Anti-coronavirus fully human broad-spectrum neutralizing antibody 76E1 and its applications

Abstract

The present invention discloses an anti-coronavirus fully human broad-spectrum neutralizing antibody and its application. Specifically, the present invention discloses a fully human monoclonal antibody against the S2 region of coronavirus S protein, a nucleic acid sequence encoding the antibody, and a method for preparing the same. The antibody can effectively and broadly bind to and neutralize various coronaviruses, and can be used for the prevention and treatment of coronavirus infection-related diseases such as SARS-CoV-2, and further discloses its potential application as a vaccine design.

Description

[Technical Field]

[0001] The present invention relates to the field of medicine, specifically to the anti-coronavirus fully human broad-spectrum neutralizing antibody 76E1 and its applications. [Background technology]

[0002] The emergence of SARS-CoV-2 has once again sparked increased attention and research on coronaviruses. Common coronaviruses, such as 229E-CoV, OC43-CoV, and NL63-CoV, only cause mild respiratory illness. However, SARS-CoV-2, SARS-CoV, and MERS-CoV have stronger infectious and pathogenic properties, posing a serious threat to human health and economic and social stability. As of August 31, 2020, a total of 25.315 million cases had been diagnosed worldwide, with 846,000 deaths (Real-time Tracking of Novel Coronavirus Pneumonia 2020 / 08 / 31). Therefore, the development of effective prevention and treatment methods is urgently needed.

[0003] Clinical intervention with monoclonal antibodies has been highly effective in preventing and treating viral infections and has been successfully used clinically to prevent respiratory syncytial virus infection. A large amount of preclinical and early clinical data indicates that monoclonal antibodies can effectively prevent and treat infections caused by various viruses. Therefore, the development of prophylactic and therapeutic neutralizing antibodies will be extremely valuable in combating the current novel coronavirus epidemic and future outbreaks of novel and sudden coronavirus infections.

[0004] The spike protein on the surface of coronaviruses is an important antigen for inducing neutralizing antibodies, which exert their neutralizing effects by blocking the binding of the S protein to receptors or inhibiting fusion of the virus with the host cell membrane. The S protein consists of two functional domains, S1 and S2. S1 binds to specific receptors on host cells to promote viral infection, and its sequence is highly diverse among various coronaviruses. S2 mediates fusion of the virus with the cell membrane and is more conserved than S1. Currently, many laboratories in China and abroad have successively isolated fully human monoclonal antibodies targeting the SARS-CoV-2 spike protein from recovered COVID-19 patients. Most of these antibodies target the receptor-binding domain (RBD) of the S protein and block the interaction between the RBD and the host receptor angiotensin-converting enzyme 2 (ACE2), thereby inhibiting viral infection of host cells. Due to differences in the RBDs of various coronaviruses, these antibodies can only neutralize SARS-CoV-2 and lack broad-spectrum neutralizing activity against other coronaviruses, or they have weak cross-neutralizing activity against SARS-CoV alone, making them ineffective against future or unexpected novel coronaviruses. Recently, numerous publications have reported that SARS-CoV-2 is mutating worldwide, and some mutations, particularly those in the RBD, render previously isolated neutralizing antibodies ineffective. The emergence of resistance sites for RBD-targeting neutralizing antibodies limits their scope of use. Therefore, it is particularly important to discover broad-spectrum coronavirus neutralizing antibodies and antibodies against neoepitopes. Currently, no antibodies targeting the conserved S2 region of novel coronaviruses have been reported, and systematic research is lacking.

[0005] Therefore, there remains a need in the art to develop more effective fully human monoclonal antibodies that can prevent and control novel coronavirus infection. Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to provide fully human broad-spectrum neutralizing antibodies that can prevent and control coronavirus infections. [Means for solving the problem]

[0007] A first aspect of the present invention provides a heavy chain variable region of an antibody, the heavy chain variable region comprising: CDR1 as shown in SEQ ID NO.:3, CDR2 as shown in SEQ ID NO.:4, and It contains three complementarity determining regions CDRs, the CDR3 of which is shown in SEQ ID NO.:5.

[0008] In another preferred example, any one of the amino acid sequences among the above amino acid sequences further includes a derivative sequence that optionally has at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid added, deleted, modified and / or substituted and can retain the binding affinity of the coronavirus S protein (preferably S2 protein).

[0009] In another preferred example, the heavy chain variable region further comprises a human-derived FR region or a mouse-derived FR region. In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.:1.

[0010] A second aspect of the invention provides an antibody heavy chain, said heavy chain having a heavy chain variable region according to the first aspect of the invention. In another preferred example, the heavy chain of the antibody further comprises a heavy chain constant region. In another preferred example, the heavy chain constant region is derived from a human, a mouse, or a rabbit.

[0011] A third aspect of the present invention provides a light chain variable region of an antibody, the light chain variable region comprising: CDR1' as shown in SEQ ID NO.:6, CDR2' having the amino acid sequence EVN, and It contains three complementarity determining regions CDRs, the CDR3' of which is shown in SEQ ID NO.:8.

[0012] In another preferred example, any one of the amino acid sequences among the above amino acid sequences further includes a derivative sequence that optionally has at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid added, deleted, modified and / or substituted and can retain the binding affinity of the coronavirus S protein (preferably S2 protein).

[0013] In another preferred example, the light chain variable region further comprises a human-derived FR region or a mouse-derived FR region. In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID NO.:2.

[0014] A fourth aspect of the invention provides a light chain of an antibody, said light chain having a light chain variable region according to the third aspect of the invention. In another preferred example, the light chain of the antibody further comprises a light chain constant region. In another preferred example, the light chain constant region is derived from a human, a mouse, or a rabbit.

[0015] A fifth aspect of the present invention provides an antibody, the antibody comprising: (1) a heavy chain variable region according to the first aspect of the present invention, and / or (2) having a light chain variable region according to the third aspect of the present invention; Alternatively, the antibody has a heavy chain according to the second aspect of the invention and / or a light chain according to the fourth aspect of the invention.

[0016] In another preferred embodiment, the antibody is a specific anti-coronavirus antibody, preferably a specific anti-S protein (preferably S2 protein) antibody. In another preferred embodiment, the antibody is selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.

[0017] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody. In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody. In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0018] In another preferred embodiment, the antibody is in the form of a drug conjugate. In another preferred example, the antibody heavy chain variable region sequence is as shown in SEQ ID NO.:1 and the antibody light chain variable region sequence is as shown in SEQ ID NO.:2.

[0019] A sixth aspect of the present invention provides a recombinant protein, the recombinant protein comprising: (i) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; and (ii) have an optional tag sequence to aid in expression and / or purification;

[0020] In another preferred example, the tag sequence includes a 6His tag, a GGGS sequence, and a FLAG tag. In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein. In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

[0021] In another preferred embodiment, the recombinant protein specifically binds to the coronavirus S protein, preferably the S2 protein, more preferably the peptide at positions 809-823 of the S protein, and most preferably the peptide at positions 809-833 of the S protein.

[0022] A seventh aspect of the present invention provides a CAR construct, wherein the scFv segment of the antigen binding region of the CAR construct is a binding region that specifically binds to a coronavirus S protein (preferably S2 protein), and the scFv has a heavy chain variable region according to the first aspect of the invention and a light chain variable region according to the third aspect of the invention.

[0023] An eighth aspect of the present invention provides a recombinant immune cell, said immune cell expressing an exogenous CAR construct according to the seventh aspect of the invention. In another preferred embodiment, the immune cells are selected from the group consisting of NK cells and T cells. In another preferred embodiment, the immune cells are derived from a human or non-human mammal (eg, a mouse).

[0024] A ninth aspect of the present invention provides an antibody drug conjugate, said antibody drug conjugate comprising: (a) an antibody portion selected from the group consisting of a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, or a combination thereof; and (b) a coupling moiety coupled to said antibody moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0025] In another preferred example, the conjugate is selected from a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biotoxin, a cytokine (e.g., IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a magnetic nanoparticle, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), a chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticle, etc. In another preferred embodiment, the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.

[0026] A tenth aspect of the present invention provides use of an active ingredient selected from the group consisting of a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, or a combination thereof, wherein the active ingredient is used in the preparation of a drug, a reagent, a detection plate or a kit.

