CN116063473B

CN116063473B - Anti-novel coronavirus Spike protein antibody and application thereof

Info

Publication number: CN116063473B
Application number: CN202211600320.1A
Authority: CN
Inventors: 袁清安; 孟庆武; 白丽莉; 赵立坤; 李延虎; 薛晓莹; 郭大庆; 张吟秋; 郑珊珊; 温振国; 张国良; 陈小军; 杨吉
Original assignee: Excyte Beijing Pharmaceutical Technology Development Co ltd
Current assignee: Excyte Beijing Pharmaceutical Technology Development Co ltd
Priority date: 2020-05-19
Filing date: 2020-05-19
Publication date: 2026-06-09
Anticipated expiration: 2040-05-19
Also published as: CN116143912B; CN116444660B; CN116063473A; CN116102644A; CN113683687A; CN116143912A; CN116162157B; CN113683687B; CN116444660A; CN116162157A; CN116023484A; CN116253797A; CN116023484B; CN116253797B; CN116102644B

Abstract

The invention provides an anti-novel coronavirus Spike protein antibody and application thereof. And screening out the specific antibody of the anti-novel coronavirus S protein from a single-chain antibody library of non-immunized fully human sequences by utilizing genetic engineering and phage surface display library technology. The affinity of the anti-virus S protein antibody and the virus S protein is between 1nM and 50nM, and the antibody has an inhibition effect on the combination of the novel coronavirus S protein and a human receptor ACE2, so that the anti-virus S protein antibody has good capability of combining the S protein and potential neutralization inhibition effect. The invention provides specific antibody candidate molecules for developing diagnostic reagents, preventive and therapeutic antibody drugs for novel coronaviruses (2019-nCoV), and other diseases such as pneumonia treatment caused by coronaviruses.

Description

Anti-novel coronavirus Spike protein antibody and application thereof

The invention relates to a Chinese patent application with the application number of CN202010426201.3, the name of which is an anti-novel coronavirus Spike protein antibody and application thereof.

Technical Field

The invention relates to the fields of genetic engineering and immunology, in particular to an anti-novel coronavirus Spike protein antibody and application thereof.

Background

The novel coronavirus (2019-nCoV) is a novel coronavirus, and is beta-CoV belonging to the family Coronaviridae of the order Corona, together with SARS-CoV, and is a non-segmented single-stranded positive-strand RNA virus, each group of which has a genome length of about 30000 nucleotides. Unlike the middle east respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus (SARS-CoV), the novel coronavirus is the 7 th of the family of coronaviruses that infect humans. Based on the homology of the gene sequences, the novel coronavirus genome has 80% similarity to SARS, and the novel coronavirus has 40% similarity to the gene sequence of MERS-CoV.

The novel coronaviruses 2019-nCoV exhibit typical coronavirus structures (FIG. 4), including 5 untranslated regions (UTRs), replicase complex (orf 1 ab), S genes, E genes, M genes, N genes, 3 UTRs, and several unidentified unstructured open reading frames.

There is a key S protein (spike protein) in coronaviruses, comprising 2 subunits S1 and S2. S1 promotes the binding of the virus to host cell receptors and contains an important C-terminal RBD domain, which is responsible for receptor binding. The novel coronavirus RBD domain has a high degree of homology with SARS. Of the 5 key sites of SARS infection, 1 is retained by the novel coronavirus, and the remaining 4 have amino acid substitutions and alterations.

The S protein (spike) of coronavirus is combined into a trimer containing about 1300 amino acids, belonging to the first class of membrane fusion proteins (CLASS I VIRAL fusion proteins), and the same class of viral membrane fusion proteins also include Env protein of HIV, HA protein of influenza, gp protein of Ebola virus, etc. The S protein determines the host range and specificity of the virus and is also an important site of action for the host neutralizing antibodies. And is also a key target point of vaccine design.

