Anti-novel coronavirus antibody and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an anti-novel coronavirus antibody and application thereof. The invention discloses an antibody for resisting novel coronavirus or an antigen binding fragment thereof, the amino acid sequence of LCDR1 of a light chain variable region VL of the antibody or the antigen binding fragment thereof is as shown in SEQ ID NO: 3, 14 or 25, the amino acid sequence of LCDR2 is DAS, GAS or AAS, the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 4, 15 or 26, and the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 3, 14 or 25. The amino acid sequences of HCDR1 of the heavy chain variable region VH of the antibody or the antigen binding fragment thereof are respectively as shown in SEQ ID NO: 5, 16 or 27, the amino acid sequences of HCDR2 are respectively as shown in SEQ ID NO: 6, 17 or 28, and the amino acid sequences of HCDR3 are respectively as shown in SEQ ID NO: 7, 18 or 29. The antibody disclosed by the invention has relatively high neutralizing activity on the novel coronavirus and broad spectrum resistance to the novel coronavirus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and in particular relates to an anti-2019-nCoV antibody and application thereof. BACKGROUND

[0002] The 2019-nCoV (SARS-CoV-2) infection can cause a more serious acute respiratory disease, and severe cases can lead to pneumonia. The disease has strong infectivity and has been prevalent worldwide in recent years.

[0003] SARS-Cov-2 belongs to the beta coronavirus genus of the coronavirus family, is a single-stranded positive RNA (ribonucleic acid) virus, and has a very small genome of about 30 KB. The replication of the RNA virus depends on the RNA polymerase carried by itself, and the RNA polymerase does not have nuclease proofreading activity, so the mismatch rate of nucleotides in the replication process of the genome is relatively high. Therefore, the RNA virus has a high mutation rate.

[0004] Based on the easy mutation of the 2019-nCoV, at present, there is still a lack of antibodies that have good neutralizing effect on both wild strains and mutant strains of the virus. SUMMARY

[0005] The primary object of the present application is to provide an anti-2019-nCoV antibody and application thereof, so as to achieve that the obtained anti-2019-nCoV antibody has high neutralizing activity to the 2019-nCoV.

[0006] To this end, the present application provides the following technical solutions.

[0007] The first aspect of the present application provides an anti-2019-nCoV antibody or antigen binding fragment thereof, wherein the LCDR1 of the light chain variable region VL has an amino acid sequence as shown in SEQ ID NO: 3, 14 or 25, the amino acid sequence of LCDR2 is DAS, GAS or AAS, the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 4, 15 or 26, the amino acid sequence of HCDR1 of the heavy chain variable region VH is as shown in SEQ ID NO: 5, 16 or 27, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 6, 17 or 28, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 7, 18 or 29.

[0008] In a preferred embodiment of the present application, the VL comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, 12 or 23, and the VH comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, 13 or 24.

[0009] In a preferred embodiment of the present application, the light chain constant region CL thereof comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8, 19 or 30, and the heavy chain constant region CH comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 9, 20 or 31.

[0010] In a preferred embodiment of the present application, wherein the antigen binding fragment is selected from a diabody; the antibody is a human monoclonal antibody, the light chain thereof comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10, 21 or 32, and the heavy chain thereof comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 11, 22 or 33.

[0011] A second aspect of the present application provides a nucleic acid comprising a nucleotide sequence encoding the anti-SARS-CoV-2 antibody or antigen binding fragment thereof as described above.

[0012] A third aspect of the present application provides a vector comprising the nucleic acid as described above.

[0013] A fourth aspect of the present application provides a host cell comprising the nucleic acid or vector as described above; wherein the host cell is a mammalian cell, including but not limited to 293F cell, CHO cell.

[0014] A fifth aspect of the present application provides a method for preparing the antibody or antigen binding fragment thereof as described above, comprising: culturing the host cell as described above under conditions such that the antibody, antigen binding fragment thereof is expressed.

