Anti-SARS-CoV-2 Spike Protein Antibody and Its Applications

Abstract

The present invention provides an anti-SARS-CoV-2 Spike protein antibody and its applications. By using genetic engineering and phage display library technology, specific antibodies against the SARS-CoV-2 S protein are screened from a non-immunized fully human single-chain antibody library. Their affinity for the viral S protein is between 1 nM and 50 nM, and the antibodies inhibit the binding of the SARS-CoV-2 S protein to the human receptor ACE2, indicating that the antiviral S protein antibodies of the present invention have good ability to bind to the S protein and potential neutralizing inhibitory effects. The present invention provides specific antibody candidate molecules for the development of diagnostic reagents, prophylactic and therapeutic antibody drugs against SARS-CoV-2 (2019-nCoV), as well as the treatment of other diseases such as pneumonia caused by coronaviruses.

Description

[0001] This invention is a divisional application of a Chinese patent application with the application number CN202010426201.3 and the invention title "Antibody against Novel Coronavirus Spike Protein and Its Application". Technical Field

[0002] This invention relates to the fields of genetic engineering and immunology. Specifically, it relates to an antibody against novel coronavirus Spike protein and its application. Background Art

[0003] The novel coronavirus (2019-nCoV) is a new coronavirus, belonging to β-CoV of the Coronaviridae family in the order Nidovirales, the same as SARS-CoV. It is a non-segmented single-stranded positive-strand RNA virus, and the length of each group of its genome is about 30,000 nucleotides. Different from Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), the novel coronavirus is the 7th in the coronavirus family that infects humans. According to the homology of gene sequences, the novel coronavirus genome has 80% similarity with SARS, and the gene sequences of the novel coronavirus and MERS-CoV have 40% similarity.

[0004] The novel coronavirus 2019-nCoV shows a typical coronavirus genus structure ( Figure 4 ), including: 5 untranslated regions (UTRs), replicase complex (orf1ab), S gene, E gene, M gene, N gene, 3 UTRs, and several unidentified non-structural open reading frames.

[0005] In coronaviruses, there is a key S protein (spike protein), which contains 2 subunits: S1 and S2. S1 can promote the binding of the virus to the host cell receptor, and it contains an important C-terminal RBD domain, which is responsible for binding to the receptor. The RBD domain of the novel coronavirus has a high homology with SARS. Among the 5 key sites infected by SARS, 1 is retained by the novel coronavirus, and the other 4 have amino acid substitutions and changes.

[0006] The S protein (spike) of coronaviruses assembles into a trimer, containing about 1300 amino acids, belonging to the first class of membrane fusion proteins (Class I viral fusion protein). Similar viral membrane fusion proteins also include the Env protein of HIV, the HA protein of influenza, and the Gp protein of Ebola virus, etc. The S protein determines the host range and specificity of the virus, and is also an important action site for host neutralizing antibodies. It is also a key target for vaccine design.

[0007] Similar to other class I viral membrane fusion proteins, the S protein contains two subunits, S1 and S2. S1 mainly contains the receptor binding domain (RBD), which is responsible for recognizing the cell receptor. S2 contains the basic elements required for the membrane fusion process, including an internal fusion peptide, two heptad repeats (HR), a membrane proximal external region (MPER) rich in aromatic amino acids, and a transmembrane (TM) region. The S1 protein can be further divided into two domains, namely the N-terminal domain (NTD) and the C-terminal domain (CTD), and the conformation of the NTD is very similar to that of the galectin protein. The RBDs of most coronavirus S proteins are located in the CTD, such as those of SARS virus and Middle East respiratory syndrome virus (MERS). Only a small part of the RBDs of β-coronaviruses are located in the NTD, such as those of mouse hepatitis virus (MHV). In addition, the NTDs of bovine coronavirus (BCoV) and human coronavirus OC43 can bind specific sugar molecules (such as sialic acid), and they are also involved in the invasion process of coronaviruses. Envelope proteins of viruses in the same family need two different regions to recognize host receptors and effectively mediate the membrane fusion process between the virus and the cell. This is one of the important differences between the coronavirus S protein and other viral membrane fusion proteins.