[0027] In another preferred embodiment, the reagent, detection plate or kit is used for detecting coronavirus. In another preferred embodiment, the medicament is used to treat or prevent a coronavirus infection. In another preferred embodiment, the reagent comprises a chip, an immunoparticle coated with an antibody.

[0028] An eleventh aspect of the present invention provides a pharmaceutical composition, said pharmaceutical composition comprising: (i) an active ingredient selected from the group consisting of a heavy chain variable region according to the first aspect of the present invention, a heavy chain according to the second aspect of the present invention, a light chain variable region according to the third aspect of the present invention, a light chain according to the fourth aspect of the present invention, or an antibody according to the fifth aspect of the present invention, a recombinant protein according to the sixth aspect of the present invention, an immune cell according to the eighth aspect of the present invention, an antibody-drug conjugate according to the ninth aspect of the present invention, or a combination thereof; and (ii) Contains a pharmaceutically acceptable carrier.

[0029] In another preferred embodiment, the pharmaceutical composition is a liquid formulation. In another preferred embodiment, the pharmaceutical composition is an injection. In another preferred embodiment, the pharmaceutical composition is used to prevent and / or treat coronavirus infection.

[0030] A twelfth aspect of the present invention provides a polynucleotide, the polynucleotide comprising: (1) a heavy chain variable region according to the first aspect of the present invention, a heavy chain according to the second aspect of the present invention, a light chain variable region according to the third aspect of the present invention, a light chain according to the fourth aspect of the present invention, or an antibody according to the fifth aspect of the present invention; or (2) a recombinant protein according to the sixth aspect of the present invention; (3) Encoding a polypeptide selected from the group consisting of the CAR constructs according to the seventh aspect of the invention. In another preferred embodiment, the polynucleotide has the sequence shown in SEQ ID NO.:8 and / or SEQ ID NO.:9.

[0031] A thirteenth aspect of the present invention provides a vector, said vector comprising a polynucleotide according to the twelfth aspect of the present invention. In another preferred embodiment, the vector comprises a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vector.

[0032] A fourteenth aspect of the present invention provides a genetically engineered host cell, the host cell comprising a vector according to the thirteenth aspect of the invention or having integrated into its genome a polynucleotide according to the twelfth aspect of the invention.

[0033] A fifteenth aspect of the present invention provides a method for detecting coronavirus in a sample, said method comprising: (1) contacting a sample with an antibody according to the fifth aspect of the invention; (2) detecting whether an antigen-antibody complex is formed, where the formation of the complex indicates the presence of coronavirus in the sample. In another preferred embodiment, the detection is for non-therapeutic and non-diagnostic purposes.

[0034] The present invention further provides a method for detecting a coronavirus S protein in a sample, the method comprising: (1) contacting a sample with an antibody according to the fifth aspect of the invention; (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of coronavirus S protein in the sample. In another preferred embodiment, the coronavirus S protein is a coronavirus S2 protein. In another preferred embodiment, the detection is for non-therapeutic and non-diagnostic purposes.

[0035] A sixteenth aspect of the present invention provides a detection plate, the detection plate comprising a substrate (support plate) and a test strip, the test strip comprising an antibody according to the fifth aspect of the present invention or an immunoconjugate according to the ninth aspect of the present invention.

[0036] A seventeenth aspect of the present invention provides a kit, said kit comprising: (1) a first container comprising an antibody according to the fifth aspect of the invention; and / or (2) a second container containing a secondary antibody against the antibody according to the fifth aspect of the present invention; Alternatively, the kit comprises a detection plate according to the sixteenth aspect of the present invention.

[0037] An eighteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, said method comprising: (a) culturing a host cell according to the fourteenth aspect of the invention under conditions suitable for expression; (b) isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.

[0038] A nineteenth aspect of the invention provides a method for treating a coronavirus infection, said method comprising administering to a subject in need thereof an antibody according to the fifth aspect of the invention, an antibody-drug conjugate of said antibody, or a CAR-T cell expressing said antibody, or a combination thereof.

[0039] A twentieth aspect of the present invention provides a vaccine composition, said composition comprising: (i) a polypeptide of the amino acid sequence of positions 809-833 of the SARS-CoV-2 S2 protein as set forth in SEQ ID NO.:10 or a subsequence thereof; and (ii) a vaccinally acceptable carrier, In (i), the partial sequence comprises the amino acid sequence of positions 809-823 of the SARS-CoV-2 S2 protein as shown in SEQ ID NO.:11.

[0040] In another preferred embodiment, the polypeptide has ≧85%, preferably ≧90%, more preferably ≧95% identity to the amino acid sequence as set forth in SEQ ID NO.:10. In another preferred example, the polypeptide has ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% identity to the amino acid sequence set forth in SEQ ID NO.:10.

[0041] In another preferred embodiment, the carrier is a pharmaceutically acceptable carrier. In another preferred embodiment, the pharmaceutically acceptable carrier comprises a liquid, preferably water, saline or a buffer. In another preferred embodiment, the carrier further comprises auxiliary substances, preferably fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances, and the like. In another preferred embodiment, the carrier further comprises a cell transfection reagent.

[0042] In another preferred embodiment, the vaccine composition is a two-component vaccine or a polycomponent vaccine. In another preferred embodiment, the vaccine composition is a vaccine for preventing novel coronavirus infection. In another preferred example, the vaccine composition may further comprise a vaccine component derived from one or more pathogens selected from the group consisting of SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, or a combination thereof.

[0043] In another preferred embodiment, the vaccine components include inactivated strains, attenuated strains, or proteins, polypeptides, nucleic acids, and the like. In another preferred embodiment, the vaccine composition further comprises an adjuvant. In another preferred embodiment, the adjuvant comprises a granular adjuvant and a non-granular adjuvant.

[0044] In another preferred embodiment, the granular adjuvant is selected from the group consisting of aluminum salts, water-in-oil emulsions, oil-in-water emulsions, nanoparticles, microparticles, liposomes, immune stimulating complexes, or combinations thereof. In another preferred embodiment, the non-granular adjuvant is selected from the group consisting of muramyl dipeptide and its derivatives, saponin, lipid A, cytokines, derived polysaccharides, bacterial toxins, microorganisms and products thereof such as Mycobacterium (Mycobacterium tuberculosis, BCG), Brevibacterium, Bordetella pertussis, propolis, or a combination thereof.

[0045] In another preferred embodiment, the vaccine composition is in the form of an injection. In another preferred embodiment, the vaccine composition is a recombinant subunit vaccine, a vector vaccine, a synthetic peptide vaccine, a nucleic acid vaccine, or a combination thereof.

[0046] A twenty-first aspect of the present invention provides an inhibitor that targets a linear epitope of SARS-CoV-2 S2 protein 809-823 or SARS-CoV-2 S2 protein 809-833 and is used to inhibit infection with the novel coronavirus. In another preferred embodiment, the inhibitor is (a) a polypeptide comprising the amino acid sequence of positions 809-833 of the SARS-CoV-2 S2 protein as shown in SEQ ID NO.:10 or a subsequence thereof, wherein the subsequence comprises the amino acid sequence of positions 809-823 of the SARS-CoV-2 S2 protein as shown in SEQ ID NO.:11.

[0047] In another preferred embodiment, the inhibitor is a polypeptide comprising the amino acid sequence of positions 809-823 of the SARS-CoV-2 S2 protein as shown in SEQ ID NO.:10. In another preferred embodiment, the inhibitor is a polypeptide comprising the amino acid sequence of positions 809-823 of the SARS-CoV-2 S2 protein as shown in SEQ ID NO.:11.

[0048] In another preferred embodiment, the polypeptide has an amino acid sequence identity of ≧85%, preferably ≧90%, more preferably ≧95% with that shown in SEQ ID NO.:10 or 11. In another preferred example, the polypeptide has an amino acid sequence identity of ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% as set forth in SEQ ID NO.:10 or 11.