[0015] A sixth aspect of the present application provides a pharmaceutical composition comprising the antibody or antigen binding fragment thereof, nucleic acid, vector or host cell as described above, and a pharmaceutically acceptable carrier or excipient.

[0016] A seventh aspect of the present application provides any one of the following uses of the anti-SARS-CoV-2 antibody or antigen binding fragment thereof, nucleic acid, vector, host cell or pharmaceutical composition as described above: (1) for the preparation of a product for detecting the presence or level of SARS-CoV-2 or S protein thereof in a sample; (2) for the preparation of a product for diagnosing SARS-CoV-2 infection or a disease caused by SARS-CoV-2 infection; (3) for detecting the presence or level of SARS-CoV-2 or S protein thereof in a sample for non-diagnostic and therapeutic purposes; (4) in the application of detecting the antigen content of the novel coronavirus vaccine; (5) in the application of quality control of the novel coronavirus vaccine; (6) in the application of preparing a drug for preventing or treating novel coronavirus infection or diseases caused by novel coronavirus infection.

[0017] By the above technical solution, the present application has at least the following advantages: The antibody against the novel coronavirus has a unique CDR region, which can effectively neutralize the prototype strain of the novel coronavirus and various mutant strains of the novel coronavirus currently prevalent worldwide, has a significant broad-spectrum neutralization ability and a broad spectrum of anti-novel coronavirus. Therefore, the antibody of the present application can be used for preparing a drug for preventing and / or treating the novel coronavirus. In addition, the antibody of the present application can be used to prepare a novel coronavirus detection reagent, so as to detect viral antigens and to find effective neutralizing epitopes.

[0018] The above description is only a summary of the technical solutions of the present application. In order to more clearly understand the technical means of the present application and to implement the content of the description, the following will describe in detail the preferred embodiments of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 A schematic diagram of flow cytometry sorting; Figure 2 A nucleic acid gel electrophoresis diagram of antibody heavy chain and light chain expression genes. DETAILED DESCRIPTION

[0020] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the following will combine the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0021] Unless otherwise specified, the percentage content involved in the present application refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0022] Unless otherwise specified, the percentage concentration involved in the present application refers to the final concentration. The final concentration refers to the proportion of the added component in the system after adding the component.

[0023] The temperature parameter in the present application, if not particularly limited, allows for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows for fluctuations within the precision range of the instrument control.

[0024] The experimental methods used in the following examples, if not specifically stated, are conventional methods.

[0025] The materials, reagents, etc. used in the following examples, if not specifically stated, are commercially available.

[0026] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Antibodies generally comprise a variable region and a constant region in each of the heavy and light chains. The variable region of the heavy and light chains of an antibody comprises the binding domain that interacts with the antigen. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq (the first component in the classical pathway of complement activation). Thus, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form the portion of the antibody that binds to the antigen.

[0027] As used herein, the term "antibody analog" refers to a derivative of an antibody structure that is produced by biological or chemical methods, by deletion, addition, or modification of chemical groups (such as amino acids) based on the structure of an antibody. These derivatives still contain structures similar to the variable region of an antibody (or the CDR region in the variable region of an antibody), and can undergo reactions similar to antigen-antibody binding through these structures.

[0028] As used herein, the term "antibody variable region" refers to one domain in the antibody heavy and light chains. It includes the light chain variable region (VL) and the heavy chain variable region (VH). In nature, the antibody variable region is encoded by V, D (only for heavy chain applicable), and J segments in the immunoglobulin (heavy and light chain) genes through gene recombination splicing. The amino acid sequences of variable regions are highly variable among different antibodies (the amino acid sequences of other regions of antibodies are relatively highly identical), and are responsible for the recognition and binding with specific antigenic determinants. In the antibody variable region, the VL domain and the VH domain both comprise framework regions (FRs) and CDR regions (comlementarity determining regions) from the amino terminus to the carboxyl terminus. A typical antibody variable region has 3 framework regions and 3 CDR regions, which are arranged in between: FR1, CDR1, FR2, CDR2, FR3, and CDR3. The framework regions FR mainly function to form the framework of the protein domain, while the CDR regions mainly function to recognize and bind with the antigen-antibody specificity. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0029] As used herein, the term "identity" when used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, is determined by the percent of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences, as determined by conventional methods, see, e.g., Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978) Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). There are a number of algorithms that are used to align sequences and determine sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the search for similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and the BLAST family of algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs are available that use these algorithms to determine identity, including, but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al. Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program from Intelligenetics, Mountain View, California.

