Anti-sars-cov-2 spike protein antibodies and uses thereof

Abstract

The application provides an anti-Spike protein antibody of a novel coronavirus and an application thereof. By means of genetic engineering and phage surface display library technology, specific antibodies against the S protein of the novel coronavirus are screened from a single-chain antibody library of non-immune full human sequences. The antibodies have an affinity to the S protein of the virus between 1nM and 50nM, the antibodies have an inhibiting effect on the combination of the S protein of the novel coronavirus and human receptor ACE2, and the anti-S protein antibodies of the application have a good ability to combine with the S protein and a potential neutralizing inhibiting effect. The application provides specific antibody candidate molecules for the development of diagnostic reagents, preventive and therapeutic antibody drugs against the novel coronavirus (2019-nCoV) and the treatment of other diseases such as infection caused by the coronavirus.

Description

[0001] This invention is a divisional application of Chinese patent application CN202010426201.3, entitled "Anti-novel coronavirus Spike protein antibody and its application". Technical Field

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

[0003] The novel coronavirus (2019-nCoV) is a new coronavirus belonging to the β-CoV family of Coronaviridae in the order Nidovirales, as well as SARS-CoV. It is a non-segmented, single-stranded, positive-sense RNA virus with each genome segment approximately 30,000 nucleotides in length. Unlike Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), the novel coronavirus is the seventh member of the coronavirus family to infect humans. Based on gene sequence homology, the novel coronavirus genome shares 80% similarity with SARS and 40% similarity with MERS-CoV.

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

[0005] Coronaviruses contain a key S protein (spike protein), which comprises two subunits: S1 and S2. S1 facilitates viral binding to host cell receptors and contains an important C-terminal RBD domain responsible for receptor binding. The RBD domain of the novel coronavirus shows high homology to that of SARS. Of the five key sites in SARS infection, one is retained by the novel coronavirus, while the remaining four have amino acid substitutions and changes.

[0006] The spike protein of coronaviruses forms a trimer containing approximately 1300 amino acids. It belongs to the Class I viral fusion protein group, similar to HIV's Env protein, influenza's HA protein, and Ebola's Gp protein. The spike protein determines the virus's host range and specificity, and is also a crucial site for host-neutralizing antibodies. It is also a key target for vaccine design.

[0007] Similar to other type 1 viral membrane fusion proteins, the S protein contains two subunits, S1 and S2. S1 primarily contains the receptor-binding domain (RBD), responsible for recognizing cellular receptors. S2 contains the essential components required for membrane fusion, including an intrinsic fusion peptide, two heptad repeats (HR), a membrane proximal external region (MPER) rich in aromatic amino acids, and a transmembrane region (TM). The S1 protein can be further divided into two domains: the N-terminal domain (NTD) and the C-terminal domain (CTD), with the NTD conformation being very similar to that of galectin proteins. In most coronaviruses, such as SARS and Middle East respiratory syndrome (MERS), the RBD of the S protein is located within the CTD. Only a small number of beta coronaviruses have their RBD located in the NTD, such as mouse hepatitis virus (MHV). Additionally, the NTDs of bovine coronavirus (BCoV) and human coronavirus OC43 can bind specific sugar molecules (such as sialic acid), and they also participate in the coronavirus invasion process. Viral envelope proteins within the same family require two distinct regions to recognize host receptors and effectively mediate viral-cell membrane fusion. This is one of the key differences between coronavirus S proteins and other viral membrane fusion proteins.

[0008] Currently identified 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 these, 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-isotype of mouse CEACAM1 protein is the receptor for MHV. The crystal structures of many coronavirus RBD-host receptor complexes have been resolved, 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 vaccines or specific drugs for infection with the novel coronavirus 2019-nCoV or the related diseases caused by it. Therefore, the development of diagnostic and therapeutic drugs for 2019-nCoV is an urgent task. Summary of the Invention

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

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

[0012] The antibody variable region amino acid sequence provided by this invention has the following pattern: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In this invention, the region division of FR and CDR is based on the Kabat nomenclature system. Here, FR1–FR4 represent four frame regions, and CDR1–CDR3 represent three hypervariable regions. FR1–FR4 can be isolated from constant region sequences (e.g., the most commonly used amino acids in human immunoglobulin light and heavy chains, subclasses, or subfamilies), or isolated from individual antibody frame regions, or derived from combinations of different frame region genes.