[0049] In another preferred embodiment, the inhibitor is a polypeptide as set forth in SEQ ID NO.:10 or 11. In another preferred embodiment, the inhibitor is (b) a small molecule compound that targets the 809-833 linear epitope of SARS-CoV-2 S2 protein or the 809-823 linear epitope of SARS-CoV-2 S2 protein. In another preferred embodiment, the inhibitor is a combination of (a) and (b). [Effects of the Invention]

[0050] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here due to space limitations. [Brief explanation of the drawings]

[0051] [Figure 1] 1 shows the binding activity of the 76E1 antibody to the full-length S protein, S1 protein, and S2 protein of the coronavirus SARS-CoV-2, where the 76E1 antibody can bind to the full-length S protein and S2 protein of SARS-CoV-2, but cannot bind to the S1 protein. [Figure 2A-D] The 76E1 antibody is capable of binding broadly to the S proteins of various coronaviruses. Figures 2A-C show the binding profiles of 76E1 / 76F6 / 28-12 to the S proteins of seven human coronavirus strains, Figure 2D shows the results of affinity analysis between 76E1 and in vitro purified coronavirus S proteins from seven strains, Figures 2E-G show the results of flow cytometry analysis of the binding activity of 76E1 to cell surface S proteins, and Figures 2H-I show the ability of the 76E1 antibody to bind broadly to various novel coronavirus mutant strains. [Figure 2E-H] The 76E1 antibody is capable of binding broadly to the S proteins of various coronaviruses. Figures 2A-C show the binding profiles of 76E1 / 76F6 / 28-12 to the S proteins of seven human coronavirus strains, Figure 2D shows the results of affinity analysis between 76E1 and in vitro purified coronavirus S proteins from seven strains, Figures 2E-G show the results of flow cytometry analysis of the binding activity of 76E1 to cell surface S proteins, and Figures 2H-I show the ability of the 76E1 antibody to bind broadly to various novel coronavirus mutant strains. [Figure 2I]The 76E1 antibody is capable of binding broadly to the S proteins of various coronaviruses. Figures 2A-C show the binding profiles of 76E1 / 76F6 / 28-12 to the S proteins of seven human coronavirus strains, Figure 2D shows the results of affinity analysis between 76E1 and in vitro purified coronavirus S proteins from seven strains, Figures 2E-G show the results of flow cytometry analysis of the binding activity of 76E1 to cell surface S proteins, and Figures 2H-I show the ability of the 76E1 antibody to bind broadly to various novel coronavirus mutant strains.

[0052] [Figure 3] This shows that the 76E1 antibody can broadly neutralize various coronaviruses and pseudoviruses of novel coronavirus mutant strains. [Figure 4] We demonstrate that the 76E1 antibody can neutralize the true virus of SARS-CoV-2. [Figure 5] This shows that the 76E1 antibody recognizes linear epitopes of S2 809-833 and 809-823. [Figure 6] The results of crystal violet staining of the 76E1 antibody, which inhibits the fusion of the virus and cell membrane, are shown.

[0053] [Figure 7] 1 shows the results of Western blot of the 76E1 antibody, which inhibits enzymatic digestion of the S2′ enzymatic digestion site. [Figure 8] The animal protection experiment protocol for the 76E1 antibody is shown. [Figure 9] We show that the 76E1 antibody protects ACE2-humanized mice from SARS-CoV-2 infection, reduces their weight loss, and reduces the viral load in the lungs of ACE2-humanized mice after SARS-CoV-2 infection. DETAILED DESCRIPTION OF THE INVENTION

[0054] Through extensive and thorough research, the inventors unexpectedly obtained a fully human monoclonal antibody, 76E1, directed against the coronavirus S2 protein. This antibody can broadly bind to the coronavirus S2 protein, has high binding neutralization activity against coronaviruses, has broad-spectrum discrimination and broad-spectrum neutralization, and can inhibit or block coronaviruses from infecting susceptible cells. In vitro and in vivo experiments confirm that the 76E1 antibody effectively prevents and controls coronavirus infection. Based on this, the present invention is completed.

[0055] In the present invention, we screened for the anti-coronavirus fully human broad-spectrum neutralizing antibody 76E1 using single-cell RT-PCR technology from PBMCs of volunteers who had recovered from COVID-19 infection. ELISA binding experiments and cell-level neutralization experiments confirmed that the 76E1 antibody has broad binding and neutralizing activity against the coronavirus S2 protein. Because the 76E1 antibody is fully human and does not contain mouse components, it has low immunogenicity and high safety, demonstrating its potential clinical application value against coronavirus infection and providing a new candidate drug for coronavirus infection in clinical settings. The antibody of the present invention binds to the coronavirus S protein, specifically to positions 809-823 or 809-833 of the S protein fusion peptide. The discovery of the 76E1 antibody epitope also provides new ideas and reference material for coronavirus vaccine design.

[0056] term In order to make the present invention more readily understandable, certain technical and scientific terms are specifically defined below. Unless expressly defined herein, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, since such methods and conditions may vary. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be limiting, and that the scope of the present invention is limited only by the appended claims.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in connection with a specifically stated numerical value, the term "about" means that the value may vary by no more than 1% from the stated value. For example, as used herein, the term "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0058] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. Biol. Chem, 243, p. 3558 (1968). As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent, including the monoclonal antibodies against a coronavirus S protein (preferably S2 protein) of the present invention and compositions thereof, to a patient having one or more disease symptoms, where the therapeutic agent is known to have a therapeutic effect on those symptoms. Typically, the amount of therapeutic agent administered to the patient is an amount that effectively alleviates one or more disease symptoms (a therapeutically effective amount).

[0059] As used herein, the term "optional" or "optionally" means that the event or circumstance described below may occur, but does not necessarily occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that a particular sequence of antibody heavy chain variable regions can, but does not necessarily, have 1, 2, or 3.

[0060] "Sequence identity" as used herein refers to the degree of identity between two nucleic acid or two amino acid sequences when compared optimally under the conditions of appropriate mutations such as exchanges, insertions, or deletions. The sequence identity between a sequence described in the present invention and a sequence having the same identity may be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0061] Coronavirus (CoV) Coronaviruses belong to the genus Coronavirus in the family Coronaviridae, order Nidovirales. Coronaviruses are a large class of RNA viruses with an envelope and a linear, single-stranded, positive-sense genome, and are widespread in nature. Coronaviruses are approximately 80-120 nm in diameter, have a methylated cap structure at the 5' end of their genome, and a poly(A) tail at the 3' end. Their genomes are approximately 27-32 kb long, making them the largest known RNA viruses. They infect only vertebrates, including humans, mice, pigs, cats, dogs, wolves, chickens, cattle, and livestock.

[0062] The 2019 novel coronavirus (SARS-CoV-2, which causes COVID-19) is the seventh known coronavirus capable of infecting humans to date; the other six are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (which causes severe acute respiratory syndrome), and MERS-CoV (which causes Middle East respiratory syndrome).

[0063] In order to obtain novel coronavirus antibody drugs with better therapeutic effects and to explore new antibody-specific epitopes, the present invention used single-cell RT-PCR technology to isolate the broad-spectrum neutralizing antibody 76E1 from human peripheral blood PBMCs. The discovery of this new antibody provides new options for the therapeutic application of broad-spectrum neutralizing antibodies, while the discovery of this new epitope provides new ideas for the development of broad-spectrum vaccines.

[0064] antibody As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical light chains (L) and two identical heavy chains (H) with identical structural characteristics. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies among heavy chains of different immunoglobulin isotypes. Each heavy and light chain also contains regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the light chain constant region facing the first constant region of the heavy chain and the light chain variable region facing the heavy chain variable region. Certain amino acid residues form an interface between the light and heavy chain variable regions.

[0065] As used herein, the term "variable" refers to differences in the sequences of certain portions of the variable regions in antibodies, which determines the binding and specificity of various particular antibodies for a particular antigen. However, variability is not evenly distributed throughout antibody variable regions. It is concentrated in three segments called the complementarity-determining regions (CDRs) or hypervariable regions of the light and heavy chain variable regions. The more conserved portions of the variable regions are called framework regions (FRs). Naturally occurring heavy and light chain variable regions each contain four FR regions, which are usually in a β-folded configuration connected by the three CDRs that form a connecting ring, and can occasionally form a partial β-folded structure. The CDRs of each chain are closely juxtaposed by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publication No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions are directly involved in binding the antibody to an antigen, but they also exhibit various effector functions, such as those involved in antibody-dependent cellular toxicity of the antibody.

[0066] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two different classes (called kappa and lambda) depending on the amino acid sequence of their constant region. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant region of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known to those skilled in the art.