[0030] Without materially affecting the activity of the antibody (retaining at least 95% activity), one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids can be substituted, added and / or deleted from the sequences of the present application by one of skill in the art to obtain variants of the sequences of the antibody or functional fragment thereof. They are all considered to be included in the scope of protection of the present application. Amino acids with similar properties are substituted in the variable region. The variant sequences described in the present application can have at least 80% identity (or homology) with the reference sequences. The sequence identity described in the present application can be measured using sequence analysis software. For example, computer program BLAST, especially BLASTP or TBLASTN, using default parameters. The amino acid sequences referred to in the present application are all shown in the manner of N-terminal to C-terminal.

[0031] As used herein, the term "conservative amino acid substitution" refers to the substitution of one amino acid for another amino acid that is biologically, chemically, or structurally similar. Biologically similar means that the substitution does not destroy the biological activity of the anti-novel coronavirus antibody or with the novel coronavirus antigen. Structurally similar means that the amino acids have similar length side chains, such as alanine, glycine or serine, or have similar size side chains. Chemically similar means that the amino acids have the same charge or are both hydrophilic or hydrophobic. For example, hydrophobic residues isoleucine, valine, leucine or methionine are substituted for one another. Or polar amino acids are substituted, for example, lysine for arginine, glutamic acid for aspartic acid, glutamine for asparagine, serine for threonine, and the like.

[0032] As used herein, the term "heavy chain constant region" includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of a CH1 domain, a hinge (e.g., an upper hinge region, an intermediate hinge region, and / or a lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen binding polypeptide for use in the present disclosure can comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present disclosure comprises a polypeptide chain comprising a CH3 domain. Furthermore, an antibody for use in the present disclosure can lack at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). As noted above, one of ordinary skill in the art will appreciate that a heavy chain constant region can be modified such that it differs in amino acid sequence from a naturally occurring immunoglobulin molecule.

[0033] Based on the amino acid sequences of the constant regions of the heavy chains, immunoglobulin molecules can be assigned to five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into subclasses, e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, and the like. Based on the amino acid sequences of the light chains, the light chains of antibodies can be assigned to lambda (l) chains and kappa (K) chains. The antibodies disclosed herein can be of any of the above classes or subclasses.

[0034] As used herein, the term "antigen binding fragment" or "antibody fragment" refers generally to an antigen-binding antibody fragment, which can include a portion of an intact antibody, generally the antigen binding or variable region, examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, scFv, dAb, Fab / c, complementarity determining regions, single-chain antibodies, diabodies, or single-domain antibody molecules, etc. Among them, "Fab fragment" is composed of one light chain and CH1 and variable region of one heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule. Fab' fragment, which has one or more cysteine residues at the C-terminus of the CH1 domain of the Fab fragment; F(ab')2 fragment, which is a bivalent fragment containing two Fab' fragments connected by a disulfide bond in the hinge region; Fd fragment, which has VH and CH1 domains; Fv fragment, which has VL and VH domains in a single arm of the antibody; dAb fragment, which is composed of VH domain or VL domain; isolated CDR region; and any of the above fragments retains antigen binding activity.

[0035] As used herein, the term "fully humanized antibody" refers to the transfer of all human-encoding antibody genes into genetically engineered antibody gene-deficient animals by transgenic or transchromosomal techniques, so that the animals express human antibodies, so as to achieve the purpose of fully humanizing antibodies. The purpose of "fully humanization" is to eliminate the immunogenicity of non-human-derived antibodies in the human body, while retaining the affinity as much as possible. It is advantageous to select human framework sequences that are most similar to the framework sequences of non-human-derived antibodies as templates for humanization. In some cases, it can be necessary to replace one or more amino acids in the human framework sequence with the corresponding residues in the non-human framework to avoid loss of affinity.