Similar to other first type of viral membrane fusion proteins, the S protein contains two subunits (subnit), S1 and S2. S1 mainly comprises a receptor binding domain (receptorbinding domain, RBD) responsible for recognizing the receptor of the cell. S2 contains the essential elements required for the membrane fusion process, including an intrinsic membrane fusion peptide (HEPTAD REPEAT, HR), an aromatic amino acid-rich membrane proximal region (membrane proximal external region, MPER), and a transmembrane region (transmembrane, TM). The S1 protein can be further divided into two regions (domains), an N-terminal domain (NTD) and a C-terminal domain (CTD), wherein the conformation of the NTD is very similar to that of the galectin protein. Most of the RBD of coronavirus S proteins are located in CTD, such as SARS virus and middle east respiratory syndrome virus (MIDDLE EAST respiratory syndrome, MERS), etc. Only a small fraction of the RBDs of the beta coronavirus are located in NTDs, such as the mouse hepatitis virus (mouse hepatitisvirus, MHV). In addition, NTD of bovine coronavirus (bovine coronavirus, BCoV) and human coronavirus OC43 can bind specific sugar molecules (sialic acid, etc.), which are also involved in the invasion process of coronavirus. The envelope proteins of the same family of viruses require two distinct regions to recognize host receptors and effectively mediate the viral and cellular membrane fusion process. This is one of the important differences between coronavirus S proteins and other viroid membrane fusion proteins.

The currently found coronavirus receptors mainly include aminopeptidase N (animopeptidase N, APN), angiotensin converting enzyme 2 (angiotensin converting enzyme II, ACE 2), dipeptidylpeptidase 4 (DPP 4), and CEACAM1 (carcinoembryonicantigen-RELATED CELL adhesion molecule). Among the APN proteins with species specificity are receptors for human coronavirus 229E, feline coronavirus (FCoV) and porcine coronavirus TGEV. Human ACE2 is a receptor for SARS virus and NL 63. Human DPP4 is a receptor for MERS virus. The alpha subtype of the mouse CEACAM1 protein is the receptor for MHV. The complex crystal structure of many coronaviruses RBD binding to host receptors has been resolved, mainly including SARS-RBD-ACE2 complex, NL63-RBD-ACE2 complex, MERS-RBD-DPP4 complex, HKU4-RBD-DPP4 complex, and MHV-RBD-mCEACAM. Alpha. Complex crystal structure.

The related diseases caused by the infection of the novel coronavirus 2019-nCoV are not provided with corresponding vaccines and specific medicines at present, so that the development of 2019-nCoV diagnosis and treatment medicines is urgent.

Disclosure of Invention

The invention aims to provide an anti-novel coronavirus Spike protein antibody and application thereof.

To achieve the object of the present invention, in a first aspect, the present invention provides an anti-coronavirus Spike protein antibody or an active fragment thereof, wherein the CDR1 of the heavy chain variable region comprises or consists of the amino acid sequence shown as SEQ ID No. 1, the CDR2 of the heavy chain variable region comprises or consists of the amino acid sequence shown as SEQ ID No. 2 or 3, the CDR3 of the heavy chain variable region comprises or consists of the amino acid sequence shown as any one of SEQ ID nos. 4 to 7, the CDR1 of the light chain variable region comprises or consists of the amino acid sequence shown as any one of SEQ ID nos. 8 to 12, the CDR2 of the light chain variable region comprises or consists of the amino acid sequence shown as any one of SEQ ID nos. 13 to 16, and the CDR3 of the light chain variable region comprises or consists of the amino acid sequence shown as any one of SEQ ID nos. 17 to 19.

The invention provides an antibody variable region amino acid sequence, which has the mode of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In the present invention, the region division of FR and CDR is based on Kabat naming system. Here, FR1 to FR4 represent 4 framework regions and CDR1 to CDR3 represent 3 hypervariable regions. FR 1-FR 4 may be isolated from constant region sequences (e.g., the most common amino acids of the human immunoglobulin light and heavy chains, subclasses, or subfamilies), or may be isolated from the framework regions of a personal antibody or may be derived from a combination of different framework region genes.