[0067] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies within the population are identical except for some possible naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. Also, unlike traditional polyclonal antibody preparations (which typically have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, an advantage of monoclonal antibodies is that they are synthesized by the hybridoma culture and are uncontaminated by other immunoglobulins. The modifier "monoclonal" reflects the character of the antibody being obtained from a substantially homogeneous population of antibodies; it should not be construed as requiring any particular method of antibody purification.

[0068] The present invention further includes monoclonal antibodies having the amino acid sequence corresponding to the anti-coronavirus S protein (preferably S2 protein) monoclonal antibody, monoclonal antibodies having the variable region chains of the anti-coronavirus S protein (preferably S2 protein) monoclonal antibody, as well as other proteins or protein conjugates and fusion expression products having these chains. Specifically, the present invention includes any proteins or protein conjugates and fusion expression products (i.e., immunoconjugates and fusion expression products) having light and heavy chains containing hypervariable regions (complementarity-determining regions, CDRs), so long as the hypervariable regions are identical to or at least 90% homologous, preferably at least 95% homologous, to the hypervariable regions of the light and heavy chains of the present invention.

[0069] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by attaching drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the anti-coronavirus S protein monoclonal antibodies or fragments thereof. The present invention further includes cell surface markers or antigens that bind to the anti-coronavirus S protein monoclonal antibodies or fragments thereof.

[0070] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment of an antibody" include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; and (iv) an Fv fragment consisting of the VH and VL domains of a single antibody arm. Fv antibodies contain the heavy and light chain variable regions of an antibody but lack the constant region, and are the minimum antibody fragments containing all of the antigen-binding site. Generally, Fv antibodies contain a polypeptide linker between the VH and VL domains, allowing them to form the structure required for antigen binding.

[0071] The present invention includes not only complete monoclonal antibodies, but also immunoreactive antibody fragments such as Fab or (Fab')2 fragments, antibody heavy chains, and antibody light chains. The terms "epitope" or "antigenic determinant" refer to a site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope generally includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or noncontiguous amino acids in a unique spatial conformation.

[0072] The terms "specifically bind," "selectively bind," "selectively bind," and "specifically bind" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, an antibody binds to a specific antigen within a range of about 10 -7 Less than M, e.g., about 10 -8 Under M, 10 -9 Less than M or 10 -10 binds with a specific affinity (KD) of less than M or less.

[0073] As used herein, the term "antigenic determinant" refers to a three-dimensional site that is non-contiguous on an antigen and that is recognized by an antibody or antigen-binding fragment of the present invention. The present invention includes not only complete antibodies, but also immunologically active antibody fragments or fusion proteins formed from antibodies and other sequences. Thus, the present invention further includes fragments, derivatives and analogs of said antibodies.

[0074] In the present invention, antibodies include murine, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies are useful antibodies, such as chimeric and humanized monoclonal antibodies, containing human and non-human portions, which can be prepared using DNA recombinant techniques well known in the art. The term "murine-derived antibody" in the present invention refers to a monoclonal antibody against coronavirus S protein prepared according to the knowledge and techniques of the art. The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by murine antibodies. The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting murine CDR sequences onto a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. Humanized antibodies retain a large amount of murine protein components and can overcome the heterologous reaction induced by chimeric antibodies. Such framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. To avoid reduced immunogenicity and reduced activity, minimal back mutations or reverse mutations can be performed on the human antibody variable region framework sequences to maintain activity.

[0075] In the present invention, antibodies can be monospecific, bispecific, trispecific, or of greater multispecificity. As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably. As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0076] The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that are primarily responsible for antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).

[0077] In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises: CDR1:GFSFKDYG(SEQ ID NO.:3), CDR2: ISGDTRGT (SEQ ID NO.: 4), and It contains three complementarity determining regions (CDRs): CDR3: AALVIVAAGDDFDL (SEQ ID NO.: 5).

[0078] In another preferred embodiment, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.:1, where the underlined sequences are the amino acid sequences of the heavy chain variable region CDR1, CDR2, and CDR3, respectively. EVQLVESGGGVVQPGGSLRLSCEAS GFSFKDYG MHWIRQTPGKGLEWISR ISGDTRGT SYVDSVKGRFIVSRDNSRNSLFLQMNSLRSEDTALYYC AALVIVAAGDDFDL WGQGTVVTVSS(SEQ ID NO.:1)

[0079] In another preferred embodiment, the nucleic acid coding sequence of the heavy chain variable region is as set forth in SEQ ID NO.:8, wherein the underlined sequences are the nucleic acid coding sequences of the heavy chain variable region CDR1, CDR2, and CDR3, respectively. GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTGCAGCCGGGGGGGTCCCTGAGGCTCTCCTGTGAAGCCTCT GGATTCAGCTTTAAAGACTATGGC ATGCACTGGATCCGTCAGACTCCAGGGAAGGGTCTGGAGTGGATCTCTCGT ATTAGTGGAGACACTAGAGGCACA TCCTATGTAGACTCTGTGAAGGGCCGATTCATCGTCTCCAGAGACAACAGCAGAAACTCCTTGTTTTTACAAATGAACAGTCTGAGAAGTGAAGACACCGCCTTGTATTACTGT GCAGCATTAGTTATTGTAGCTGCCGGCGATGATTTTGATCTC TGGGGCCAAGGGACAGTGGTCACCGTTTCTTCA(SEQ ID NO.:8)

[0080] In a preferred embodiment of the present invention, the heavy chain of the antibody comprises the heavy chain variable region and a heavy chain determining region, and the heavy chain variable region may be of mouse or human origin. As used herein, "light chain variable region" and "V L The terms " and " are used interchangeably.

[0081] In a preferred embodiment of the invention, the light chain variable region of the antibody according to the invention comprises: CDR1':SSDIGSYNF(SEQ ID NO.:6), CDR2':EVN, and CDR3': has a complementarity determining region CDR selected from the group consisting of CSYGGRNNLI (SEQ ID NO.:7).

[0082] In another preferred embodiment, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.:2, where the underlined sequences are the amino acid sequences of the light chain variable region CDR1', CDR2', and CDR3, respectively. QSALTQPLSVSGSPGQSVTISCTGS SSDIGSYNF VSWYRQYPGKAPKVMIY EVN KRPSGVPVRFSGSKSGNTASLTVSGLQHEDEADYYC CSYGGRNNLI FGGGTKLTVL(SEQ ID NO.:2)

[0083] In another preferred embodiment, the nucleic acid coding sequence of the light chain variable region is as set forth in SEQ ID NO.:9, wherein the underlined sequences are the nucleic acid coding sequences of the light chain variable region CDR1', CDR2', and CDR3', respectively. CAGTCTGCCCTGACTCAGCCTCTCTCAGTGTCCGGGTCTCCTGGACAGTCCGTCACCATCTCCTGCACTGGATCC AGCAGTGACATTGGGAGTTATAATTTT GTCTCCTGGTATCGACAATATCCAGGCAAAGCCCCCAAAGTCATGATCTAT GAGGTCAAT AAGCGGCCCTCGGGGGTCCCTGTTCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCTGACCGTCTCTGGGCTCCAACATGAGGATGAGGCTGACTATTACTGC TGCTCATATGGAGGCCGCAACAATTTGATT TTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO.:9)

[0084] In a preferred embodiment of the present invention, the light chain of the antibody comprises the above-mentioned light chain variable region and light chain constant region, and the light chain constant region may be derived from a mouse or a human.

[0085] The function of the antibody of the present invention is determined by the specific gene sequences of the antibody light and heavy chain variable region genes, which can bind broadly to the coronavirus S2 protein and prevent coronaviruses from infecting susceptible cells. Using the antibody variable region genes or complementarity-determining region (CDR) genes, various forms of genetically engineered antibodies can be engineered and produced in any expression system using prokaryotic and eukaryotic cells.

[0086] In the present invention, the terms "antibody of the invention," "protein of the invention," or "polypeptide of the invention" are used interchangeably and all refer to an antibody that specifically binds to a coronavirus S protein (preferably the S2 protein), such as a protein or polypeptide having a heavy chain variable region (amino acid sequence encoded by the nucleotide sequence as shown in SEQ ID NO.:8) and / or a light chain variable region (amino acid sequence encoded by the nucleotide sequence as shown in SEQ ID NO.:9), which may or may not contain a starting methionine.