[0036] As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., that contain only the single antibody, except for possible mutations that can be present in minor amounts. Thus, the term "monoclonal" indicates the character of the antibody as not being a mixture of different antibodies. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other antibodies. The term "monoclonal" should not be construed as requiring production of the antibody by any particular method. The term monoclonal antibody specifically includes chimeric antibodies, humanized antibodies, and human antibodies.

[0037] As used herein, the terms "pseudovirus" and "virus-like particle" both have the same meaning and are used interchangeably; it refers to a virus-like particle self-assembled from viral proteins, which does not encapsidate nucleic acid or encapsidate other nucleic acid, so that the pseudovirus or virus-like particle, although able to infect host cells, does not have the ability to replicate autonomously. Therefore, it has high biosafety compared to the real virus. The packaging system of the pseudovirus generally consists of two parts, namely the packaging component and the expression component. The packaging component is constructed by removing the genetic information required for packaging, reverse transcription and integration from the viral (e.g., HIV-1) genome, which provides the necessary proteins for the pseudovirus particle; the expression component is complementary to the packaging component, which contains the genetic information required for packaging, reverse transcription and integration, and also contains the exogenous gene of interest. Co-transfecting the packaging component and the vector component into host cells can harvest the pseudovirus particles in the cell supernatant.

[0038] As used herein, the term "neutralizing antibody" refers to an antibody having neutralizing activity. The term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment that binds to an antigenic protein on the virus, thereby preventing the virus from infecting cells and / or the maturation of viral progeny and / or the release of viral progeny, an antibody or antibody fragment having neutralizing activity can prevent the amplification of the virus, thereby inhibiting or eliminating the infection of the virus.

[0039] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In some embodiments, vectors include, but are not limited to: (1) plasmids; (2) phagemids; (3) cosmids; (4) artificial chromosomes, such as yeast artificial chromosomes, bacterial artificial chromosomes, or artificial chromosomes derived from P1; (5) bacteriophages, such as lambda phage or M13 phage; (6) animal viruses, such as retroviruses, adenoviruses, adeno-associated viruses, spore cyst viruses, poxviruses, baculoviruses.

[0040] In linking the nucleic acid of the present disclosure to a vector, the nucleic acid can be linked to the control elements on the vector either directly or indirectly, as long as the control elements are capable of controlling the translation and expression of the nucleic acid, etc. Of course, the control elements can be directly from the vector itself, or can be exogenous, i.e., not from the vector itself. Of course, the polynucleotide is operably linked to the control elements. "Operably linked" herein means that the exogenous gene is linked to the vector such that the control elements, such as transcriptional control sequences and translational control sequences, etc., within the vector are able to perform their intended function of regulating the transcription and translation of the exogenous gene. Of course, the polynucleotides encoding the heavy and light chains of the antibody can be inserted into different vectors independently, or commonly inserted into the same vector.

[0041] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced. The expression vector can be introduced into a host cell to construct a recombinant cell, which is then used to express the antibody or antigen binding fragment provided by the present disclosure. The corresponding antibody can be obtained by culturing the recombinant cell. In some embodiments, the host cell includes, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293F.

[0042] In the present application, the term "pharmaceutical composition" generally refers to a pharmaceutical composition suitable for administration to a patient, which can comprise the antibody, nucleic acid molecule, vector or cell described in the present application, and can also comprise one or more pharmaceutically acceptable adjuvants, such as one or more of the following: carriers, protective agents, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives.

[0043] The present application obtains four humanized monoclonal antibodies against the novel coronavirus by single B cell flow sorting-antibody gene amplification pairing expression technology screening.