The anti-novel coronavirus Spike protein antibody of the invention is:

i) The heavy chain variable region comprises or consists of an amino acid sequence as shown in any one of SEQ ID NOS.20-25, and the light chain variable region comprises or consists of an amino acid sequence as shown in any one of SEQ ID NOS.26-33;

ii) the antibody of i) is a derivative antibody of i) with equivalent functions by substituting, deleting or adding one or more amino acids;

iii) An antibody derived from i) having 70%, 80%, 85%, 90% or 97% or more sequence homology to the antibody of i) and having the same function.

Preferably any one of antibodies CS1 to CS 8:

CS1 heavy chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 20, and light chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 26;

CS 2-heavy chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 21, and light chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 27;

CS3 heavy chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 22, and light chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 28;

CS4 heavy chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 22, and light chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 29;

CS5 heavy chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 23, and light chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 30;

CS6 heavy chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 22, and light chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 31;

CS7 heavy chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 24, and light chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 32;

CS8 heavy chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 25, and light chain variable region comprises or consists of the amino acid sequence shown as SEQ ID NO. 33.

In a second aspect, the present invention provides antibodies engineered against the novel coronavirus Spike protein antibodies described above, or active fragments thereof, including but not limited to single chain antibodies, fab, miniantibodies, chimeric antibodies, whole antibody immunoglobulins IgG1, igG2, igA, igE, igM, igG4, igD, or the like.

The anti-novel coronavirus Spike protein antibody provided by the invention binds to the novel coronavirus (2019-nCoV) Spike protein with an affinity of 1nM-50 nM. The antibodies inhibit binding of novel coronavirus Spike proteins to human ACE2.

In a third aspect, the present invention provides a gene encoding the above antibody.

In view of the degeneracy of codons, genes encoding the antibodies of the invention may be modified in their coding regions, without altering the amino acid sequence, to obtain genes encoding the same antibodies. Those skilled in the art can artificially synthesize engineered genes to increase the expression efficiency of antibodies according to the codon preference of the host expressing the antibodies.

In a fourth aspect, the present invention provides biological materials comprising the genes, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, viral vectors, engineered bacteria or transgenic cell lines, and the like.

In a fifth aspect, the invention provides any one of the following uses of the antibody, gene encoding the antibody or biological material containing the gene:

1) The application of the novel coronavirus Spike protein in preparing a disease treatment drug or composition taking the novel coronavirus Spike protein as a target, wherein the drug is preferably a diagnostic and therapeutic drug for diseases caused by the novel coronavirus 2019-nCoV Spike protein;

2) The application in preparing a medicine or a composition for preventing or treating novel coronavirus 2019-nCoV infection or related diseases caused by the infection;

3) The application in preparing a cell therapeutic drug or a composition for preventing or treating novel coronavirus 2019-nCoV infection or related diseases caused by the infection;

4) Application in preparing novel coronavirus 2019-nCoV detection and diagnosis reagent or kit;

5) Use in the preparation of a related formulation of CAR-T therapy targeting a novel coronavirus Spike protein;

6) For detection (including non-diagnostic purposes) of novel coronaviruses 2019-nCoV;

7) For preventing or treating novel coronavirus 2019-nCoV infection or related diseases caused by the infection;

8) For CAR-T treatment.

In a sixth aspect, the invention provides a medicament or composition comprising said anti-novel coronavirus Spike protein antibody or active fragment thereof.

In a seventh aspect, the present invention provides a detection reagent or kit comprising said anti-novel coronavirus Spike protein antibody or active fragment thereof.

The antibody provided by the invention is a whole antibody or various other forms of genetically engineered antibodies. For example, the anti-novel coronavirus Spike protein antibody may be a whole antibody or an antibody fragment. The antibody molecules themselves may be used in therapy and diagnosis. Antibodies can be labeled, cross-linked or conjugated and expressed in fusion with other protein or polypeptide molecules to form complexes (e.g., cytotoxic substances, radioactive toxins, and/or chemical molecules, etc.) for diagnostic and therapeutic use.