[0087] In another preferred example, the antibody is a mouse or human-mouse chimeric anti-coronavirus S protein (preferably S2 protein) monoclonal antibody, and its heavy chain constant region and / or light chain constant region may be humanized. More preferably, the humanized heavy chain constant region or light chain constant region is a heavy chain constant region or light chain constant region of human IgG1, IgG2, or the like.

[0088] In general, the antigen-binding properties of an antibody can be explained by three specific regions located in the variable regions of the heavy and light chains, called CDRs (variable regions). These sections are divided into four framework regions (FRs), whose amino acid sequences are relatively conserved and not directly involved in binding reactions. These CDRs form a cyclic structure, and the β-fold formed by the FRs between them is spatially adjacent. The heavy chain CDR and the corresponding light chain CDR constitute the antigen-binding site of the antibody. The amino acids that make up the FR or CDR region can be determined by comparing the amino acid sequences of antibodies of the same species.

[0089] The heavy and / or light chain variable regions of the antibodies of the present invention are of particular interest because at least some of them are involved in binding to antigen. Thus, the present invention includes molecules comprising the light and / or heavy chain variable regions of these monoclonal antibodies containing CDRs, so long as the CDRs share 90% or more (preferably 95% or more, and most preferably 98% or more) homology with the CDRs identified herein.

[0090] The present invention encompasses not only intact monoclonal antibodies, but also immunologically active antibody fragments or fusion proteins formed between antibodies and other sequences. Accordingly, the present invention also encompasses fragments, derivatives, and analogs of the antibodies. For example, in the modification of the Fc fragment based on the antibody of the present invention, three mutations, M252Y, S254T, and T256E, are introduced into the CH2 region to extend the half-life of the antibody.

[0091] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain essentially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative, or analog of the invention can be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) have been substituted (such substituted amino acid residues may or may not be encoded by the genetic code), or (ii) a polypeptide having a substitution at one or more amino acid residues, or (iii) a polypeptide formed by fusing the mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a fusion protein formed with a leader sequence, a secretory sequence, a sequence for purifying the polypeptide, a protein sequence, or a 6His tag). Following the teachings of the present specification, these fragments, derivatives, and analogs are well within the skill of those in the art.

[0092] The antibody of the present invention refers to a polypeptide comprising the above-described CDR region and having coronavirus S protein (preferably S2 protein)-binding activity. The term also encompasses mutant forms of polypeptides comprising the above-described CDR region that have the same function as the antibody of the present invention. These mutant forms include, but are not limited to, deletion, insertion, and / or substitution of one or more amino acids (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, and most preferably 1 to 10) and addition of one or more amino acids (usually 20 or less, preferably 10 or less, and more preferably 5 or less) at the C-terminus and / or N-terminus. For example, substitution with amino acids with similar or similar performance generally does not alter the function of the protein. As another example, addition of one or more amino acids at the C-terminus and / or N-terminus also generally does not alter the function of the protein. The term also encompasses active fragments and active derivatives of the antibody of the present invention.

[0093] Variant forms of the polypeptide include homologous sequences, conservative variants, allelic variants, naturally occurring mutants, induced mutants, proteins encoded by DNA capable of hybridizing to the coating DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained by using antisera against the antibody of the present invention.

[0094] The present invention further provides other polypeptides, such as fusion proteins, comprising human antibodies or fragments thereof. In addition to most full-length polypeptides, the present invention also includes fragments of the antibodies of the present invention. Typically, such fragments comprise at least about 50 contiguous amino acids of an antibody of the present invention, preferably at least about 60 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

[0095] In the present invention, "conservative variants of the antibodies of the invention" refer to polypeptides formed by substituting up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids having the same or similar properties compared to the amino acid sequence of an antibody of the invention. These conservative variant polypeptides are best generated by making amino acid substitutions according to Table A. [Table 1]

[0096] The present invention further provides a polynucleotide molecule encoding the antibody, or a fragment thereof, or a fusion protein thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO.:8 or 9, or may be a degenerate variant. As used herein, the term "degenerate variant" refers to a nucleic acid sequence that encodes the same amino acid sequence as the polypeptide of the present invention but differs from the coding region sequence shown in SEQ ID NO.:8 or 9.

[0097] A polynucleotide encoding a mature polypeptide of the present invention includes a coding sequence encoding only the mature polypeptide, a coding sequence for the mature polypeptide and various additional coding sequences, a coding sequence for the mature polypeptide (and any additional coding sequences) and non-coding sequences. The term "polynucleotide encoding a polypeptide" includes a polynucleotide that encodes the polypeptide, or further includes a polynucleotide that contains additional coding and / or non-coding sequences.

[0098] The present invention further relates to polynucleotides that hybridize to the above sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% homology between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the above polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refers to (1) hybridization and elution at lower ionic strength and higher temperatures, e.g., 0.2×SSC, 0.1% SDS, and 60°C; (2) the addition of a denaturing agent during hybridization, e.g., 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, and 42°C; or (3) hybridization that occurs only when the identity between the two sequences is at least 90%, more preferably 95%. Furthermore, polypeptides encoded by hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides set forth in SEQ ID NO.:1 and / or SEQ ID NO.:2.

[0099] The full-length nucleotide sequence of an antibody of the present invention or a fragment thereof can usually be obtained by PCR amplification, recombinant methods, or artificial synthesis. Especially when the fragment is short, a viable method is to synthesize the relevant sequence using artificial synthesis. Generally, multiple small fragments are first synthesized and then joined to obtain a very long fragment. Furthermore, the coding sequence of the heavy chain and an expression tag (e.g., 6His) can be fused to form a fusion protein.

[0100] Once the relevant sequence is obtained, recombinant methods can be used to obtain the relevant sequence in large quantities. Typically, this is achieved by cloning it into a vector, then transforming it into cells, and then isolating the relevant sequence from the host cells grown by conventional methods. The biological molecules (nucleic acids, proteins, etc.) involved in the present invention include biological molecules present in isolated form.

[0101] Currently, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) can be obtained entirely by chemical synthesis. The DNA sequences can then be introduced into a variety of existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequences of the present invention by chemical synthesis.

[0102] The present invention further relates to vectors containing the appropriate DNA sequences and appropriate promoter or control sequences, which can be used to transform appropriate host cells so as to express the proteins.

[0103] Host cells can be prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells. Representative examples include bacterial cells of Escherichia coli, Streptomyces, and Chinese hamster ovary, fungal cells such as yeast, insect cells of Drosophila S2 or Sf9, and animal cells such as CHO, COS7, and 293 cells.

[0104] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be obtained by treating the cells with CaCl2 after the exponential growth phase, a process well known in the art. Another method is to use MgCl2. Transformation can also be carried out by electroporation, if necessary. When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical methods such as microinjection and electroporation, liposome packaging, etc. can be selected.

[0105] The resulting transformant can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used for culture can be selected from a variety of conventional media. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced using an appropriate method (e.g., temperature shift or chemical induction), and the cells are further cultured for a certain period of time.

[0106] The recombinant polypeptide in the method can be expressed intracellularly or at the cell membrane, or can be secreted extracellularly. If necessary, the recombinant protein can be isolated and purified using a variety of isolation methods using physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation, treatment with protein precipitants (salting out), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0107] The antibodies of the present invention can be used alone, conjugated or coupled to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase)-modifying moiety, or any combination of these substances.

[0108] Detectable markers for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0109] Therapeutic agents that can be coupled include, but are not limited to, insulin, IL-2, interferon, calcitonin, GHRH peptides, gut peptide analogs, albumin, antibody fragments, cytokines, and hormones.

[0110] Additionally, therapeutic agents that can be bound or coupled to the antibodies of the present invention include, but are not limited to, 1. radionuclides, 2. biological toxins, 3. cytokines such as IL-2, 4. gold nanoparticles / nanorods, 5. viral particles, 6. liposomes, 7. nanomagnetic particles, 8. prodrug-activating enzymes, 10. chemotherapeutic agents (e.g., cisplatin) or nanoparticles of any form, etc.

[0111] The present invention further provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the antibody of the present invention, an active fragment thereof, or a fusion protein thereof, and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, the pH of which is typically about 5 to 8, preferably about 6 to 8, depending on the nature of the formulated substance and the disease being treated. The formulated pharmaceutical composition can be administered by any conventional route, including, but not limited to, oral, respiratory, intratumoral, intravenous, or topical administration.