[0008] The currently discovered coronavirus receptors mainly include the following: aminopeptidase N (APN), angiotensin converting enzyme II (ACE2), dipeptidylpeptidase 4 (DPP4), and CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule). Among them, the species-specific APN protein is the receptor for human coronavirus 229E, feline coronavirus (FCoV), and porcine coronavirus TGEV. Human ACE2 is the receptor for SARS virus and NL63. Human DPP4 is the receptor for MERS virus. The α subtype of the mouse CEACAM1 protein is the receptor for MHV. Crystal structures of complexes of many coronavirus RBDs binding to host receptors have been resolved, mainly including the crystal structures of SARS-RBD-ACE2 complex, NL63-RBD-ACE2 complex, MERS-RBD-DPP4 complex, HKU4-RBD-DPP4 complex, and MHV-RBD-mCEACAM1α complex.

[0009] There are currently no corresponding vaccines and specific drugs for the infection caused by the novel coronavirus 2019-nCoV and the related diseases caused by its infection. Therefore, the development of drugs for the diagnosis and treatment of 2019-nCoV has become an urgent task. Summary of the Invention

[0010] The object of the present invention is to provide an antibody against the Spike protein of the novel coronavirus and its application.

[0011] To achieve the object of the present invention, in a first aspect, the present invention provides an antibody against the Spike protein of the novel coronavirus or its active fragment, wherein the CDR1 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of the same, the CDR2 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2 or 3 or consists of the same, the CDR3 of the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 4-7 or consists of the same; the CDR1 of the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 8-12 or consists of the same, the CDR2 of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13-16 or consists of the same, and the CDR3 of the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 17-19 or consists of the same.

[0012] The amino acid sequence of the antibody variable region provided by the present invention has the pattern: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In the present invention, the region division of FR and CDR is based on the Kabat nomenclature system. Here, FR1 to FR4 represent 4 framework regions, and CDR1 to CDR3 represent 3 hypervariable regions. FR1 to FR4 can be separated from the constant region sequence (such as the most commonly used amino acids of human immunoglobulin heavy and light chain classes, subclasses or subfamilies), or can be separated from the individual antibody framework region or from different framework region gene combinations.

[0013] The anti-SARS-CoV-2 Spike protein antibody of the present invention is:

[0014] i) The heavy chain variable region contains the amino acid sequence shown in any one of SEQ ID NO: 20-25 or consists of it, and the light chain variable region contains the amino acid sequence shown in any one of SEQ ID NO: 26-33 or consists of it;

[0015] ii) An antibody derived from i) by substituting, deleting or adding one or several amino acids and having the same function;

[0016] iii) An antibody derived from i) having a sequence homology of 70%, 80%, 85%, 90% or more than 97% with the antibody of i) and having the same function.

[0017] Preferably any one of the antibodies CS1 to CS8:

[0018] CS1: The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 20 or consists of it, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 26 or consists of it;

[0019] CS2: The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 21 or consists of it, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 27 or consists of it;

[0020] CS3: The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 22 or consists of it, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 28 or consists of it;

[0021] CS4: The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 22 or consists of it, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 29 or consists of it;

[0022] CS5: The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 23, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 30;

[0023] CS6: The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 22, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 31;

[0024] CS7: The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 24, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 32;

[0025] CS8: The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 25, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 33.

[0026] In a second aspect, the present invention provides an antibody obtained by modifying the above-mentioned anti-SARS-CoV-2 Spike protein antibody or its active fragment, and the antibody includes but is not limited to single-chain antibody, Fab, minibody, chimeric antibody, whole antibody immunoglobulin IgG1, IgG2, IgA, IgE, IgM, IgG4 or IgD, etc.

[0027] The anti-SARS-CoV-2 Spike protein antibody provided by the present invention binds to the SARS-CoV-2 (2019-nCoV) Spike protein with an affinity of 1 nM - 50 nM. This antibody inhibits the binding of the SARS-CoV-2 Spike protein to human ACE2.

