibodies against SARS-CoV-2 using a single B cell in this invention has the advantages of simple and quick operation, and the human antibodies produced have high affinity and specificity. Attached Figure Description

[0043] Figure 1 The image shows the ELISA experimental results of Example 1. Detailed Implementation

[0044] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below, but should not be construed as limiting the scope of the present invention.

[0045] To provide a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. In the embodiments, all original reagent materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions 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 containing at least one antigen-binding site that binds immune-specifically to a specific target antigen. Therefore, the term "antibody" includes, but is not limited to, full-length antibodies and / or variants thereof, fragments thereof, peptide bodies and variants thereof, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two complete antibodies, human antibodies, humanized antibodies, and antibody mimics of the structure and / or function of antibodies, or designated fragments or portions thereof, including single-chain antibodies and fragments thereof. Antibody binding to a target can elicit a variety of effects, such as, but not limited to, regulating, reducing, increasing, antagonizing, activating, mitigating, slowing, blocking, inhibiting, eliminating, and / or interfering with at least one target activity or binding, or receptor activity or binding, in vitro, in situ, and / or in vivo. Therefore, the antibodies disclosed herein encompass antibody fragments capable of binding to biomolecules (e.g., antigens or receptors) or portions thereof, including but not limited to Fab, Fab' and F(ab')2, pFc', Fd, single-domain antibodies (sdAb), variable fragments (Fv), single-chain variable fragments (scFv), or disulfide-linked Fv (sdFv); bifunctional antibodies or bivalent bifunctional antibodies; linear antibodies; single-chain antibody molecules; and 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 derived from a group of substantially the same type of antibody; that is, the individual antibodies constituting the group are identical, except for the possibility of a small number of naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike polyclonal antibody preparations which contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, a key advantage of monoclonal antibodies is that they can be synthesized without contaminating other antibodies. The modifier "monoclonal" should not be interpreted as requiring any particular method to produce the antibody.

[0048] As used in this article, the terms HCDR and LCDR have the same meaning as the heavy chain complementarity determination region.

[0049] As used herein, monoclonal antibodies include “chimeric” antibodies, wherein a portion of the heavy chain and / or light chain is identical or homologous to the 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 is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and these antibody fragments exhibit the desired biological activity.

[0050] As used in this article, the term "SARS-CoV-2" is also referred to as "novel coronavirus," referring to the newly emerging virus that causes novel coronavirus pneumonia (COVID-19).

[0051] As used in this article, the S protein refers to the spike protein on the coronavirus. SARS-CoV-2 uses the spike protein on its surface to recognize ACE2 on the surface of human cells and infect host cells. Blocking the S protein on the surface of the coronavirus SARS-CoV-2 can effectively inhibit viral adhesion to target cell receptors and prevent the virus from invading cells.

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

[0053] As used herein, the term "antigen-binding fragment" includes any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments capable of binding to the S protein of SARS-CoV-2.

[0054] On the one hand, the present invention provides a fully human monoclonal antibody against SARS-CoV-2 or a bioactive 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 6Y13

[0056] 6Y13-HCDR1: GGSINTYY SEQ ID No: 1;

[0057] 6Y13-HCDR2: ISYSRST SEQ ID No: 2;

[0058] 6Y13-HCDR3: ARRDPQYNLFDH SEQ ID No: 3;

[0059] 6Y13-LCDR1:SGSIASNY SEQ ID No: 4;

[0060] 6Y13-LCDR:2: EDN SEQ ID No: 5; and

[0061] 6Y13-LCDR3: QSYDSSNLIWV SEQ ID No: 6;

[0062] In some embodiments, the amino acid sequence of the heavy chain variable region of antibody 6Y13 is as shown in SEQ ID NO:7, or the sequence is modified by substituting, deleting or adding one or more amino acids to form an amino acid sequence with equivalent function.

[0063] QVQLQESGPGLVKPSETLSLTCTVSGGSINTYYWSWIRQPPGKGLEYIGHISYSRSTTSNPSLKSRVTISVHTSKNQFSLKLSSVTAADTAVYYCARRDPQYNLFDHWGQGTLVTVSSSEQ ID No: 7

[0064] and

[0065] The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:8, or an amino acid sequence with equivalent function formed by replacing, deleting or adding one or more amino acids to this sequence: NFMLTQPHSVSESPGKTVTISCTRTSGSIASNYVQWYQQRPGSAPTTVMFEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNLIWVFGGGTKLTVLV SEQ ID No:8.