[0112] The pharmaceutical composition of the present invention can be used directly to bind to the coronavirus S protein (preferably S2 protein) molecule, and therefore can be used to extend the half-life of the drug, and further can be used simultaneously with other therapeutic agents.

[0113] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001 to 99 wt%, preferably 0.01 to 90 wt%, more preferably 0.1 to 80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The drug formulation must be consistent with the method of administration. The pharmaceutical compositions of the present invention can be prepared in the form of injections by conventional methods, for example, using saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions must be prepared under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 10 mg / kg body weight daily. Furthermore, the polypeptides of the present invention can also be used in combination with other therapeutic agents.

[0114] When using pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is typically at least about 10 μg / kg body weight and in most cases does not exceed about 8 mg / kg body weight, and preferably the dosage is about 10 μg / kg body weight to about 1 mg / kg body weight. Of course, the specific dosage must take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0115] Detection Applications and Kits The antibodies of the present invention can be used in detection applications, such as, for example, detecting a sample, thereby providing diagnostic information. In the present invention, the sample used includes cells, tissue samples, and biopsy specimens. The term "biopsy" used in the present invention includes all types of biopsy known to those skilled in the art. Therefore, the biopsy used in the present invention can include tissue samples prepared by endoscopy or organ puncture or needle biopsy.

[0116] Samples for use in the present invention include fixed or preserved cell or tissue samples. The present invention further provides a kit comprising the antibody of the present invention (or a fragment thereof), and in a preferred embodiment of the present invention, the kit comprises a container, an instruction manual, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0117] SARS-CoV-2 S2 protein 809-833 linear epitope The monoclonal antibody provided by the present invention binds to the SARS-CoV-2 S2 protein 809-823 linear epitope (antigenic epitope peptide), preferably to the SARS-CoV-2 S2 protein 809-833 linear epitope (antigenic epitope peptide), the specific sequence of which is as follows: SARS-CoV-2 S2 protein 809-833 linear epitope (antigenic epitope peptide): PSKPSKRSFIEDLLFNKVTLADAGF(SEQ ID NO.:10) SARS-CoV-2 S2 protein 809-823 linear epitope (antigenic epitope peptide): PSKPSKRSFIEDLLF(SEQ ID NO.:11) Here, 815(R), 819(E), and 823(F) are the most important epitopes for the function of the antibody 76E1 of the present invention, and 820(D) and 822(L) are secondary important epitopes.

[0118] Based on the epitope, the present invention further provides a vaccine composition, which comprises: (i) a polypeptide comprising the amino acid sequence of positions 809-833 of the SARS-CoV-2 S2 protein as set forth in SEQ ID NO.:10 or a partial sequence thereof (the amino acid sequence of positions 809-823 of the SARS-CoV-2 S2 protein as set forth in SEQ ID NO.:11); and (ii) a vaccinally acceptable carrier.

[0119] Furthermore, the present invention provides an inhibitor that targets a linear epitope of SARS-CoV-2 S2 protein 809-823 or SARS-CoV-2 S2 protein 809-833 and is used to inhibit infection with the new coronavirus.

[0120] In another preferred embodiment, the inhibitor is (a) a polypeptide comprising the amino acid sequence of positions 809-833 of the SARS-CoV-2 S2 protein as shown in SEQ ID NO.:10 or a partial sequence thereof (including the amino acid sequence of positions 809-823 of the SARS-CoV-2 S2 protein as shown in SEQ ID NO.:11).

[0121] In another preferred embodiment, the inhibitor is (b) a small molecule compound that targets the SARS-CoV-2 S2 protein 809-833 linear epitope. In another preferred embodiment, the inhibitor is a combination of (a) and (b).

[0122] The main advantages of the present invention are: (1) The fully human monoclonal antibody of the present invention can specifically recognize and bind to the S protein of coronaviruses, has extremely high neutralizing activity against coronaviruses, and can neutralize seven strains of coronaviruses that infect humans, namely 229E-CoV, OC43-CoV, NL63-CoV, HKU1-CoV, SARS-CoV-2, SARS-CoV, and MERS-CoV, and can effectively inhibit or prevent coronaviruses from infecting susceptible cells. (2) The fully human monoclonal antibodies of the present invention have broad binding activity and broad neutralizing activity against various coronaviruses and can effectively neutralize various coronaviruses.

[0123] (3) The present invention is a fully human monoclonal antibody 76E1 that does not contain any mouse-derived portions, and is therefore less immunogenic and highly safe for humans, and can avoid antibody-mediated immune rejection reactions derived from other types of antibodies, such as human anti-mouse antibodies. (4) The fully human monoclonal antibody 76E1 of the present invention binds to the fusion peptide on the coronavirus S protein, particularly the S2 protein, which is a linear epitope of the human amino acid sequence at amino acid positions 809-823 (e.g., amino acids 809-833) of the SARS-CoV-2 S2 protein. The discovery of the 76E1 antibody epitope also provides some ideas and reference information for the design of coronavirus vaccines.

[0124] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are used only to illustrate the present invention and do not limit the scope of the present invention. In the following examples, experimental methods without specific conditions are generally in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are calculated as weight percentages and weight parts.

[0125] In the examples or test examples of the present invention, experiments for which no specific conditions are given are generally carried out according to conventional conditions or conditions provided by raw material / product manufacturers, and reagents for which no specific source is given are conventional reagents purchased on the market.

[0126] The following describes the steps for preparing neutralizing fully human monoclonal antibodies capable of neutralizing coronavirus S protein of the present invention and analyzing the antibody characteristics. Example 1. Single-cell RT-PCR method for obtaining antibody genes and antibody expression 1.1. Peripheral blood mononuclear cells (PBMC) acquisition Peripheral blood was collected from healthy volunteers and separated into 10 s by conventional Ficoll-Paque (Lympholyte®-H (CEDARLANE) company) density gradient centrifugation. 7 Obtain peripheral blood mononuclear cells (PBMCs) as described above.

[0127] Ficoll separation method: (1) Blood was collected into a 50 ml centrifuge tube (previously containing 1 ml of 4% sodium citrate), and 20 ml of whole blood was collected and mixed evenly by inverting 8-10 times (i.e., to make the final concentration of sodium citrate 0.4%). (2) Add an equal volume of RPMI1640 (containing sodium citrate) and mix evenly. (3) Using a 15 ml transparent centrifuge tube, plate 3 ml of lymphocyte separation fluid and carefully add 6 ml of blood sample on top to form a separation interface (or add 8 ml of blood sample to 4 ml of separation fluid). (4) Centrifuge at 800 g for 20 minutes at room temperature (2000 rpm for 20 minutes). (5) Carefully aspirate the interface layer cells and transfer them to a new tube. (6) Add RPMI 1640 (containing sodium citrate) and dilute until the liquid density is reduced. Centrifuge at 800g / 2000rpm for 10 minutes. Discard the supernatant. (7) Wash the cells 2-3 times with RPMI 1640 and store them.

[0128] 1.2. Acquisition of spike protein S-specific memory B cells FITC-CD19 / APC-IgG / BV421-S protein was used as a marker, BD Horizon™ Fixable Viability Stain 780-APC-Cy7 was used to remove dead cells, and CD4 / CD14 / CD8-percp5.5 was used to remove macrophages, T cells, etc. Specific B cells were isolated by flow cytometry and transferred to a 96-well RT-PCR plate at one cell per well to obtain S protein-specific memory B cells. (1) The SARS-CoV-2 S protein was expressed in a mammalian CHO expression system. The S protein sequence was derived from nCoV-SH01 (GenBank: MT121215.1) and synthesized by Shanghai GENEray. (2) Label the SARS-CoV-2 S protein with biotin and detect the biotin-labeled S protein. (3) Labeling of sorted cells: PBMC cells were divided into experimental and control groups, and markers were added according to the cell number. They were stained and labeled in the dark, resuspended in PBS, and then filtered through a 40 μm BD Falcon filter membrane. (4) Selection of specific B cells: Screen lymphocytes from PBMCs using BD FACS Influx according to anterior and lateral angles, and then obtain S protein-specific memory B cells through the control and compensation of different control groups. These are then sorted into 96-well plates, one cell per well, for RT-PCR (reverse transcription PCR), and the plates are placed on dry ice.