[0013] The antibody against the Spike protein of the novel coronavirus of the present invention is as follows:

[0014] i) The heavy chain variable region contains or is composed of any of the amino acid sequences shown in SEQ ID NO:20-25, and the light chain variable region contains or is composed of any of the amino acid sequences shown in SEQ ID NO:26-33.

[0015] ii) Antibodies derived from i) with the same function but with one or more amino acids replaced, deleted, or added;

[0016] iii) Antibodies derived from i) that have 70%, 80%, 85%, 90% or 97% or more sequence homology with and have equivalent function to antibodies from i).

[0017] Preferred antibody is any one of CS1 to CS8:

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

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

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

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

[0022] CS5: The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:30.

[0023] CS6: The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:22, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:31.

[0024] CS7: The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:24, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:32.

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

[0026] Secondly, the present invention provides antibodies obtained by modifying the above-mentioned anti-novel coronavirus Spike protein antibody or its active fragment, wherein the antibody includes, but is not limited to, single-chain antibodies, Fab, micro antibodies, chimeric antibodies, whole antibody immunoglobulins IgG1, IgG2, IgA, IgE, IgM, IgG4 or IgD, etc.

[0027] The antibody against the novel coronavirus Spike protein provided by this invention binds to the novel coronavirus (2019-nCoV) Spike protein with an affinity of 1 nM-50 nM. This antibody inhibits the binding of the novel coronavirus Spike protein to human ACE2.

[0028] Thirdly, the present invention provides a gene encoding the aforementioned antibody.

[0029] Considering the degeneracy of codons, the gene encoding the antibody described in this invention can be modified in its coding region without altering the amino acid sequence to obtain a gene encoding the same antibody. Those skilled in the art can artificially synthesize and modify genes based on the codon preference of the host expressing the antibody to improve antibody expression efficiency.

[0030] Fourthly, the present invention provides biological materials containing the aforementioned gene, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0031] Fifthly, the present invention provides any 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 disease treatment drug or composition targeting the Spike protein of the novel coronavirus; preferably, the drug is a diagnostic and therapeutic drug for diseases caused by the Spike protein of the novel coronavirus 2019-nCoV;

[0033] 2) Use in the preparation of medicaments or compositions for the prevention or treatment of infection with or related to the novel coronavirus 2019-nCoV;

[0034] 3) Use in the preparation of cell therapy drugs or compositions for the prevention or treatment of novel coronavirus 2019-nCoV infection or related diseases caused by it;

[0035] 4) Application in the preparation of reagents or kits for the detection and diagnosis of the novel coronavirus 2019-nCoV;

[0036] 5) Application in the preparation of formulations for CAR-T therapy targeting the Spike protein of the novel coronavirus;

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

[0038] 7) For the prevention or treatment of infection with the novel coronavirus 2019-nCoV or related diseases;

[0039] 8) Used for CAR-T therapy.

[0040] In a sixth aspect, the present invention provides a medicine or composition containing the antibody against the novel coronavirus Spike protein or an active fragment thereof.

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

[0042] The antibodies provided by this invention are whole antibodies or various other forms of genetically engineered antibodies. For example, the antibody against the Spike protein of the novel coronavirus can be a whole antibody or an antibody fragment. The antibody molecule itself can be used for treatment and diagnosis. Antibodies can be labeled, cross-linked, or conjugated, and fused with other protein or polypeptide molecules to form complexes (such as cytotoxic substances, radiotoxins, and / or chemical molecules) for diagnosis and treatment.

[0043] Furthermore, this invention provides an independent gene encoding an antibody, an expression vector, vector transfection control technology for host cells and host cells, an antibody expression process, and antibody recovery from cell culture supernatant. This invention also provides antibody-containing components and pharmacologically acceptable delivery molecules or solutions. The therapeutic components are sterile and can be lyophilized at low temperatures.