[0028] In a third aspect, the present invention provides a gene encoding the above-mentioned antibody.

[0029] Considering the degeneracy of codons, the gene encoding the antibody of the present invention can modify the gene sequence encoding the above-mentioned antibody in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody. Those skilled in the art can artificially synthesize and modify the gene according to the codon preference of the antibody-expressing host to improve the expression efficiency of the antibody.

[0030] In a fourth aspect, the present invention provides a biological material containing the gene, and the biological material includes but is not limited to recombinant DNA, expression cassette, transposon, plasmid vector, phage vector, viral vector, engineering bacteria or transgenic cell line, etc.

[0031] In a fifth aspect, the present invention provides any one of the following applications of the antibody, the gene encoding the antibody or the biological material containing the gene:

[0032] 1) Use in the preparation of a therapeutic drug or composition for treating diseases targeting the novel coronavirus Spike protein; preferably, the drug is a diagnostic and therapeutic drug for diseases caused by the 2019-nCoV Spike protein of the novel coronavirus.

[0033] 2) Use in the preparation of a drug or composition for preventing or treating infection with the novel coronavirus 2019-nCoV or related diseases caused by its infection.

[0034] 3) Use in the preparation of a cell therapy drug or composition for preventing or treating infection with the novel coronavirus 2019-nCoV or related diseases caused by its infection.

[0035] 4) Use in the preparation of a detection and diagnostic reagent or kit for the novel coronavirus 2019-nCoV.

[0036] 5) Use in the preparation of a related preparation for CAR-T therapy targeting the novel coronavirus Spike protein.

[0037] 6) For the detection (including non-diagnostic purposes) of the novel coronavirus 2019-nCoV.

[0038] 7) For preventing or treating infection with the novel coronavirus 2019-nCoV or related diseases caused by its infection.

[0039] 8) For CAR-T therapy.

[0040] In a sixth aspect, the present invention provides a drug or composition containing the anti-novel coronavirus Spike protein antibody or its active fragment.

[0041] In a seventh aspect, the present invention provides a detection reagent or kit containing the anti-novel coronavirus Spike protein antibody or its active fragment.

[0042] The antibody provided by the present invention is a whole antibody or various other forms of genetically engineered antibodies. For example, the anti-novel coronavirus Spike protein antibody can be a whole antibody or an antibody fragment. The antibody molecule itself can be used for treatment and diagnosis. The antibody can be labeled, crosslinked or conjugated and fused with other protein or polypeptide molecules for expression to form a complex (such as a cytotoxic substance, a radioactive toxin and / or a chemical molecule, etc.) for diagnosis and treatment.

[0043] Furthermore, the present invention provides an independent gene encoding the antibody, an expression vector, related control technologies for transfecting the host cell with the vector, and the host cell, an antibody expression process and the recovery of the antibody from the cell culture supernatant. The present invention also provides components containing the antibody and a pharmaceutically acceptable delivery molecule or solution. The therapeutic component is sterile and can be lyophilized at low temperature.

[0044] The present invention provides an antibody against the Spike protein of novel coronavirus (2019-nCoV), and this antibody also exerts its function by hindering the binding of the Spike protein of novel coronavirus (2019-nCoV) to ACE2. All the interference functions possessed by the antagonists of the Spike protein of novel coronavirus (2019-nCoV) fall within the protection scope of the present invention.

[0045] The sequences shown as SEQ ID NO:1-33 in the present invention include "conservative sequence modifications", that is, nucleotide and amino acid sequence modifications that do not significantly affect and change the binding characteristics of the antibody or the antibody containing the amino acid sequence. The conservative sequence modifications include nucleotide or amino acid substitutions, additions or deletions. In the art, families of amino acid residues with similar side chains have been defined. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (such as threonine, valine, isoleucine) and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to replace the non-essential amino acid residues in the human antibody against the Spike protein of novel coronavirus (2019-nCoV) with another amino acid residue from the same side chain family.

[0046] Antibodies containing the specific amino acid composition of the present invention, including antibodies encoded by substantially similar sequences with conservative sequence modifications or containing similar sequences with conservative sequence modifications, all fall within the protection scope of the present invention.