[0044] The four antibodies screened by the present application all belong to neutralizing antibodies, which are some antibodies produced by B lymphocytes, can bind to antigens on the surface of pathogenic microorganisms, thereby preventing pathogenic microorganisms from adhering to target cell receptors and invading host cells. In the treatment of COVID-19, neutralizing antibodies are considered to be antibodies that can recognize the S protein on the surface of the virus and prevent it from binding to host cell receptors, which can compete with cells to bind to RBD on the S protein of the virus. The spike protein (S protein) of the novel coronavirus is composed of a trimer, which contains about 1300 amino acids. The S protein determines the host range and specificity of the virus, and is also an important action site for host neutralizing antibodies.

[0045] In addition, the antibodies screened by the present application are fully humanized antibodies, which can eliminate the immunogenicity of non-human antibodies in the human body, while retaining the affinity as much as possible.

[0046] In order to obtain broad-spectrum neutralizing antibodies against the novel coronavirus, peripheral blood samples (a total of 29 samples) were collected from 10 healthy volunteers who had completed the inactivated COVID-19 vaccination, PBMCs were separated by Ficoll density gradient centrifugation, and IgG+ memory B cells specific to SARS-CoV-2 spike protein were obtained by flow cytometry sorting. Subsequently, RT-PCR amplification of the variable regions of the heavy and light chains of the antibodies, IMGT sequence alignment and recombinant expression were performed on single cells, and 1438 strains of fully human monoclonal antibodies with correct pairing were obtained. After screening wild type and various epidemic variants (including BF.7, BA.4 / 5, XBB.1.5, BQ.1.1, CH.1.1, EG.5.1, etc.) by pseudovirus neutralization experiment, 8 candidate antibodies with broad-spectrum neutralization activity were finally identified.

[0047] The scheme of the present application will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained by purchase.

[0048] Example 1: Screening of Anti-SARS-CoV-2 Fully Human Monoclonal Antibodies 1. Volunteer recruitment and blood sample collection Ten healthy volunteers who have completed COVID-19 vaccination (inactivated vaccine) were recruited, and 10-20 mL of their peripheral blood was collected after signing the informed consent form, and blood samples were collected using EDTA anticoagulant tubes (BD Vacutainer®, Cat# 366643) for a total of 29 samples.

[0049] 2. Single B cell sorting 2.1 Sample processing The peripheral blood sample was separated by Ficoll-Paque PLUS (GE Healthcare, Cat# 17-1440-02) density gradient centrifugation to obtain PBMCs, and the operation steps were as follows: First, centrifuge the sample at 1500 rpm for 10 min, and the balance error should be within 0.1 g. Pay attention to aseptic operation, prepare ficoll, PBS, 15 mL, 50 mL centrifuge tube and other related reagents and materials, and freeze the box to room temperature in advance. Absorb the supernatant after centrifugation into an EP tube, and label it (name, date) on the tube body. Absorb the remaining material into a centrifuge tube, dilute it 1:1 with PBS. Add ficoll to the existing material in the tube at a ratio of 1:1, and slowly add it with the syringe inserted into the bottom of the tube. Then centrifuge the centrifuge tube at 25°C, 600 g, 30 min, with an acceleration of 1 and a deceleration of 0. Absorb the middle lymphocyte layer into a clean centrifuge tube, and dilute it with PBS 5 times. Then centrifuge at 500 g for 5 min. Discard the supernatant and repeat the previous step. Add 20 mL of PBS to the cell pellet to resuspend the cells and obtain a cell suspension. Take 3 mL of cell suspension and add it to a 15 mL centrifuge tube. Centrifuge the 15 mL centrifuge tube and the original 50 mL centrifuge tube (about 17 mL of PBMC cell suspension remaining) at 380 g for 7-8 min. The 15 mL centrifuge tube is centrifuged and the supernatant is discarded, 1 mL of cell freezing solution (90% FBS + 10% DMSO) is added, and the resuspended mixture is added to the freezing tube and labeled; the 50 mL centrifuge tube is centrifuged and the supernatant is discarded, 5 mL of FBS is added to resuspend the PBMCs, and they are divided into 5 tubes and labeled. Place the PBMC freezing tube in the freeze box cooled to room temperature in advance, and store it at -80°C for more than 24 hours, and store it in liquid nitrogen for long-term storage.