[0066] ELISA experiments have verified that the fully human monoclonal antibody against SARS-CoV-2 described in this invention can target and bind to the S protein of the SARS-CoV-2 virus. The antibody described in this invention is a fully human monoclonal antibody. Compared to murine antibodies, fully human antibodies are genetically derived entirely from humans, without components from other species. They do not exhibit toxic side effects such as anti-mouse or anti-antibody reactions in the human body, possess better biocompatibility, and are more suitable and have greater potential as a large-molecule drug for treating influenza viruses.

[0067] On the other hand, the present invention provides a gene encoding the fully human monoclonal antibody against SARS-CoV-2 described herein. In some embodiments, the gene comprises a nucleotide sequence encoding the amino acids shown above.

[0068] In some specific embodiments, the nucleotide sequence is as follows (the following sequences are merely exemplary; those skilled in the art can design other nucleotide sequences that can be translated into the desired amino acid sequence based on the specific amino acid sequence):

[0069] The nucleotide sequence encoding the variable region of the antibody 6Y13 heavy chain is as follows:

[0070] Caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctctggtggctccatcaatacttactactggagctggatccggcagcccccagggaagggactggagtacattgggcatatctcttacagtcggagcaccacctcca acccctccctcaagagtcgagtcaccatatcagtacacacgtccaagaaccagttctccctgaagctgagctctgtgaccgctgcggacacggccgtgtattactgtgcgaggcgagacccccaatataatttgttcgaccactggggccagggaaccctggtcaccgtctcctcaSEQ ID No: 9

[0071] The nucleotide sequence encoding the variable region of the 6Y13 light chain of antibody is as follows:

[0072] aattttatgctgactcagccccactctgtgtcggagtctccggggaagacggtaaccatctcctgcacccgcaccagtggcagcattgccagcaactatgtgcagtggtaccagcagcgcccgggcagtgcccccaccactgtgatgtttgaggataaccaaagaccctct ggggtccctgatcggttctctggctccatcgacagctcctccaactctgcctccctcaccatctctggactgaagactgaggacgaggctgattactactgtcagtcttatgatagcagcaatcttatttgggtgttcggcggagggaccaagctgaccgtcctaGTASEQ ID No: 10.

[0073] In the heavy chain variable region and light chain variable region sequences of the antibody of the present invention, the underlined portion shows the CDR region.

[0074] On the other hand, the present invention provides a vector containing the genes described above.

[0075] In another aspect, the present invention provides cells containing the genes or vectors described above.

[0076] In another aspect, the present invention provides a method for generating the anti-SARS-COV-2 fully human monoclonal antibody or a bioactive fragment derived from the monoclonal antibody that can specifically bind to SARS-COV-2. The method includes culturing genetically engineered cells 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 cells, collecting and purifying the anti-SARS-COV-2 fully human monoclonal antibody.

[0077] Existing technologies include methods for preparing human monoclonal antibodies against SARS-CoV-2 using phage display technology. While these methods offer advantages such as low production costs and the elimination of cumbersome procedures like immunization and cell fusion, they also have significant drawbacks. Antibodies obtained from non-immune antibody libraries often lack sufficient affinity, are limited by the conversion rate of exogenous genes, and the antibody library's capacity is insufficient to cover the antibody diversity of animals. This invention isolates B cells that secrete functional antibodies from the patient's blood, then extracts RNA and synthesizes cDNA, clones the gene that secretes the target antibody, and finally recombines and expresses a fully human monoclonal antibody. This technology is simple and rapid, producing human antibodies with high affinity and specificity. Furthermore, the improved technology for isolating monoclonal antibodies with virus-neutralizing or tumor-killing functions from memory B cells further reduces cumbersome operations and costs.

[0078] On the other hand, the present invention provides a pharmaceutical composition comprising the fully human monoclonal antibody against SARS-CoV-2 as described in the present invention or a bioactive fragment derived from the monoclonal antibody capable of specifically binding to SARS-CoV-2.

[0079] On the other hand, the present invention provides the use of the aforementioned fully human monoclonal antibody against SARS-CoV-2, or a bioactive fragment derived from the monoclonal antibody capable of specifically binding to SARS-CoV-2, or the aforementioned pharmaceutical composition, in the preparation of a medicament for treating diseases caused by the SARS-CoV-2 virus.