[0044] This invention provides an antibody against the 2019-nCoV Spike protein, which also works by blocking the binding of the 2019-nCoV Spike protein to ACE2. The interfering functions of 2019-nCoV Spike protein antagonists all fall within the scope of protection of this invention.

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

[0046] Antibodies containing the specific amino acid composition of this invention, including those encoded by similar sequences modified with conserved sequences or those containing similar sequences modified with conserved sequences, all fall within the scope of protection of this invention.

[0047] The present invention provides a bispecific or multispecific molecule comprising the antibody or the antigen-binding site of the antibody provided by the present invention.

[0048] The present invention provides a fusion protein of an antibody with other proteins and / or peptides, comprising a complex of the antibody provided by the present invention and other protein or peptide molecules having a certain function.

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

[0050] The novel coronavirus (2019-nCoV) Spike protein antibody provided by this invention has promising therapeutic applications, mainly due to its specific binding activity to the novel coronavirus (2019-nCoV) Spike protein. ELISA and flow cytometry analysis showed that the antibody exhibits good target specificity.

[0051] This invention utilizes genetic engineering and phage surface display library technology to screen specific antibodies against the SARS-CoV-2 spike protein from a non-immune, fully human single-chain antibody library. Octet Blitz assays showed that these antibodies exhibited an epigenetic affinity for the viral spike protein between 1 nM and 50 nM, and also inhibited the binding of the SARS-CoV-2 spike protein to the human receptor ACE2, indicating that the antiviral spike protein antibodies of this invention possess good binding ability to the spike protein and potential neutralizing and inhibitory effects. This invention provides specific antibody candidate molecules for the development of diagnostic reagents, preventive and therapeutic antibody drugs against SARS-CoV-2 (2019-nCoV), as well as for the treatment of infections caused by other coronaviruses. Attached Figure Description

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

[0053] in, Figure 1A-Figure 1B The results are shown for cell line ID8 with PE-labeled anti-human Fc and FITC-labeled anti-mouse Fc added only.

[0054] Figures 1C-1K The results of flow cytometry analysis were performed to detect antibodies and to detect the results of binding to ID8 cells overexpressing the novel coronavirus (2019-nCoV).

[0055] Figures 2A-2E The results of Octet Blitz detection of antibody binding to the Spike protein of the novel coronavirus (2019-nCoV) are shown in a preferred embodiment of the present invention.

[0056] in, Figure 2A The results show the binding of ACE2 to RBD and the full-length trimeric S protein.

[0057] Figure 2B This indicates that antibody CS1 binds to RBD and can competitively bind to ACE2.

[0058] Figure 2C The results show that antibodies CS1 and CS2 can bind to RBD simultaneously.

[0059] Figure 2D The results show the competitive binding of antibodies CS1 and CS8 to RBD.

[0060] Figure 2E This indicates that antibody CS1 can bind to RBD simultaneously with CS2-CS7.

[0061] Figure 3A The results of antibody CS1 competitively inhibiting ACE2 binding to ID8 in cells overexpressing the novel coronavirus (COVID-19) at different concentration gradients in a preferred embodiment of the present invention.

[0062] Figure 3B The results of antibody CS2 competitively inhibiting ACE2 binding to ID8 in cells overexpressing the novel coronavirus (COVID-19) at different concentration gradients in a preferred embodiment of the present invention.

[0063] Figure 3C The results of antibody CS3 competitively inhibiting ACE2 binding to ID8 in cells overexpressing the novel coronavirus (COVID-19) at different concentration gradients in a preferred embodiment of the present invention.

[0064] Figure 3D The results of antibody CS4 competitively inhibiting ACE2 binding to ID8 in cells overexpressing the novel coronavirus (COVID-19) at different concentration gradients in a preferred embodiment of the present invention.

[0065] Figure 3E The results of antibody CS5 competitively inhibiting ACE2 binding to ID8 in cells overexpressing the novel coronavirus (COVID-19) at different concentration gradients in a preferred embodiment of the present invention.