[0129] 1.3. Obtaining antibody genes and constructing vectors The cDNA of the isolated single memory B cells was obtained by RT-PCR, followed by nested PCR to obtain the variable regions of the antibody genes. The antibody gene sequences were then searched for by running the cDNA on an agarose gel and recovering and sequencing the gel blocks containing paired heavy and light chains. The antibody genes were then ligated into the corresponding IgH, Igκ, and Igλ expression vectors. The fully human antibody expression vectors IgH, Igκ, and Igλ (expressing the antibody heavy, kappa, and lambda chains, respectively) were kindly provided by the Patrick Wilson laboratory.

[0130] 1.4. Antibody Expression and Purification CHO cells are transiently transfected for fully human antibody expression. On day 1 of transfection (day -1), ExpiCHO-S™ cells are transfected at a final density of 3 x 10 6 -4×10 6 Divide the cells into viable cells / mL and grow them overnight. The next day (day 0), measure the viable cell density and viability. The cell density should be approximately 7 x 10 6 -10×10 6 Viability must be 95-99% to proceed with transfection. Final density 6 x 10 viable cells / mL must be reached. 6Dilute the cells to 100% viable cells / mL. Prepare the ExpiFectamine™ CHO / plasmid DNA complex using pre-chilled reagent (4°C). After incubating the ExpiFectamine™ CHO / plasmid DNA complex at room temperature for 1-5 minutes, slowly transfer the solution to a CHO cell culture flask, gently shaking the culture flask during the addition. Culture the cells in an orbital shaker (in a 37°C incubator with 8% CO2 deflux air conditions). Culture for 7-11 days until half of the cells have died. Collect the supernatant and begin producing antibodies.

[0131] Antibodies were purified using Protein G Agarose 4FF packing (purchased from GE). First, the collected CHO cell suspension was centrifuged at 4000 rpm for 30 minutes at 4°C, and the collected supernatant was filtered through a 0.45 μm filter for purification. A gravity-fed spin column was used, and Protein G Agarose 4FF packing was added. The packing was stabilized with 3 column volumes of 20% ethanol, followed by equilibration with 5 column volumes of binding buffer. The sample was then loaded, equilibrated with 10 column volumes of binding buffer, and finally eluted with 3 column volumes of elution buffer. A neutralization buffer was added to the eluted antibody solution to adjust the pH of the eluted sample to approximately 7.5. The antibody solution was then dialyzed three times against 5 L of 1xPBS, concentrating the antibody and allowing it to be stored at -80°C.

[0132] 1.5. Experimental Results: Using single-cell RT-PCR and nested-PCR, we isolated matching antibody heavy and light chain variable region genes with a molecular weight of approximately 400 bp. The agarose gel was then collected and sequenced. The sequencing results were compared at http: / / www.ncbi.nlm.nih.gov / igblast and http: / / www.imgt.org / to obtain the antibody germline gene information and the hypervariable region information of the antibody heavy and light chain genes, followed by the construction of an expression vector and subsequent expression and purification. Finally, using this technology, we successfully isolated a fully human monoclonal antibody that broadly neutralizes coronaviruses, named 76E1.

[0133] The gene sequence of the heavy chain variable region of the fully human antibody 76E1 is as follows, where the underlined sequences are the sequences of the hypervariable regions in the heavy chain gene variable region, and are, in order, the heavy chain gene CDR1, CDR2, and CDR3 sequences. GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTGCAGCCGGGGGGGTCCCTGAGGCTCTCCTGTGAAGCCTCT GGATTCAGCTTTAAAGACTATGGC ATGCACTGGATCCGTCAGACTCCAGGGAAGGGTCTGGAGTGGATCTCTCGT ATTAGTGGAGACACTAGAGGCACA TCCTATGTAGACTCTGTGAAGGGCCGATTCATCGTCTCCAGAGACAACAGCAGAAACTCCTTGTTTTTACAAATGAACAGTCTGAGAAGTGAAGACACCGCCTTGTATTACTGT GCAGCATTAGTTATTGTAGCTGCCGGCGATGATTTTGATCTC TGGGGCCAAGGGACAGTGGTCACCGTTTCTTCA(SEQ ID NO.:8)

[0134] The amino acid sequence of the heavy chain variable region of the fully human antibody 76E1 is as follows, where the underlined sequences are the heavy chain amino acid sequences CDR1, CDR2, and CDR3, respectively. EVQLVESGGGVVQPGGSLRLSCEAS GFSFKDYG MHWIRQTPGKGLEWISR ISGDTRGT SYVDSVKGRFIVSRDNSRNSLFLQMNSLRSEDTALYYC AALVIVAAGDDFDL WGQGTVVTVSS(SEQ ID NO.:1)

[0135] The gene sequence of the light chain variable region of the fully human antibody 76E1 is as follows, where the underlined sequences are the sequences of the hypervariable regions in the variable region of the light chain gene, and are, in order, the CDR1', CDR2', and CDR3' sequences. CAGTCTGCCCTGACTCAGCCTCTCTCAGTGTCCGGGTCTCCTGGACAGTCCGTCACCATCTCCTGCACTGGATCC AGCAGTGACATTGGGAGTTATAATTTT GTCTCCTGGTATCGACAATATCCAGGCAAAGCCCCCAAAGTCATGATCTAT GAGGTCAAT AAGCGGCCCTCGGGGGTCCCTGTTCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCTGACCGTCTCTGGGCTCCAACATGAGGATGAGGCTGACTATTACTGC TGCTCATATGGAGGCCGCAACAATTTGATT TTCGGCGGAGGGACCAAGCTGACCGTCCTA(SEQ ID NO.:9)

[0136] The amino acid sequence of the light chain variable region of fully human antibody 76E1 is as follows, where the underlined sequences are the light chain amino acid CDR1', CDR2' and CDR3' sequences, in that order. QSALTQPLSVSGSPGQSVTISCTGS SSDIGSYNF VSWYRQYPGKAPKVMIY EVN KRPSGVPVRFSGSKSGNTASLTVSGLQHEDEADYYC CSYGGRNNLI FGGGTKLTVL(SEQ ID NO.:2)

[0137] Example 2. Antibody Characterization 2.1. ELISA detection of antibody-bound antigen activity ELISA was used to detect whether the expressed antibodies recognized various coronavirus S proteins. Various coronavirus S proteins were purchased from Beijing SinoBiological Co., Ltd. 28-12 was a negative control antibody prepared in our laboratory. An S protein ELISA plate was coated overnight at 4°C with 100 μL of 0.5 μg / mL antibody per well. The next day, the plate was washed three times with PBST. 2% BSA was added to the plate, 200 μL per well, and blocked for 2 hours at 37°C. The plate was washed three times with PBST again. 76E1 and the control antibody were serially diluted three-fold to an initial test concentration of 10 μg / mL. 100 μL of sample was loaded per well at 37°C for 2 hours. The plate was washed three times with PBST. 100 μL of Goat Anti-Human IgG (Fc specific)-Peroxidase antibody (Sigma) was diluted 1:8000 and incubated at 37°C for 1 hour in 100 μL per well. Wash the plate three times with PBST. Add 100 μL / well of the substrate TMB to develop color. If the color is too light, incubate at 37°C for 15 minutes in the dark. Stop the reaction by adding 50 μL of 2M H2SO4 per well. 450 Measure and process the data.

[0138] 2.2. Biofilm Layer Interferometry (BLI) Affinity Detection This experiment was performed using the OCTET RED 96 instrument at the Molecular Platform of the Biochemistry and Cell Research Institute. First, the AHC sensor was placed in a 96-well black plate containing PBS and allowed to soak for at least 10 minutes. Sample 76E1 was added at a concentration of 20 μg / ml. A gradient of antigen was added, with two-fold serial dilutions from 200 nM to 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM. The antibody-antigen, regeneration buffer, and PBS buffer were added to another black plate, which was then placed on the instrument. The program was set up and started: baseline 120 s → loading Ab → baseline 300 s → association 240 s → disassociation 900 s → regeneration 900 s → baseline 5 s → regeneration 5 s → baseline 5 s → regeneration 5 s → baseline 5 s. The data was analyzed using software to calculate the antigen-antibody affinity (KD) value.