Further, the invention provides independent genes encoding antibodies, expression vectors, related control techniques for vector transfection into host cells and host cells, antibody expression procedures and recovery of antibodies in cell culture supernatants. The invention also provides compositions comprising antibodies and pharmacologically acceptable delivery molecules or solutions. The therapeutic components are sterile and can be lyophilized.

The present invention provides an antibody against the Spike protein of novel coronavirus (2019-nCoV), which also acts by blocking the binding of the Spike protein of novel coronavirus (2019-nCoV) to ACE 2. The interference function of the novel coronavirus (2019-nCoV) Spike protein antagonist falls within the protection scope of the invention.

The sequences shown in SEQ ID NOS.1-33 in the present invention include "conservative sequence modifications", i.e., nucleotide and amino acid sequence modifications that do not significantly affect or alter the binding characteristics of the antibody or 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 (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace an unnecessary amino acid residue in a human anti-novel coronavirus (2019-nCoV) Spike protein antibody with another amino acid residue from the same side chain family.

Antibodies comprising a specific amino acid composition of the invention include antibodies that are substantially encoded by or comprise a similar sequence modified by a conserved sequence, and fall within the scope of the invention.

The invention provides a bispecific or multispecific molecule comprising an antibody or antigen-binding portion of an antibody provided herein.

The invention provides a fusion protein of an antibody and other proteins and/or polypeptides, comprising a complex of the antibody provided by the invention and other protein or polypeptide molecules with certain functions.

Furthermore, the fusion protein is a recombinant expression vector constructed by connecting antibody genes with immunotoxin or cytokine genes, and recombinant fusion protein molecules are obtained through mammalian cells or other expression systems.

The novel coronavirus (2019-nCoV) Spike protein antibody provided by the invention has good therapeutic application prospect, and is mainly expressed as having specific binding activity with the novel coronavirus (2019-nCoV) Spike protein. ELISA detection and flow cytometry detection show that the antibody has good target specificity.

The invention utilizes genetic engineering and phage surface display library technology to screen out specific antibody of anti-new coronavirus S protein from the single-chain antibody library of non-immune fully human sequence. The apparent affinity of the antigen and the virus S protein is 1nM-50nM, and the antigen has an inhibition effect on the binding of the novel coronavirus S protein and a human receptor ACE2, which shows that the anti-virus S protein antibody has good binding capacity and potential neutralization inhibition effect on the S protein. The invention provides specific antibody candidate molecules for developing diagnostic reagents, preventive and therapeutic antibody drugs for novel coronaviruses (2019-nCoV), and other diseases such as pneumonia treatment caused by coronaviruses.

Drawings

FIGS. 1A-1K are flow cytometry charts of antibody-binding over-expression of novel coronavirus (2019-nCoV) cell ID8 in a preferred embodiment of the invention. The binding of the antibody to ID8 was detected by FACS analysis using a Beckman flow cytometer CytoFlex, and the antibody was converted to scFv-Fc form and purified by expression and added to 200,000 ID8 cells at a final concentration of 10. Mu.g/ml for incubation. The fluorescent secondary antibody was PE-labeled anti-human Fc, FITC-labeled anti-murine Fc.

Among them, FIGS. 1A-1B show the results of adding only PE-labeled anti-human Fc and FITC-labeled anti-murine Fc, respectively, to cell line ID 8.

FIG. 1C-FIG. 1K shows the results of flow cytometry detection of antibodies and binding of over-expressed novel coronavirus (2019-nCoV) cell ID 8.

FIGS. 2A-2E show the binding of the Octet Blitz detection antibody to the Spike protein of novel coronaviruses (2019-nCoV) in a preferred embodiment of the present invention.

Among them, figure 2A shows the results of ACE2 binding RBD and full length trimeric S protein.

Fig. 2B shows the results of binding of antibody CS1 to RBD and competing for binding to ACE 2.