[0047] The present invention provides a bispecific or multispecific molecule, which comprises the above-mentioned antibody provided by the present invention or a molecule of the antigen-binding site of the antibody.

[0048] The present invention provides a fusion protein of an antibody and other proteins and / or polypeptides, which comprises a complex of the above-mentioned antibody provided by the present invention and other protein or polypeptide molecules with certain functions.

[0049] Furthermore, the fusion protein is obtained by constructing a recombinant expression vector by ligating the antibody gene with an immunotoxin or cytokine gene, and obtaining a recombinant fusion protein molecule through mammalian cells or other expression systems.

[0050] The novel coronavirus (2019-nCoV) Spike protein antibody provided by the present invention has good prospects for therapeutic applications, mainly manifested by having specific binding activity with the novel coronavirus (2019-nCoV) Spike protein. The antibody was detected by ELISA and flow cytometry, and the results showed good target specificity.

[0051] The present invention uses genetic engineering and phage surface display library technology to screen specific antibodies against the S protein of the new coronavirus from a non-immunized single-chain antibody library of fully human sequences. As determined by Octet Blitz, their apparent affinity for the viral S protein is between 1 nM and 50 nM, and they have an inhibitory effect on the binding of the new coronavirus S protein to the human receptor ACE2, indicating that the antiviral S protein antibodies of the present invention have good ability to bind the S protein and potential neutralizing inhibitory effects. The present invention provides specific antibody candidate molecules for the research and development of diagnostic reagents, preventive and therapeutic antibody drugs against the new coronavirus (2019-nCoV), and the treatment of other diseases such as pneumonia caused by coronaviruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figures 1A - 1K It is a flow cytometry diagram of the antibody binding to the overexpressed novel coronavirus (2019-nCoV) cell ID8 in a preferred embodiment of the present invention. Analysis was performed using a Beckman flow cytometer CytoFlex, and FACS was used to detect the binding of the antibody to ID8; the antibody was converted into the scFv-Fc form and expressed and purified, and added to 200,000 ID8 cells at a final concentration of 10 μg / ml for incubation. The fluorescent secondary antibody is PE-labeled anti-human Fc and FITC-labeled anti-mouse Fc.

[0053] Among them, Figures 1A - 1B are the results of adding only PE-labeled anti-human Fc and FITC-labeled anti-mouse Fc to the cell line ID8 respectively.

[0054] Figures 1C - 1K Flow cytometry was performed to detect the antibody and the results of binding to the overexpressed novel coronavirus (2019-nCoV) cell ID8.

[0055] Figures 2A - 2E It is the binding result of the antibody to the novel coronavirus (2019-nCoV) Spike protein detected by Octet Blitz in a preferred embodiment of the present invention.

[0056] Among them, Figure 2A are the results of ACE2 binding to RBD and the full-length trimeric S protein.

[0057] Figure 2B are the results of the antibody CS1 binding to RBD and being able to compete with ACE2 for binding.

[0058] Figure 2C It is the result of the simultaneous binding of antibodies CS1 and CS2 to the RBD.

[0059] Figure 2D It is the result of the competitive binding of antibodies CS1 and CS8 to the RBD.

[0060] Figure 2E It is the result of the simultaneous binding of antibody CS1 and CS2 - CS7 to the RBD.

[0061] Figure 3A It is the result of antibody CS1 competitively inhibiting the binding of ACE2 to overexpressing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell ID8 at different concentration gradients in the preferred embodiment of the present invention.

[0062] Figure 3B It is the result of antibody CS2 competitively inhibiting the binding of ACE2 to overexpressing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell ID8 at different concentration gradients in the preferred embodiment of the present invention.

[0063] Figure 3C It is the result of antibody CS3 competitively inhibiting the binding of ACE2 to overexpressing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell ID8 at different concentration gradients in the preferred embodiment of the present invention.

[0064] Figure 3D It is the result of antibody CS4 competitively inhibiting the binding of ACE2 to overexpressing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell ID8 at different concentration gradients in the preferred embodiment of the present invention.