[0050] 2.2 Standard operating procedure for human PBMC staining and sorting 2.2.1 Prepare the materials: Buffer1 (PBS+2% FBS), buffer2 PBS+2% FBS+1 mM EDTA); 37°C water bath, flow tube, 40 pm cell strainer (blue), 50 mL centrifuge tube, 1.5 mL EP tube.

[0051] 2.2.2 Operation steps: 1) Cell recovery, preheat the water bath to 37°C, and preheat 9 mL buffer1 at the same time. Take the cells from the liquid nitrogen tank or -80 refrigerator, put the lower half of the frozen cells into the water bath for quick melting (do not immerse the frozen tube into the water bath, there is a risk of contamination), add to 9 mL preheated buffer2, mix gently, then centrifuge at 300 g for 8 min, discard the supernatant, and take the cell pellet.

[0052] 2) Add 1 mL buffer2 to mix the cell pellet, add 7 mL buffer2, mix, then filter with a 40 pm cell strainer, and gently rinse the tube wall with 2 mL buffer2, then filter. Mix 50 pL for counting (usually 10-20 times dilution), and the rest is centrifuged at 300 g for 8 min, the supernatant is discarded, and the cell pellet is taken. Calculate the number and viability of cells.

[0053] 3) Fluorescent staining: negative and single staining tubes, about 1 x 10 6 cells per 1.5 mL EP tube; the remaining cells are used as sample tubes, and fluorescent staining is performed according to the experimental purpose (see Table 1 below); after staining on ice for 40 min, add 3 mL buffer1 to the sample tube, and add 1 mL buffer1 to the single staining tube, mix, then centrifuge at 300 g for 8 min, and discard the supernatant. Repeat the washing of the sample tube once (add 3 mL buffer1, centrifuge at 300 g for 8 min, and discard the supernatant).

[0054] 4) Add 0.5-1 mL buffer1 to the sample tube to resuspend (control the buffer volume according to the cell density, do not be too high or too low), add 500 pL to the negative and single staining tubes, mix, then pass through a 40 pm cell strainer, and place on an ice box. Carry a 96-well sorting plate, ice box, marker pen, and sealing film for machine sorting. The antigens used for machine sorting and their related information are shown in Table 1 below.

[0055] Table 1 Basic information of antigens 2.3 Cell sorting The stained cell sample is sorted by BD FACSAria™ Fusion, and the gate strategy is as follows: Lymphocyte population -> De-clump (single cells) -> Live cells -> CD19 + B cells -> IgM - / IgD - IgG + -> CD27 + Antigen-specific memory B cells. In the sorting process, live cells were identified as live / dead cells, and lymphocytes were sorted according to forward and side scatter. Doublets were excluded based on SSC-A vs. FSC-A and FSC-A vs. FSC-H. Dead cells were excluded based on zombie-A vs. SSC-A. Total B lymphocytes were identified as CD19 + , and IgM / IgD-IgG + secreting B cells were sorted, and finally single memory B cells were selected based on CD27+Antigen488+. The results are shown in Figure 1 .

[0056] As shown in Figure 1 , B cells accounted for a high proportion, IgG + accounted for a normal proportion; CD27 was obviously grouped; P6 gates were sorted to obtain single cells.