FIG. 2C is a graph showing the results that antibodies CS1 and CS2 can bind to RBD simultaneously.

FIG. 2D is the result of antibodies CS1 and CS8 competing for RBD binding.

FIG. 2E is a graph showing the results that antibody CS1 can bind to RBD simultaneously with CS2-CS 7.

FIG. 3A shows the results of competing inhibition of ACE2 binding to over-expressed novel coronavirus (COVID-19) cell ID8 by antibody CS1 at different concentration gradients in a preferred embodiment of the invention.

FIG. 3B shows the results of competing inhibition of ACE2 binding to over-expressed novel coronavirus (COVID-19) cell ID8 by antibody CS2 at different concentration gradients in a preferred embodiment of the invention.

FIG. 3C shows the results of competing inhibition of ACE2 binding to over-expressed novel coronavirus (COVID-19) cell ID8 by antibody CS3 at different concentration gradients in a preferred embodiment of the invention.

FIG. 3D shows the results of competing inhibition of ACE2 binding by antibody CS4 over the novel coronavirus (COVID-19) cell ID8 under different concentration gradients in a preferred embodiment of the invention.

FIG. 3E shows the results of competing inhibition of ACE2 binding by antibody CS5 over the novel coronavirus (COVID-19) cell ID8 under different concentration gradients in a preferred embodiment of the invention.

FIG. 3F shows the results of competing inhibition of ACE2 binding to the novel coronavirus (COVID-19) cell ID8 by antibody CS6 under different concentration gradients in a preferred embodiment of the invention.

FIG. 3G shows the results of competing inhibition of ACE2 binding by antibody CS7 over the novel coronavirus (COVID-19) cell ID8 under different concentration gradients in a preferred embodiment of the invention.

FIG. 3H shows the results of competing inhibition of ACE2 binding by antibody CS8 over the novel coronavirus (COVID-19) cell ID8 under different concentration gradients in a preferred embodiment of the invention.

FIG. 4 is a schematic diagram of the structure of novel coronaviruses 2019-nCoV.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.

Example 1 screening of antibodies against novel coronavirus (2019-nCoV) Spike proteins from a Natural human antibody phage surface-presented library

To obtain novel coronavirus (2019-nCoV) Spike protein-specific human antibodies, panning was performed using a solid phase screening method. The Spike RBD protein (mFc tag, sino biological, cat# 40592-V05H) was first coated. A pool of human antibodies was thawed and contained 100 million phage particles expressing different antibodies. The overnight coated RBD protein target wells were washed with PBS, 250 ul/well, 2 washes, phage particles added to RBD protein target wells, room Temperature (RT), 1h. Adding 0.2M glycine-HCl (pH 2.2) as eluent, 100 ul/hole, standing for about 10min, blowing with gun for 2 times, adding 1M Tris-HCl (pH 8.0) as neutralizing solution, and mixing. The neutralized mixtures were taken and added to 10ml of TG1 (OD ₆₀₀. About.0.6-0.8) respectively, mixed well and incubated at 37℃for 30min for dip-dyeing. Taking a phage-TG1 infection mixture, adding about 20ul to 180 ul-2 YT, uniformly mixing, marking as 20 ul-human, taking out 20ul bacterial liquid from 20 ul-human, adding to 180 ul-2 YT, uniformly mixing, marking as 2 ul-human, taking out 100ul bacterial liquid from 20 ul-human and 2 ul-human respectively, coating a 90mm dish, marking as 10 ul-human and 1 ul-human, and culturing at 37 ℃ overnight for counting the first round of output phage of a natural human antibody library. The remaining TG1 stock was resuspended in 600ul 2YT at 2400g for 10min, and plated on 180mm dishes (2 YTAG) at 30℃overnight. Counting the colony number of 10ul and 1ul labeled plates on a small plate of 90mm the next day, calculating the first round of output of a natural human antibody library, scraping off the output fungus coating on a large plate by using about 2.5ml 2YT, sucking out to a 5ml centrifuge tube, uniformly mixing, sucking out 900ul fungus liquid, adding 300ul of 50% glycerol, uniformly mixing, preserving the fungus to-80 ℃, namely 1st output-human-fungus liquid, sucking out 300ul fungus liquid, supplementing about 100ul of 2YT, uniformly mixing, temporarily preserving at 4 ℃ for fungus inoculation and preparing the first round of screened fungus.