[0066] Figure 3F The results of antibody CS6 competitively inhibiting ACE2 binding to ID8 in cells overexpressing the novel coronavirus (COVID-19) at different concentration gradients in a preferred embodiment of the present invention.

[0067] Figure 3G The results of antibody CS7 competitively inhibiting ACE2 binding to ID8 in cells overexpressing the novel coronavirus (COVID-19) at different concentration gradients in a preferred embodiment of the present invention.

[0068] Figure 3H The results of antibody CS8 competitively inhibiting ACE2 binding to ID8 in cells overexpressing the novel coronavirus (COVID-19) at different concentration gradients in a preferred embodiment of the present invention.

[0069] Figure 4 This is a structural diagram of the novel coronavirus 2019-nCoV. Detailed Implementation

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

[0072] To obtain human antibodies specific to the Spike protein of the novel coronavirus (2019-nCoV), a solid-phase screening method was used. First, a Spike-coated RBD protein (mFc tag, Sino Biological, catalog number 40592-V05H) package was prepared. A human antibody library containing 10 billion phage particles expressing different antibodies was thawed. The RBD protein target wells coated overnight were washed with PBS (250 μL / well, washed twice). Phage particles were added to the RBD protein target wells and incubated at room temperature (RT) for 1 h. Elution buffer (0.2 M glycine-HCl, pH 2.2) was added (100 μL / well), and the mixture was allowed to stand for approximately 10 min, mixing twice by pipetting during this period. Neutralization buffer (1 M Tris-HCl, pH 8.0) was added (42 μL / well), and the mixture was mixed. The neutralized mixture was then added to 10 ml of TG1 (OD100) solution. 600Take approximately 0.6-0.8 μL of the phage-TG1 infection mixture, mix well, and incubate at 37°C for 30 min for saturation. Take approximately 20 μL of the phage-TG1 infection mixture, add it to 180 μL of 2YT, mix well, and record this as 20 μL-human. Take 20 μL of bacterial culture from the 20 μL-human mixture, add it to 180 μL of 2YT, mix well, and record this as 2 μL-human. Take 100 μL of bacterial culture from the 20 μL-human and 2 μL-human mixtures respectively, spread them onto 90 mm plates, and record this as 10 μL-human and 1 μL-human. Incubate overnight at 37°C for statistical analysis of the first round of output phage from the human natural antibody library. For the remaining TG1 bacterial culture, incubate at 2400g for 10 min, discard the supernatant, resuspend in approximately 600 μL of 2YT, spread on 180 mm plates (2YTAG), and incubate overnight at 30°C. The following day, the number of colonies on 10ul and 1ul labeled plates on 90mm petri dishes was counted to calculate the first round of output from the human natural antibody library. At the same time, about 2.5ml of 2YT was used to scrape off the output bacterial growth on the large petri dish, which was then transferred to a 5ml centrifuge tube, mixed, and 900ul of bacterial culture was aspirated and added to 300ul of 50% glycerol. The culture was mixed and stored at -80℃, i.e., the first output-human-bacterial culture. In addition, 300ul of bacterial culture was aspirated, about 100ul of 2YT was added, mixed, and temporarily stored at 4℃ for inoculation and preparation of the phage after the first round of screening.