[0057] Example 2: Antibody gene amplification and transient expression The target cells in Example 1 above were single-cell sorted into a 96-well plate, and 4 μL TCL lysis buffer + 1% β-Mercaptoethanol (QIAGEN, Cat# 1031576) was added to each well. RNeasy Micro Kit (QIAGEN, Cat# 74004) was used to extract total RNA from each single cell. Reverse transcription was performed using SuperScript™ IV VILO™ MasterMix (Thermo Fisher, Cat# 11756050) to obtain cDNA. Using specific primers (described in the reference Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning), the heavy chain VH and light chain VL variable regions were amplified using Phusion High-Fidelity DNA Polymerase (Thermo Fisher, Cat# F530S) as the PCR enzyme, and the specific amplification process was as follows: (1) Amplification of heavy chain variable region gene fragments After the 96-well plate of double-stranded cDNA was subjected to plate centrifugation, 1 μL of template was taken for each well. Each component was configured according to the amplification system shown in Table 2 below, and the amplification product of the heavy chain variable region was obtained by amplification according to the amplification procedure shown in Table 3 below.

[0058] Table 2 Amplification system of heavy chain variable region After the addition of the system, the film was pasted, and was named VH, and was subjected to plate centrifugation after marking the date, and was subjected to machine operation according to the following procedure: Table 3 Amplification procedure of heavy chain variable region (2) Amplification of light chain variable region gene fragment After the 96-well plate of double-stranded cDNA was subjected to plate centrifugation, 1 μL of template was taken for each well. Each component was configured according to the amplification system shown in Table 4 below, and the amplification product of the light chain variable region was obtained by amplification according to the amplification procedure shown in Table 5 below: Table 4 Amplification system of light chain variable region After the addition of the system, the film was pasted, and was named VL+CL, and was subjected to plate centrifugation after marking the date, and was subjected to machine operation according to the following procedure: Table 5 Amplification procedure of light chain variable region The obtained antibody heavy chain and light chain expression genes were subjected to nucleic acid electrophoresis, i.e., 2xLoading was first configured, i.e., 10xLoading was diluted to obtain, and 5 μL of 2xLoading buffer was added in eight rows, 2 μL of sample was added, and agarose gel electrophoresis (1.2%) was performed, and Marker 5000 was used for Marker gradient dispensing (5 μL / 4 μL / 3 μL / 2 μL / 1 μL), and the results are shown in Figure 2 As shown in Figure 2 , the spliced expression frame gene fragments were subjected to gel running verification, and the size of the heavy chain expression frame was about 3300 bp, and the size of the light chain was about 2600 bp.

[0059] The above amplification product was subjected to IMGT alignment VDJ sequence, and the aligned BCR heavy chain and light chain variable region sequences were cloned into an expression vector (such as pCAGGS or pcDNA3.4) according to the conventional method of molecular cloning to obtain a recombinant plasmid, and Expi293F cells (Thermo Fisher, Cat# A14527) were transfected with PEI for transient expression, and the expression supernatant was collected after 48-72 hours of culture and was purified using Protein A affinity magnetic beads to obtain the antibody.

[0060] Example 3: Neutralization activity assay of antibodies against SARS-CoV-2 pseudovirus Neutralization experiments were performed using SARS-CoV-2 pseudovirus, with a HIV vector carrying Luciferase as the virus system, and SARS-CoV-2 S protein as the envelope. The target cells (HEK293T-hACE2) were infected, with 3 dilution gradients in each well, starting from a dilution factor of 1:5 to 5-fold increments, and the maximum dilution being 1:125.

[0061] According to the TCID50 value of the virus, it was diluted to 1.3 x 10 4 TCID50 / mL, 50 μL of virus diluent was added to the sample well and the virus control well, shaken to mix, neutralized at 37 °C for 1 hour, and then the cell expression supernatant obtained in Example 2 above was added to release the cells to 4 x 10 4 cells / 100 μL, 100 μL of cell diluent was added to each well, shaken to mix, and incubated in a 37 °C CO2 incubator for 24 hours, and then the Bright-Glo™ Luciferase Assay System (Promega, Cat# E2620) was used to measure the fluorescence intensity. The end point criterion was defined as follows: if the neutralization titer of the supernatant antibody was >1:125, it was determined to have neutralization activity. The results are shown in Table 6.