To further harvest specific antibody clones with higher affinity, more rounds of panning were required. To this end, log phase E.coli (e.g., strain TG 1) infected with M13 phage was infected with phage antibody solution eluted from the first round of panning to obtain an infection solution, and a series of 10-fold gradient dilutions (usually diluted to one part per million of stock solution and spread with the last three gradients) were taken in small amounts to determine the titer of output of the eluate from the first round (titer), which titer is also referred to as the first round of maximum diversity, typically the output titer after the first round of panning was below 10E6 cfu. The rest of the infection solution was spread on a bacterial culture plate containing the corresponding antibiotics all over night to obtain colonies, the colony layer was scraped off and resuspended in medium, a sufficient amount of the resuspension containing the first round of output diversity was taken to a shake flask containing a sufficient amount of liquid medium (2 YT-CG,2YT medium, carbenicillin and glucose, final concentrations 100 μg/ml and 2%) respectively, the resuspension was diluted to below od600=0.1 and cultivation was started until the log phase, i.e. the OD600 reached around 0.5. To allow these antibodies obtained in the first round of panning to reappear to the phage particle surface, 10ml of bacterial liquid was taken, helper phage M13K07 was added to give a multiplicity of infection MOI of 20:1, and the mixture was allowed to stand at 37℃for 30 minutes (this stage was phage rescue). Centrifugation was performed, and cells were resuspended in 50ml of an expression medium (2 YT-AK, carbenicillin and KANAMYCIN were added to the 2YT medium, and the final concentrations were 100. Mu.g/ml and 30. Mu.g/ml, respectively) and incubated at 30℃for 200 rpm overnight. The culture supernatant was harvested by centrifugation the next day, 1/5 volume of PEG8000/NaCl (PEG-800020%, naCl 2.5M) was added, thoroughly mixed, and incubated on ice for 1 hour. High speed centrifugation (11500 Xg) for 30 minutes to harvest phage antibody particles. The pellet was resuspended in 1ml PBS and centrifuged again at high speed to remove bacterial debris. The supernatant is the amplification solution after the first round of panning, and each antibody clone contained in the supernatant is amplified by more than ten thousand times. This amplification solution was used for the second round of panning experiments. The second round of panning operation was identical to the first round except that the addition was made to 6 (6/6) runs each when washed with PBST/PBS. In the third wheel, the number of washes can be further increased to 10/10. Multiple rounds of panning will generally be effective to enrich for specific clones, with significantly reduced diversity but with higher affinity for subsequent monoclonal screening.

In order to obtain specific monoclonal antibodies, a monoclonal phage enzyme-linked assay (Monophage ELISA) is performed. For this purpose, single colonies which were well isolated in the second and/or third rounds of gradient dilution were individually inoculated into 96-well plates containing 2YT-AG (93 colonies were inoculated per plate, leaving three wells as negative controls), and cultured overnight, i.e., as a master plate (MASTER PLATE). Bacterial liquid of each hole in the mother board is inoculated into a new culture board to grow to a logarithmic phase, phage rescue is carried out, and antibodies of each clone are expressed on the surface of phage. BCMA antigen (1 μg/ml) was coated on a common 96-well plate while another plate was coated with the same concentration of human Fc. Each separately expressed monoclonal phage antibody bacterial solution was added to the corresponding wells of RBD plates and mFc plates, followed by addition of the appropriate secondary antibody and horseradish peroxidase (HRP) -conjugated tertiary antibody, substrate development, and absorbance (450 nM) read. The method for judging the RBD positive clone is that the RBD plate is negative (not more than 1.5 times of the absorption value of the plate negative hole), the RBD plate is positive (more than 3 times of the absorption value of the plate negative hole), and the hole absorption value of the RBD plate is higher than the absorption value of the corresponding hole on the mFc plate. The clones corresponding to 88 wells, analyzed, showed positive only for RBD antigen and negative for mFc, collectively referred to as hits.