[0065] Figure 3E It is the result of antibody CS5 competitively inhibiting the binding of ACE2 to overexpressing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell ID8 at different concentration gradients in the preferred embodiment of the present invention.

[0066] Figure 3F It is the result of antibody CS6 competitively inhibiting the binding of ACE2 to overexpressing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell ID8 at different concentration gradients in the preferred embodiment of the present invention.

[0067] Figure 3G It is the result of antibody CS7 competitively inhibiting the binding of ACE2 to overexpressing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell ID8 at different concentration gradients in the preferred embodiment of the present invention.

[0068] Figure 3H It is the result of antibody CS8 competitively inhibiting the binding of ACE2 to overexpressing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell ID8 at different concentration gradients in the preferred embodiment of the present invention.

[0069] Figure 4 It is a schematic diagram of the structure of the novel coronavirus 2019-nCoV. Detailed implementation mode

[0070] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Unless otherwise specified, the examples are carried out under conventional experimental conditions, such as those described in the Molecular Cloning Laboratory Manual by Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended in the manufacturer's instructions.

[0071] Example 1 Screening for antibodies against the Spike protein of the novel coronavirus (2019-nCoV) from a natural human antibody phage display library

[0072] To obtain human antibodies specific for the Spike protein of the novel coronavirus (2019-nCoV), panning was performed using the solid-phase screening method. First, coat the RBD protein of Spike (mFc tag, Sino biological, catalog number 40592-V05H). Thaw a vial of the human antibody library, which contains 10 billion phage particles expressing different antibodies. Wash the wells coated with the RBD protein overnight with PBS, 250 μl / well, wash 2 times; add the phage particles to the wells of the RBD protein, at room temperature (RT), for 1 h. Add the elution buffer 0.2 M glycine-HCl (pH 2.2), 100 μl / well, let stand for about 10 min, and pipette and mix 2 times during this period; add the neutralization buffer 1 M Tris-HCl (pH 8.0), 42 μl / well, and mix. Take the neutralized mixture and add it to 10 ml of TG1 (OD 600Mix well (about 0.6 - 0.8), incubate statically at 37°C for 30 min for infection. Take about 20 μl of the phage-TG1 infection mixture, add it to 180 μl of 2YT, mix well, and label it as 20 μl - human; take 20 μl of the bacterial solution from 20 μl - human, add it to 180 μl of 2YT, mix well, and label it as 2 μl - human; take 100 μl of the bacterial solution from 20 μl - human and 2 μl - human respectively, spread it on a 90 mm petri dish, and label it as 10 μl - human and 1 μl - human; culture overnight at 37°C for counting the output phage of the first round of the human natural antibody library. For the remaining TG1 bacterial solution, centrifuge at 2400 g for 10 min, discard the supernatant, resuspend it with about 600 μl of 2YT, spread it on an 180 mm petri dish (2YTAG), and culture overnight at 30°C. The next day, count the number of colonies on the 90 mm small petri dishes labeled 10 μl and 1 μl to calculate the output of the first round of the human natural antibody library; at the same time, scrape off the output bacterial lawn on the large petri dish with about 2.5 ml of 2YT, aspirate it into a 5 ml centrifuge tube, mix well, aspirate 900 μl of the bacterial solution, add 300 μl of 50% glycerol, mix well, and store the bacterial strain at -80°C, which is the 1st output - human - bacterial solution; in addition, aspirate 300 μl of the bacterial solution, supplement about 100 μl of 2YT, mix well, and temporarily store it at 4°C for inoculating bacteria and preparing the phage after the first round of screening.