[0073] To obtain more specific antibody clones with higher affinity, further rounds of panning are required. For this purpose, the phage antibody solution eluted from the first round of panning is used to infect logarithmic-phase *E. coli* (such as the TG1 strain) capable of being infected by M13 phage, yielding an infected solution. A small amount is then subjected to a series of 10-fold serial dilutions (usually diluted to 1 / 1,000,000,000 of the original solution, with the last three dilutions used for plating) to determine the output titer of the first round of elution. This titer is also known as the first-round maximum diversity, and typically the output titer after the first round of panning is below 10E6 CFU. All remaining infection fluid was spread onto bacterial culture plates containing the appropriate antibiotics and incubated overnight to obtain colonies. The colony layer was scraped off and resuspended in culture medium. A sufficient volume of the resuspending containing the diversity of the first round of output was transferred to a shake flask containing sufficient liquid culture medium (2YT-CG, 2YT medium with added Carbenicillin and glucose, final concentrations of 100 μg / ml and 2%, respectively). The resuspending was diluted to below OD600 and incubated until the logarithmic phase, when OD600 reaches approximately 0.5. To ensure that the antibodies obtained in the first round of panning reappear on the surface of the phage particles, 10 ml of bacterial culture was taken, and helper phage M13K07 was added to achieve a multiplicity of infection (MOI) of 20:1. The mixture was then incubated at 37°C for 30 minutes (this stage is phage rescue). Centrifuge and resuspend the bacterial cells in 50 ml of expression medium (2YT-AK, 2YT medium with added Carbenicillin and Kanamycin, final concentrations of 100 μg / ml and 30 μg / ml, respectively), and incubate overnight at 30°C and 200 rpm. The next day, centrifuge to harvest the culture supernatant, add 1 / 5 volume of PEG8000 / NaCl (PEG-8000 20%, NaCl 2.5M), mix thoroughly, and incubate on ice for 1 hour. Centrifuge at high speed (11500×g) for 30 minutes to harvest phage antibody particles. Resuspend the precipitate in 1 ml of PBS solution and centrifuge again at high speed to remove bacterial debris. The supernatant is the amplification solution after the first round of panning, containing each antibody clone amplified more than ten thousand times. This amplification solution can be used for the second round of panning experiments. The second round of panning is exactly the same as the first round, except that the washing with PBST / PBS is increased to 6 times each (6 / 6). In the third round, the number of washes can be further increased to 10 / 10. Multiple rounds of panning usually effectively enrich specific clones, significantly reducing diversity but increasing their affinity, which facilitates subsequent single-clone screening.

[0074] To obtain specific monoclonal antibodies, a monoclonal phage ELISA assay is required. For this purpose, well-separated single colonies obtained from second and / or third rounds of serial dilutions are individually inoculated into 96-well plates containing 2YT-AG (93 colonies per plate, leaving three wells as negative controls) and incubated overnight; this is the master plate. The bacterial culture from each well of the master plate is then inoculated into new culture plates and grown to the logarithmic phase for phage rescue, allowing antibody expression of each clone to occur on the phage surface. A standard 96-well ELISA plate is coated with BCMA antigen (1 μg / ml), and another ELISA plate is coated with the same concentration of human Fc. The bacterial culture of each individually expressed monoclonal phage antibody is added to the corresponding wells of both the RBD and mFc plates, followed by the addition of appropriate secondary antibodies and horseradish peroxidase (HRP)-conjugated triple antibodies. Substrate development is performed, and the absorbance is read (450 nM). The method for identifying RBD-positive clones is as follows: clones that are negative on mFc plates (with absorbance not exceeding 1.5 times that of the corresponding negative wells on the same plate) and positive on RBD plates (with absorbance exceeding 3 times that of the corresponding negative wells on the same plate), and whose absorbance values ​​are higher than those of the corresponding wells on the mFc plate. Analysis revealed 88 clones that were positive only for the RBD antigen and negative for mFc; these clones are collectively referred to as hits.

[0075] These hist bacterial cultures were inoculated from the corresponding wells of the mother plate into 3 ml of 2YT-CG and incubated overnight at 37°C and 200 rpm. The next day, phage DNA was extracted, and the sequence of the single-stranded antibody region containing each hit was determined using specific primers. The coding region DNA sequence was translated into amino acid sequences, and multiple sequence comparison (CLUSTALW, website link https: / / www.genome.jp / tools-bin / clustalw) was performed to determine clone specificity. Analysis showed that these 88 hits belonged to 26 different clones, among which 8 antibodies showed good binding activity to the novel coronavirus (2019-nCoV) Spike protein, thus obtaining the fully human antibody variable region sequence against the novel coronavirus (2019-nCoV) Spike protein. The heavy chain variable region CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:1, the heavy chain variable region CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:2 or 3, and the heavy chain variable region CDR3 contains or is composed of the amino acid sequence shown in any of SEQ ID NO:4-7; the light chain variable region CDR1 contains or is composed of the amino acid sequence shown in any of SEQ ID NO:8-12, the light chain variable region CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:13-16, and the light chain variable region CDR3 contains or is composed of the amino acid sequence shown in any of SEQ ID NO:17-19.