These hist bacteria were inoculated onto 3ml of 2YT-CG from the corresponding wells of the master plate, and incubated at 37℃for 200 rpm overnight. The next day phagemid DNA was extracted and the sequence of the single-chain antibody region containing each hit was determined with specific primers. Coding region DNA sequences were translated into amino acid sequences and subjected to multiple sequence comparisons (CLUSTALW, website linking https:// www.genome.jp/tools-bin/CLUSTALW) to determine clone specificity. Through analysis, the 88 hits are 26 different clones in sequence, wherein 8 antibodies have good binding activity to the Spike protein of the novel coronavirus (2019-nCoV), and thus the fully human antibody variable region sequence of the Spike protein of the novel coronavirus (2019-nCoV) is obtained. CDR1 of the heavy chain variable region comprises or consists of an amino acid sequence shown as SEQ ID NO. 1, CDR2 of the heavy chain variable region comprises or consists of an amino acid sequence shown as SEQ ID NO.2 or 3, CDR3 of the heavy chain variable region comprises or consists of an amino acid sequence shown as any one of SEQ ID NO. 4-7, CDR1 of the light chain variable region comprises or consists of an amino acid sequence shown as any one of SEQ ID NO. 8-12, CDR2 of the light chain variable region comprises or consists of an amino acid sequence shown as any one of SEQ ID NO. 13-16, and CDR3 of the light chain variable region comprises or consists of an amino acid sequence shown as any one of SEQ ID NO. 17-19.

Further, the heavy chain variable region comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS.20-25, and the light chain variable region comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS.26-33.

Example 2 functional validation of antibodies

To verify whether the obtained clone of novel coronavirus (2019-nCoV) Spike protein antibody binds to novel coronavirus (2019-nCoV) Spike protein antigen and novel coronavirus (2019-nCoV) Spike protein expressed on the surface of cell membrane, the gene of novel coronavirus (2019-nCoV) Spike protein single chain antibody was cloned into 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 or detected using (HRP or fluorescein) -labeled anti-human Fc antibodies.

After the scFv-Fc protein was obtained, the binding of the antibodies to the novel coronavirus (2019-nCoV) Spike protein and RBD was examined using Octet Blitz, confirming the specific binding of the novel coronavirus (2019-nCoV) Spike protein antibodies (FIGS. 2A-2E). CS1 is a monoclonal antibody containing variable regions of SEQ ID NO. 20 and 26, CS2 is a monoclonal antibody containing variable regions of SEQ ID NO. 21 and 27, CS3 is a monoclonal antibody containing variable regions of SEQ ID NO. 22 and 28, CS4 is a monoclonal antibody containing variable regions of SEQ ID NO. 22 and 29, CS5 is a monoclonal antibody containing variable regions of SEQ ID NO. 23 and 30, CS6 is a monoclonal antibody containing variable regions of SEQ ID NO. 22 and 31, CS7 is a monoclonal antibody containing variable regions of SEQ ID NO. 24 and 32, and CS8 is a monoclonal antibody containing variable regions of SEQ ID NO. 25 and 33. CS1 apparent affinity is 1.2nM, CS2 apparent affinity is 2.1nM, CS3 apparent affinity is 23.2nM, CS4 apparent affinity is 48nM, CS5 apparent affinity is 4.1nM, CS6 apparent affinity is 35.2nM, CS7 apparent affinity is 12.3nM, and CS8 apparent affinity is 2.6nM.

Cell line ID8, which over-expressed the novel coronavirus (2019-nCoV) Spike protein, was detected with a flow cytometer, indicating that the antibody specifically binds to the cell membrane surface over-expressed the novel coronavirus (2019-nCoV) Spike protein (FIGS. 1A-1K).