[0073] To further harvest specific antibody clones with relatively high affinity, more rounds of panning are required. For this purpose, the phage antibody solution eluted in the first round of panning is used to infect Escherichia coli (such as strain TG1) in the logarithmic phase that can be infected by M13 phage to obtain an infection solution. A small amount is taken for a series of 10-fold serial dilutions (usually diluted to one millionth of the original solution, and the last three dilutions are taken for plating) to measure the titer of the output of the eluate in the first round (titer), which is also called the maximum diversity in the first round. Usually, the output titer after the first round of panning is below 10E6 cfu. The remaining infection solution is all plated on a bacterial culture plate containing the corresponding antibiotic and cultured overnight to obtain colonies; the colony layer is scraped off and resuspended in the medium. A sufficient amount of the resuspended solution containing the diversity of the first-round output is taken into a shake flask containing a sufficient amount of liquid medium (2YT-CG, 2YT medium added with Carbenicillin and glucose, with final concentrations of 100 μg / ml and 2% respectively), and the resuspended solution is diluted to below OD600 = 0.1 and then cultured until the logarithmic phase, that is, OD600 reaches about 0.5. To present these antibodies obtained in the first round of panning on the surface of phage particles again, 10 ml of the bacterial solution is taken and the helper phage M13K07 is added to make the multiplicity of infection MOI 20:1, and it is left standing at 37 °C for 30 minutes (this stage is phage rescue). Centrifuge, resuspend the cells in 50 ml of expression medium (2YT-AK, 2YT medium added with Carbenicillin and Kanamycin, with final concentrations of 100 μg / ml and 30 μg / ml respectively), and culture overnight at 30 °C, 200 rpm. The next day, the culture supernatant is harvested by centrifugation, 1 / 5 volume of PEG8000 / NaCl (PEG-8000 20%, NaCl 2.5 M) is added, mixed well, and incubated on ice for 1 hour. Centrifuge at high speed (11500×g) for 30 minutes to harvest phage antibody particles. The precipitate is resuspended in 1 ml of PBS solution, and centrifuged at high speed again to remove bacterial debris. The supernatant is the amplification solution after the first round of panning, and each antibody clone contained therein has been amplified by more than ten thousand times. This amplification solution can be used for the second round of panning experiment. The operation of the second round of panning is exactly the same as that of the first round except that when washing with PBST / PBS, it is increased to 6 times (6 / 6) each. In the third round, the washing times can be further increased to 10 / 10. Multiple rounds of panning usually effectively enrich specific clones, with significantly reduced diversity but relatively high affinity, which is convenient for subsequent monoclonal screening.

[0074] To obtain specific monoclonal antibodies, a monoclonal phage ELISA experiment is required. For this purpose, well-separated single colonies obtained in the second and / or third round of serial dilutions are individually inoculated into 96-well culture plates containing 2YT-AG (93 colonies are inoculated per plate, leaving three wells as negative controls), and cultured overnight to obtain the master plate. The bacterial solution in each well of the master plate is inoculated into a new culture plate and grown to the logarithmic phase for phage rescue, so that the antibodies of each clone are expressed on the surface of the phage. BCMA antigen (1 μg / ml) is coated on a common 96-well ELISA plate, and another ELISA plate is coated with human Fc at the same concentration. The individually expressed monoclonal phage antibody bacterial solutions are added to the corresponding wells of the RBD plate and the mFc plate respectively, and then appropriate secondary antibodies and a tertiary antibody conjugated with horseradish peroxidase (HRP) are added. The substrate develops color, and the absorbance value (450 nM) is read. The method for judging RBD-positive clones is as follows: negative on the mFc plate (not exceeding 1.5 times the absorbance value of the negative wells on its own plate), positive on the RBD plate (more than 3 times higher than the absorbance value of the negative wells on its own plate), and the absorbance value of its well is higher than that of the corresponding well on the mFc plate. After analysis, the clones corresponding to 88 wells showed positive only for the RBD antigen and negative for mFc. These clones are collectively referred to as hits.