[0062] Table 6 Neutralization titers of various antibodies against wild-type and mutant strains of novel coronavirus As shown in Table 6, the above 8 antibodies all have neutralization activity against all the novel coronavirus strains tested.

[0063] Example 4: Antibody sequencing and analysis The antibodies ZN-3M-3, JH-6M-2, and TZM-1M-1 clones with neutralization activity screened in Example 3 were extracted for plasmids (QIAGEN Plasmid Mini Kit, Cat# 12125), and Sanger sequencing was performed to obtain the sequences of the antibodies. The results are shown in Tables 7-9.

[0064] Table 7 Antibody ZN-3M-3 sequence Table 8 Antibody JH-6M-2 sequence Table 9 Antibody TZM-1M-1 sequence The above merely describes preferred embodiments of the present application, and is not intended to limit the present application in any form. Although the present application has been disclosed with preferred embodiments as above, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed methods and technical contents to make equivalent embodiments with equivalent changes, without departing from the technical solution of the present application. Any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application, without departing from the technical solution of the present application, still belongs to the scope of the technical solution of the present application.

Claims

1. An antibody against the novel coronavirus or an antigen-binding fragment thereof, wherein the amino acid sequence of LCDR1 in the light chain variable region VL of the antibody or antigen-binding fragment is as shown in SEQ ID NO: 3, 14 or 25, the amino acid sequence of LCDR2 is DAS, GAS or AAS, the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 4, 15 or 26, and the amino acid sequence of HCDR1 in the heavy chain variable region VH of the antibody or antigen-binding fragment is as shown in SEQ ID NO: 5, 16 or 27, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 6, 17 or 28, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 7, 18 or 29.

2. The antibody against the novel coronavirus or its antigen-binding fragment according to claim 1, wherein the VL comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, 12 or 23, and the VH comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 2, 13 or 24.

3. The antibody against the novel coronavirus or its antigen-binding fragment according to claim 1 or 2, wherein the light chain constant region CL contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8, 19 or 30, and the heavy chain constant region CH contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 9, 20 or 31.

4. The antibody against the novel coronavirus according to claim 1 or 2, or the antigen-binding fragment thereof, wherein the antigen-binding fragment is selected from double-chain antibodies; the antibody is a human monoclonal antibody, wherein its light chain contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 10, 21 or 32, and its heavy chain contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 11, 22 or 33.

5. A nucleic acid comprising a nucleotide sequence encoding an antibody against the novel coronavirus or an antigen-binding fragment thereof according to any one of claims 1-4.

6. A vector comprising the nucleic acid of claim 5.

7. A host cell comprising the nucleic acid of claim 5 or the vector of claim 6; in, The host cell is a mammalian cell, including but not limited to 293F cells and CHO cells.

8. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-4, comprising: The host cells according to claim 7 are cultured under conditions that enable the expression of the antibody and its antigen-binding fragment.

9. A pharmaceutical composition comprising an antibody against the novel coronavirus as described in any one of claims 1-4 or an antigen-binding fragment thereof, a nucleic acid as described in claim 5, a vector as described in claim 6 or a host cell as described in claim 7, and a pharmaceutically acceptable carrier or excipient.

10. The following applications of the antibody against the novel coronavirus according to any one of claims 1-4 or its antigen-binding fragment, the nucleic acid according to claim 5, the vector according to claim 6, the host cell according to claim 7, or the pharmaceutical composition according to claim 9: (1) Use in the preparation of products for detecting the presence or level of the novel coronavirus or its S protein in a sample; (2) Use in the preparation of products for diagnosing or diagnosing novel coronavirus infection or disease caused by novel coronavirus infection; (3) Application in detecting the presence or level of the novel coronavirus or its S protein in samples for non-diagnostic and non-therapeutic purposes; (4) Application in detecting the antigen content of novel coronavirus vaccines; (5) Application in the quality control of novel coronavirus vaccines; (6) Use in the preparation of medicines for the prevention or treatment of novel coronavirus infection or diseases caused by novel coronavirus infection.

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