[0080] On the other hand, the present invention provides a kit for detecting SARS-CoV-2 virus levels, which contains the fully human anti-SARS-CoV-2 monoclonal antibody described in the present invention or a bioactive fragment derived from the monoclonal antibody that can specifically bind to SARS-CoV-2; in some embodiments, the kit further contains a second antibody and an enzyme or fluorescent or radiolabeled for detection, as well as a buffer; the second antibody is, for example, an anti-antibody against the monoclonal antibody described in the present invention.

[0081] To provide a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. In the embodiments, all original reagent materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0082] Example 1

[0083] (1) Constructing an NTH-3T3 cell line stably expressing CD40L (3T3-CD40L)

[0084] 3T3-CD40L feeder cells were established using lentivirus. The lentiviral expression vector pLVX-CD40L was constructed and transfected into 293T cells. Viral supernatant was collected on the fourth day after transfection. NIH-3T3 cells were activated, cultured for 3 passages, and then infected with lentivirus. The cells were cultured and passaged 3 times. Cells with FITC fluorescence intensity near the MFI were sorted using flow cytometry and reintroduced into culture flasks. They were then cultured and analyzed at 37°C in a 5% CO2 incubator. Specifically, 3T3 cells expressing CD40L and 3T3 cells transfected with the empty vector pLVX (containing ZxGreen) were stained with anti-CD40L containing APC, and then analyzed by flow cytometry. The results showed that all 3T3-CD40L feeder cells expressed CD40L. When the cells reached 80%–90% confluence, they were digested and collected at a concentration of 1 × 10⁻⁶ cells / mL. 7 Cells were placed in a radiation instrument for 5000 rads of radiation, and then resuspended in cryopreservation solution at a concentration of 3.5 × 10⁻⁶ cells / mL. 7 Cells were aliquoted into 1 mL vials and cryopreserved in liquid nitrogen (can be stored for 2 years).

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

[0086] PBMCs from recovered SARS-CoV-2 patients were isolated and cryopreserved using lymphocyte separation fluid, with 10–50 × 10⁻⁶ cells per tube. 6 Cells were cryopreserved in liquid nitrogen. PBMC flow cytometry staining solution was prepared, and its components are shown in Table 1 below.

[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] Thaw PBMCs, add the above PBMC flow cytometry staining solution, and sort them on a flow cytometer to separate CD19. + IgM – IgA – IgD– Memory B cells must have a purity of over 90%. If the purity is below 90%, the sorting process must be repeated. Prepare the mixed culture medium for activating B cells as shown in Table 2 below:

[0090] Table 2

[0091] Components volume Complete IMDM medium 336mL <![CDATA[IL-2(10,000U mL -1 )]]> 3.5mL <![CDATA[IL-21(100μg mL -1 )]]> 175μL The 3T3-CD40L obtained in step (1) 10mL

[0092] Memory B cells were added to a mixed culture medium, mixed thoroughly, and then infinitesimally diluted in 384-well plates, with one cell per well (50 μl volume). The plates were then incubated statically at 37°C in a 5% CO2 incubator. After 13 days, the supernatant was used for ELISA to obtain human monoclonal antibodies.

[0093] (3) Experiment on binding of human monoclonal antibody to the surface antigen S protein of SARS-CoV-2 virus

[0094] The surface antigen S protein was purchased from Sinocare Biotech and is immunogenic. Anti-S protein antibodies can detect SARS-CoV-2 influenza virus. An ELISA experiment was performed on the human monoclonal antibody obtained from the supernatant. Specifically:

[0095] (1) Coat 100 ng / 100 μL of HA protein of SARS-CoV-2 virus in a 96-well microplate, 100 μL per well;

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

[0097] (3) Wash three times with PBST solution, add 200 μL of 5% skim milk powder solution to each well, and saline at 37°C. ℃ Incubate for 1 hour;

[0098] (4) Wash three times with PBST solution, add 100 μl of normal human serum (negative control) or supernatant that is not infected with the virus, and repeat three times for each.