[0075] These hist bacterial solutions were respectively inoculated from the corresponding holes on the master plate into 3 ml of 2YT-CG, and cultured overnight at 37°C with a rotation speed of 200 revolutions per minute. The phagemid DNA was extracted the next day, and the sequences of the single-chain antibody regions containing each hit were determined using specific primers. The DNA sequences of the coding regions were translated into amino acid sequences, and multiple sequence alignments (CLUSTALW, website link: https: / / www.genome.jp / tools-bin / clustalw) were performed on them to determine the cloning specificity. After analysis, these 88 hits belonged to 26 different clones in terms of sequence. Among them, 8 antibodies had good binding activity to the Spike protein of the novel coronavirus (2019-nCoV). Thus, the sequences of the variable regions of the fully human antibodies against the Spike protein of the novel coronavirus (2019-nCoV) were obtained. The CDR1 of the heavy-chain variable region contains the amino acid sequence shown in SEQ ID NO:1 or consists of it, the CDR2 of the heavy-chain variable region contains the amino acid sequence shown in SEQ ID NO:2 or 3 or consists of it, and the CDR3 of the heavy-chain variable region contains the amino acid sequence shown in any one of SEQ ID NO:4 - 7 or consists of it; the CDR1 of the light-chain variable region contains the amino acid sequence shown in any one of SEQ ID NO:8 - 12 or consists of it, the CDR2 of the light-chain variable region contains the amino acid sequence shown in SEQ ID NO:13 - 16 or consists of it, and the CDR3 of the light-chain variable region contains the amino acid sequence shown in any one of SEQ ID NO:17 - 19 or consists of it.

[0076] Furthermore, the heavy-chain variable region contains the amino acid sequence shown in any one of SEQ ID NO:20 - 25 or consists of it, and the light-chain variable region contains the amino acid sequence shown in any one of SEQ ID NO:26 - 33 or consists of it.

[0077] Example 2 Verification of Antibody Function

[0078] To verify whether the obtained clones of the antibodies against the Spike protein of the novel coronavirus (2019-nCoV) bind to the Spike protein antigen of the novel coronavirus (2019-nCoV) and the Spike protein expressed on the cell membrane surface, the gene of the single-chain antibody against the Spike protein of the novel coronavirus (2019-nCoV) was cloned into the eukaryotic expression vector pFH. In this vector, the scFv gene and the Fc gene of human IgG are fused to express a protein in the form of scFv-Fc, which can be affinity purified using Potein-A and can also be detected using an anti-human Fc antibody labeled with (HRP or fluorescein).

[0079] After obtaining the scFv-Fc protein, the binding of the antibody to the Spike protein and RBD of the novel coronavirus (2019-nCoV) was detected by Octet Blitz, which confirmed the specific binding of the antibody to the Spike protein of the novel coronavirus (2019-nCoV). Figures 2A - 2E ) CS1 is a monoclonal antibody containing the variable regions of SEQ ID NO: 20 and 26; CS2 is a monoclonal antibody containing the variable regions of SEQ ID NO: 21 and 27; CS3 is a monoclonal antibody containing the variable regions of SEQ ID NO: 22 and 28; CS4 is a monoclonal antibody containing the variable regions of SEQ ID NO: 22 and 29; CS5 is a monoclonal antibody containing the variable regions of SEQ ID NO: 23 and 30; CS6 is a monoclonal antibody containing the variable regions of SEQ ID NO: 22 and 31; CS7 is a monoclonal antibody containing the variable regions of SEQ ID NO: 24 and 32; CS8 is a monoclonal antibody containing the variable regions of SEQ ID NO: 25 and 33. The apparent affinity of CS1 is 1.2 nM, the apparent affinity of CS2 is 2.1 nM, the apparent affinity of CS3 is 23.2 nM, the apparent affinity of CS4 is 48 nM, the apparent affinity of CS5 is 4.1 nM, the apparent affinity of CS6 is 35.2 nM, the apparent affinity of CS7 is 12.3 nM, and the apparent affinity of CS8 is 2.6 nM.

[0080] Flow cytometry was used to detect the antibody against the cell line ID8 overexpressing the Spike protein of the novel coronavirus (2019-nCoV), indicating that the antibody specifically binds to the overexpressed Spike protein of the novel coronavirus (2019-nCoV) on the cell membrane surface. Figures 1A - 1K )

[0081] Flow cytometry was used to detect the antibody against the cell line ID8 that inhibits the binding of ACE2 to the overexpressed Spike protein of the novel coronavirus (2019-nCoV). The results showed that the antibody could partially inhibit the binding of ACE2 to the cell line ID8 overexpressing the Spike protein of the novel coronavirus (2019-nCoV), and the inhibitory effect increased with the increase in concentration. Figures 3A - 3H )

[0082] Example 3

[0083] The Cell binding method based on FACS analysis was used to detect the competition of the antibody with ACE2 for binding to the cell line ID8 specifically expressing the 2019-nCoV Spike protein.