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

[0077] Example 2 Antibody Function Verification

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

[0079] After obtaining the scFv-Fc protein, the binding of the antibody to the novel coronavirus (2019-nCoV) Spike protein and RBD was detected using Octet Blitz, confirming the specific binding of the antibody to the novel coronavirus (2019-nCoV) Spike protein. 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; and CS8 is a monoclonal antibody containing the variable regions of SEQ ID NO:25 and 33. The apparent affinity of CS1 was 1.2 nM, that of CS2 was 2.1 nM, that of CS3 was 23.2 nM, that of CS4 was 48 nM, that of CS5 was 4.1 nM, that of CS6 was 35.2 nM, that of CS7 was 12.3 nM, and that of CS8 was 2.6 nM.

[0080] Flow cytometry analysis was performed on cell line ID8, which overexpresses the 2019-nCoV Spike protein, demonstrating that the antibody specifically binds to the overexpressed 2019-nCoV Spike protein on the cell membrane surface. Figures 1A-1K ).

[0081] Flow cytometry analysis was used to detect the inhibitory effects of antibodies on the binding of ACE2 to the ID8 cell line overexpressing the 2019-nCoV Spike protein. The results showed that all antibodies could partially inhibit the binding of ACE2 to the ID8 cell line overexpressing the 2019-nCoV Spike protein, and the inhibitory effect increased with increasing concentration. Figures 3A-3H ).

[0082] Example 3

[0083] Cell binding assay based on FACS analysis was used to detect the competitive binding of antibodies to the ID8 cell line, which specifically expresses the 2019-nCoV Spike protein, against ACE2.

[0084] The lowest concentration of ACE2-mFC protein in saturated ID8 cells was determined to be 0.02 μg / ml.

[0085] 1. Collect ID8 cells: Collect 0.5 × 10⁸ cells. 6 cells / tube.

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

[0087] 3. Antibody binding: Add antibodies CS1-CS8 (0-40 μg / ml) at different concentration gradients 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 and incubate on ice for 30 min.

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

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

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

[0092] 8. Analytical assay: After resuspending the cells in 100-200 μl of PBS, analyze them using a Beckman CytoFlex analyzer.

[0093] Experimental results showed that different antibodies had varying effects on blocking ACE2 and Spike proteins. At an antibody concentration of 20 μg / ml, the blocking efficiency was 10.81% for CS1, 16.78% for CS2, 41.94% for CS3, 5.51% for CS4, 4.36% for CS5, 24.89% for CS6, 27.26% for CS7, and 93.12% for CS8.

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

Claims

1. An antibody CS5 against the Spike protein of the 2019-nCoV novel coronavirus or its antigen-binding fragment, characterized in that, Antibody CS5 is a monoclonal antibody containing the heavy chain variable region shown in SEQ ID NO:23 and the light chain variable region shown in SEQ ID NO:

30.

2. The antibody obtained by modifying the antibody CS5 or its antigen-binding fragment according to claim 1, wherein the antibody is a single-chain antibody, Fab antibody, micro antibody, chimeric antibody or whole antibody.

3. The antibody according to claim 2, characterized in that, The complete antibody is an immunoglobulin IgG1, IgG2, IgA, IgE, IgM, IgG4, or IgD.

4. The gene encoding the antibody CS5 as described in claim 1 or 2.

5. A biological material containing the gene of claim 4, wherein the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or transgenic cell line.

6. Any of the following applications of the antibody CS5 of claim 1 or 2, the gene of claim 4, or the biological material of claim 5: 1) Application in the preparation of reagents or kits for the detection and diagnosis of the novel coronavirus 2019-nCoV; 2) For non-diagnostic purposes, detection of the novel coronavirus 2019-nCoV.

7. A composition containing the antibody CS5 as described in claim 1 or 2.

8. A detection reagent or kit containing the antibody CS5 as described in claim 1 or 2.

Images