The detection of the antibody pair by a flow cytometer inhibits the ACE2 from binding to the cell line ID8 of the Spike protein of the over-expressed novel coronavirus (2019-nCoV), and the result shows that the antibody can partially inhibit the ACE2 from binding to the cell line ID8 of the Spike protein of the over-expressed novel coronavirus (2019-nCoV) and the inhibition effect is enhanced along with the increase of the concentration (figures 3A-3H).

Example 3

Cell binding method based on FACS ANALYSIS detects that antibody competes with ACE2 for binding to Cell line ID8 specifically expressing 2019-nCoV Spike protein.

The minimum concentration of ACE2-mFC protein of saturated ID8 cell Spike protein was determined to be 0.02 μg/ml.

1. ID8 cells were collected, 0.5X10 ⁶ cells/tube were collected.

2. Cells were rinsed once with 1ml staining buffer (PBS containing 1% w/v BSA), centrifuged at 350Xg for 5min at 4℃and resuspended in 95. Mu. L STAINING buffer after centrifugation.

3. Antibody binding antibodies CS1-CS8 (0-40. Mu.g/ml) were added at different concentration gradients and incubated on ice for 60min.

4. ACE2-mFC binding human ACE2-mFC protein was added to a concentration of 0.02. Mu.g/ml, respectively, and incubated on ice for 30min.

5. Rinsing the cells, namely adding 1ml staining buffer parts of the cell suspension, centrifuging for 5 minutes at the temperature of 4 ℃ after uniform mixing, removing the supernatant, and rinsing for 2 times again. After centrifugation, the cells were resuspended in 100. Mu. L STAINING buffer.

6. The sample tube was added with 5. Mu.l of the Biolegend direct antibody (APC anti-Mousw IgG Fc Antibody, biolegend, 405308) and incubated for 15-20min on ice in the absence of light.

7. Rinsing the cells, namely adding 1ml staining buffer parts of the cell suspension, centrifuging for 5 minutes at the temperature of 4 ℃ after uniform mixing, removing the supernatant, and rinsing again.

8. After resuspension of cells with 100-200. Mu.l PBS, the assay was performed using Beckman CytoFlex.

Experimental results show that different antibodies have different effects of blocking ACE2 and Spike proteins. At an antibody concentration of 20 μg/ml, the CS1 blocking effect was 10.81%, the CS2 blocking effect was 16.78%, the CS3 blocking effect was 41.94%, the CS4 blocking effect was 5.51%, the CS5 blocking effect was 4.36%, the CS6 blocking effect was 24.89%, the CS7 blocking effect was 27.26%, and the CS8 blocking effect was 93.12%.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. The novel anti 2019-nCoV coronavirus Spike protein antibody CS1 or antigen-binding fragment thereof is characterized in that the antibody CS1 is a monoclonal antibody containing a heavy chain variable region shown as SEQ ID NO. 20 and a light chain variable region shown as SEQ ID NO. 26.

2. The antibody CS1 or an antigen-binding fragment thereof of claim 1, which is a single chain antibody, fab, minibody, chimeric antibody, or whole antibody.

3. The antibody of claim 2, wherein the whole antibody is an immunoglobulin IgG1, igG2, igA, igE, igM, igG4 or IgD.

4. A gene encoding the antibody CS1 as claimed in claim 1 or 2.

5. A biological material comprising the gene according to claim 4, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineering bacterium or a transgenic cell line.

6. The antibody CS1 of claim 1 or 2, the gene of claim 4 or any of the following uses of the biological material of claim 5:

1) Application in preparing novel coronavirus 2019-nCoV detection and diagnosis reagent or kit;

2) Detection of novel coronaviruses 2019-nCoV for non-diagnostic purposes.

7. A composition comprising the antibody CS1 as claimed in claim 1 or 2.

8. A detection reagent or kit comprising the antibody CS1 as claimed in claim 1 or 2.