[0099] (5) After incubating at 37°C for 1 hour, wash three times with PBST solution;

[0100] (6) Dilute the HRP-containing anti-human IgG antibody (abcam) at a ratio of 1:5000 and add 100 μL to each well of the ELISA plate;

[0101] (7) After incubating at 37°C for 1 hour, wash three times with PBST solution;

[0102] (8) Add 100 μL of TMB substrate solution (Thermo Scientific) to each well and incubate at 37°C for 5 minutes;

[0103] (9) Add 100 μL of 2M sulfuric acid as a stop solution to each well, and immediately measure the absorbance at 450 nm using a microplate reader. The results are as follows: Figure 1 As shown, the ELISA experiment demonstrates that the human monoclonal antibody obtained in this invention can target and bind to the S protein of the SARS-CoV-2 virus.

[0104] Example 2: Cloning of humanized monoclonal antibody gene

[0105] B cells obtained in Example 1 that secrete antibodies binding to the SARS-CoV-2 virus were lysed, and the lysate was used for reverse transcription of RNA to obtain the PCR template cDNA of the human antibody gene. Primers for cloning the antibody gene were designed and synthesized, and the heavy and light chain genes of the antibody were cloned using the cDNA as a template, and sent to Genewiz 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 dNTPs (Invitrogen, 18427-088), 1 μL 0.1 MDTT (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), add DEPC water to 14 μL / well.

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

[0109] (4) cDNA is stored at -20℃.

[0110] (5) Primer design and synthesis:

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

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

[0113] (8) Agarose gelation was performed on the amplification products.

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

[0115] The nucleotide sequence of the heavy chain variable region of antibody 6Y13 is as shown in SEQ ID No:9:

[0116] Caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctctggtggctccatcaatacttactactggagctggatccggcagcccccagggaagggactggagtacattgggcatatctcttacagtcggagcaccacctcc aacccctccctcaagagtcgagtcaccatatcagtacacacgtccaagaaccagttctccctgaagctgagctctgtgaccgctgcggacacggccgtgtattactgtgcgaggcgagacccccaatataatttgttcgaccactggggccagggaaccctggtcaccgtctcctca SEQ ID No: 9;

[0117] The amino acid sequence of the variable region of the antibody 6Y13 heavy chain is as shown in SEQ ID No:7:

[0118] QVQLQESGPGLVKPSETLSLTCTVSGGSINTYYWSWIRQPPGKGLEYIGHISYSRSTTSNPSLKSRVTISVHTSKNQFSLKLSSVTAADTAVYYCARRDPQYNLFDHWGQGTLVTVSSSEQ ID No: 7;

[0119] The nucleotide sequence of the variable region of the light chain of antibody 6Y13 is as shown in SEQ ID No:10.

[0120] aattttatgctgactcagccccactctgtgtcggagtctccggggaagacggtaaccatctcctgcacccgcaccagtggcagcattgccagcaactatgtgcagtggtaccagcagcgcccgggcagtgcccccaccactgtgatgtttgaggataaccaaagaccctct ggggtccctgatcggttctctggctccatcgacagctcctccaactctgcctccctcaccatctctggactgaagactgaggacgaggctgattactactgtcagtcttatgatagcagcaatcttatttgggtgttcggcggagggaccaagctgaccgtcctaGTASEQ ID No: 10;

[0121] The amino acid sequence of the variable region of the light chain of antibody 6Y13 is shown in SEQ ID No:8.

[0122] NFMLTQPHSVSESPGKTVTISCTRTGSSIASNYVQWYQQRPGSAPTTVMFEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNLIWVFGGGTKLTVLV SEQ ID No: 8.

[0123] Correspondingly, the CDR region sequences of each antibody are shown below:

[0124] Antibody 6Y13

[0125] 6Y13-HCDR1: GGSINTYY SEQ ID No: 1;

[0126] 6Y13-HCDR2: ISYSRST SEQ ID No: 2;

[0127] 6Y13-HCDR3: ARRDPQYNLFDH SEQ ID No: 3;

[0128] 6Y13-LCDR1:SGSIASNY SEQ ID No: 4;

[0129] 6Y13-LCDR:2: EDN SEQ ID No: 5; and

[0130] 6Y13-LCDR3: QSYDSSNLIWV SEQ ID No: 6;

[0131] The results showed that the supernatant contained antibodies that could bind to the SARS-CoV-2 virus.