[0084] It was determined that the lowest concentration of the ACE2-mFC protein saturating the Spike protein of ID8 cells was 0.02 μg / ml.

[0085] 1. Collect ID8 cells: Collect 0.5×10 6 cells / tube.

[0086] 2. Rinse the cells: Rinse the cells once with 1 ml of staining buffer (PBS containing 1% w / v BSA), centrifuge at 350 x g for 5 min at 4°C, and resuspend with 95 μl of staining buffer after centrifugation.

[0087] 3. Antibody binding: Add antibodies CS1 - CS8 at different concentration gradients (0 - 40 μg / ml) respectively, and incubate on ice for 60 min.

[0088] 4. ACE2 - mFC binding: Add human ACE2 - mFC protein to a concentration of 0.02 μg / ml respectively, and incubate on ice for 30 min.

[0089] 5. Rinse the cells: Add 1 ml of staining buffer to the cell suspension, mix well and centrifuge at 350 g for 5 min at 4°C, discard the supernatant, and rinse twice more. Resuspend the cells with 100 μl of staining buffer after centrifugation.

[0090] 6. Add 5 μl of Biolegend directly labeled antibody (APC anti - Mousw IgG Fc Antibody, Biolegend, 405308) to the sample tube, and incubate in the dark on ice for 15 - 20 min.

[0091] 7. Rinse the cells: Add 1 ml of staining buffer to the cell suspension, mix well and centrifuge at 350 g for 5 min at 4°C, discard the supernatant, and rinse once more.

[0092] 8. Detection by flow cytometry: After resuspending the cells with 100 - 200 μl of PBS, analyze by Beckman CytoFlex flow cytometry.

[0093] The experimental results show that different antibodies have different effects on blocking the binding of ACE2 and Spike protein. When the antibody concentration is 20 μg / ml, the blocking effect of CS1 is 10.81%, CS2 is 16.78%, CS3 is 41.94%, CS4 is 5.51%, CS5 is 4.36%, CS6 is 24.89%, CS7 is 27.26%, and CS8 is 93.12%.

[0094] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made thereto, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. An anti-SARS-CoV-2 Spike protein antibody CS3 or its active fragment, characterized in that, Antibody CS3 is a monoclonal antibody containing the variable regions of SEQ ID NO: 22 and 28.

2. An antibody obtained by modifying the antibody CS3 or its active fragment according to claim 1, wherein the antibody is a single-chain antibody, Fab, minibody, chimeric antibody, whole antibody immunoglobulin IgG1, IgG2, IgA, IgE, IgM, IgG4 or IgD.

3. A gene encoding the antibody CS3 according to claim 1 or 2.

4. A biological material containing the gene according to claim 3, wherein the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, phage vector, viral vector, engineered bacterium or transgenic cell line.

5. Any of the following applications of the antibody CS3 according to claim 1 or 2, the gene according to claim 3 or the biological material according to claim 4: 1) Application in the preparation of a disease treatment drug or composition targeting the Spike protein of novel coronavirus; 2) Application in the preparation of a drug or composition for preventing or treating novel coronavirus 2019-nCoV infection or related diseases caused by its infection; 3) Application in the preparation of a cell therapy drug or composition for preventing or treating novel coronavirus 2019-nCoV infection or related diseases caused by its infection; 4) Application in the preparation of a detection reagent or kit for novel coronavirus 2019-nCoV; 5) Application in the preparation of a related preparation for CAR-T therapy targeting the Spike protein of novel coronavirus; 6) Detection of novel coronavirus 2019-nCoV for non-diagnostic purposes.

6. A drug or composition containing the antibody CS3 according to claim 1 or 2.

7. A detection reagent or kit containing the antibody CS3 according to claim 1 or 2.

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