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the implementation process and features of the present invention, and are not intended to limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention without departing from the spirit and scope of the present invention. Any modifications or partial substitutions should be covered within the protection scope of the present invention. SEQUENCE LISTING <110> Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences <120> Anti-SARS-CoV-2 fully human monoclonal antibody, its preparation method and application <130> CP121011051C <160> 10 <170> PatentIn version 3.3 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <400> 1 Gly Gly Ser Ile Asn Thr Tyr Tyr 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial sequence <400> 2 Ile Ser Tyr Ser Arg Ser Thr 1 5 <210> 3 <211> 12 <212> PRT <213> Artificial sequence <400> 3 Ala Arg Arg Asp Pro Gln Tyr Asn Leu Phe Asp His 1 5 10 <210> 4 <211> 8 <212> PRT <213> artificial sequence <400> 4 Ser Gly Ser Ile 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 Ile Trp Val 1 5 10 <210> 7 <211> 118 <212> PRT <213> artificial sequence <400> 7 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Asn Thr Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45 Gly His Ile Ser Tyr Ser Arg Ser Thr Thr Ser Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val His Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Arg Asp Pro Gln Tyr Asn Leu Phe Asp His Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 8 <211> 113 <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<00​​​​​​​​​​​​​​Ser Asn Leu Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 Val <210> 9 <211> 354 <212> DNA <213> Artificial sequence <400> 9 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcaat acttactact ggagctggat ccggcagccc 120 ccagggaagg gactggagta cattgggcat atctcttaca gtcggagcac cacctccaac 180 ccctccctca agagtcgagt caccatatca gtacacacgt ccaagaacca gttctccctg 240 aagctgagct ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag gcgagacccc 300 caatataatt tgttcgacca ctggggccag ggaaccctgg tcaccgtctc ctca 354 <210> 10 <211> 339 <212> DNA <213> Artificial sequence <400> 10<​​​​ccgggcagtg cccccaccac tgtgatgttt gaggataacc aaagaccctc tggggtccct 180 gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240 ctgaagactg aggacgaggc tgattactac tgtcagtctt atgatagcag caatcttatt 300 tgggtgttcg gcggagggac caagctgacc gtcctagta 339

Claims

1. An isolated anti-SARS-CoV-2 antibody or its antigen-binding fragment, having the following three heavy chain complementarity-determining regions and three light chain complementarity-determining regions: Antibody 6Y13 6Y13-HCDR1: GGSINTYY; 6Y13-HCDR2: ISYSRST; 6Y13-HCDR3: ARRDPQYNLFDH; 6Y13-LCDR1: SGSIASNY; 6Y13-LCDR:2: EDN; and 6Y13-LCDR3: QSYDSSNLIWV.

2. The isolated anti-SARS-CoV-2 antibody or its antigen-binding fragment according to claim 1, wherein the sequences of the heavy chain variable region and the light chain variable region of antibody 6Y13 are respectively; 6Y13 heavy chain variable region: QVQLQESGPGLVKPSETLSLTCTVSGGSINTYYWSWIRQPPGKGLEYIGHISYSRSTTSNPSLKSRVTIS VHTSKNQFSLKLSSVTAADTAVYYCARRDPQYNLFDHWGQGTLVTVSS, 6Y13 light chain variable region: NFMLTQPHSVSESPGKTVTISCTRTSGSIASNYVQWYQQRPGSAPTTVMFEDNQRPSGVPDRFSGSI DSSSSNSASLTISGLKTEDEADYYCQSYDSSNLIWVFGGGTKLTVLV.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1 or 2, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

4. An isolated nucleic acid molecule, characterized in that: Its encoding is the antibody or antigen-binding fragment thereof as described in any one of claims 1-3.

5. A carrier, characterized in that: It includes the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that: It includes the carrier described in claim 5.

7. A kit comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-3.

8. A detection reagent comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-3.

9. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-3 in the preparation of a reagent for detecting SARS-CoV-2 virus levels, wherein the reagent is for use in enzyme-linked immunosorbent assay (ELISA), immunoblotting, flow cytometry, immunohistochemistry, or immunoPCR.

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

11. Use of the antibody against SARS-CoV-2 or its antigen-binding fragment as described in any one of claims 1-3, or the pharmaceutical composition as described in claim 10, in the preparation of a medicament for the prevention, treatment, or relief of at least one symptom or indication of SARS-CoV-2 infection.

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

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

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