[0139] 2.3. Pseudovirus neutralization experiment Human coronavirus S protein plasmids, such as full-length SARS-CoV, SARS-CoV-2, and MERS-CoV, and the pNL4-3 plasmid were co-transfected into HEK293T cells in 10cm culture dishes. The liquid was replaced after 6 hours. After 48 hours, the supernatant was collected and diluted in complete medium. The antibody was serially diluted, mixed with an equal amount of virus, and incubated at 37°C for 1 hour. The antibody and virus mixture was transferred to HEK293T cells stably expressing human ACE2 or cells expressing the corresponding coronavirus receptor. The cells were incubated at 37°C for 48 hours. The supernatant was removed, and the cells were thoroughly lysed with lysis buffer. Luciferase activity (Promega) was measured. Neutralization efficiency was calculated by comparing the luciferase values ​​of the experimental group with those of the virus control group. The calculation formula is as follows: Inhibitory activity percentage (%) = (mean virus control value - reading of test well) / (mean virus control value - mean cell control value) x 100 I C 50 The values ​​are calculated by the Prism software.

[0140] 2.4. Syncytium formation experiment Plasmids encoding SARS-CoV, MERS-CoV, and SARS-CoV-2 S proteins were transfected into Hela cells, respectively. Simultaneously, separate Hela cells were transfected with plasmids encoding hACE2 and hDpp4, respectively. 48 hours after transfection, Hela cells expressing S proteins were mixed with various concentrations of antibodies and incubated at 37°C for 1 hour. ACE2-expressing Hela cells were mixed 1:1 with the above cell-antibody mixture expressing SARS-CoV or SARS-CoV-2 S proteins and cultured at 37°C for 12 hours. Similarly, hDpp4-expressing Hela cells were mixed 1:1 with the above cell-antibody mixture expressing MERS-CoV S proteins and cultured at 37°C for 12 hours. Cells were fixed with 4% PFA for 15 minutes. Crystal violet staining was performed for 2 hours. Images were collected and analyzed using an Olympus IX73 confocal microscope.

[0141] 2.5. Enzyme digestion inhibition experiment Freshly purified SARS-CoV, MERS-CoV, and SARS-CoV-2 S proteins were prepared and incubated with 76E1 and control antibody 28-12 at a 1:5 ratio for 1 hour at room temperature. TPCK-Trypsin was added at a trypsin:HA mass ratio of 1:100. Enzymatic digestion was performed at room temperature (22°C). The time of trypsin addition was recorded as 0 minutes. Samples were taken at 10, 20, and 40 minutes, respectively. Protein loading buffer was then immediately added, and the samples were boiled at 100°C for 10 minutes. Western blot analysis was performed using a 6xHis antibody to detect His chromogenicity and determine the HA protease cleavage status.

[0142] 2.6. True virus neutralization experiment (1) Cell inoculation: Vero-E6 cells (coronavirus) or RD cells (OC43) in the logarithmic growth phase were inoculated into a 96-well plate, 100 μl per well, 4 × 10 per well. 4 Inoculate with cells.

[0143] (2) Neutralization experiment: Dilution of test samples: Add 60 μl of the 10 μg / ml pre-diluted sample to column 1 (column BG) of a 96-well plate, add 60 μl of virus dilution to make the final antibody concentration 5 μg / ml, and add 60 μl of serially 3-fold diluted samples to the remaining wells. Column 1 contains the cell control (CC) and adds 120 μl of serum-free medium, and column 8 contains 60 μl of serum-free medium for the virus control (VC).

[0144] Virus dilution: The titer of the virus stock solution is 2.5 x 10 5 TCID50 / ml, take 200 μl of virus stock solution, add 25 ml of serum-free medium, mix thoroughly, and dilute the virus to 100 TCID50 / 50 μl. Virus dropwise addition: Virus (except cell control) is dropped vertically into a 96-well plate, and a sample volume of 60 μl / well is added to obtain a final virus-antibody mixture of 120 μl.

[0145] Neutralization: The loaded cell culture plate was mixed evenly on a shaker and placed in a 37°C incubator for 1 hour to neutralize. After neutralization, the supernatant was aspirated from the inoculated cell culture plate, and 100 μl of the virus-serum mixture was added per well. The plate was then incubated in a CO2 incubator at 37°C for 1 hour to allow for infection. After viral infection, the supernatant was aspirated from the culture plate, and the plaque formation experiment sample was added to a maintenance medium containing 1% methylcellulose (DMEM medium containing 2% FBS) and incubated in a CO2 incubator at 37°C for 72-96 hours. The results were observed and recorded on the fourth day using an inverted microscope. The supernatant was discarded, and the plaque formation experiment sample was fixed with formaldehyde, stained with crystal violet, and analyzed by counting the plaques. To test for 100% inhibition, CPE was observed under an inverted microscope on the second day and the results were recorded.

[0146] 2.7.Animal Protection Experiments Prevention experiment with 76E1 in mice: Ten-week-old hACE transgenic female mice were placed in the animal testing room of a biosafety level 3 laboratory and divided into three groups of 4-6 mice per group. On day 0, mice in the three groups were inoculated with 50 mg / kg, 150 mg / kg of 76E1 antibody, or PBS as a control, respectively. Twenty-four hours later, the mice were anesthetized with 0.5% sodium pentobarbital and administered 3.7 x 10^4 SARS-CoV-2 viral particles via the nasal cavity. Weights were measured continuously for three days, and on day 3, the mice were euthanized and lung tissue was dissected and collected for viral RNA measurement and histopathological examination.

[0147] Viral RNA was extracted from lung tissue using Trizol Reagent (Invitrogen), and the RNA was reverse-transcribed into cDNA using a reverse transcription kit (Tiangen, China). Real-time quantitative PCR was performed using the Super Real Pre Mix Plus SYBR Green kit and the SARS-CoV-2 N gene-specific primers described above to measure the viral RNA content in lung tissue. Mouse lungs were fixed in 4% paraformaldehyde solution. Hematoxylin-eosin (H&E) was used to stain tissue paraffin sections. Sections were observed under an Olympus microscope.

[0148] 2.8.Experimental Results (1) 76E1 antibody that can bind only to the S2 region of SARS-CoV-2 To investigate the epitope location of the 76E1 antibody that binds to the SARS-CoV-2 S protein, we used ELISA to verify the binding activity of the 76E1 antibody to the full-length S protein, S1, and S2 protein, respectively. As shown in Figure 1, the 76E1 antibody can bind to the full-length S protein and S2 protein, but not to S1. Unlike most previously reported antibodies that target the receptor-binding region, the 76E1 antibody primarily targets S2, which means that its mechanism of action is also different from that of antibodies that target the RBD.

[0149] (2) 76E1 antibody, which can bind broadly to various coronavirus S proteins The S2 region is located in the stalk of the S protein, and its sequence is highly conserved among various coronaviruses. To study the ability of the 76E1 antibody to bind to various coronavirus S proteins, we used an ELISA method to verify its binding activity to various purified soluble coronavirus S proteins in vitro. 76F6, an antibody targeting the RBD, was used as a negative control antibody. 28-12, an anti-influenza fully human antibody owned by the inventors' laboratory, was used as a negative isotype control antibody. As shown in Figure 2A-C, the 76E1 antibody can broadly bind to the S proteins of seven human-infecting coronavirus strains: 229E-CoV, OC43-CoV, NL63-CoV, HKU1-CoV, SARS-CoV-2, SARS-CoV, and MERS-CoV. The RBD antibody 76F6 can only bind to SARS-CoV-2, while the negative control antibody 28-12 does not bind to various coronavirus S proteins.

[0150] Simultaneously, we analyzed the affinity of 76E1 with the in vitro purified coronavirus S proteins of the seven strains. 76E1 and the seven coronavirus S proteins all showed higher affinity, reaching the nM level or higher (Figure 2, D). To further investigate the binding activity of 76E1 to cell surface S proteins, we transfected various S proteins into CHO cells and detected the binding activity of 76E1 to the S proteins on the cell membrane surface by flow cytometry. The results showed that 76E1 could bind to all seven cell surface-expressed S proteins, while 76F6 could only bind to the SARS-CoV-2 S protein (Figure 2, E). However, 76E1 could not bind to the pre-fusion form of the trimeric S protein, indicating that 76E1 recognizes specific conformations of the S protein during structural changes (Figure 2, F, G). As shown in Figure 2H, the 76E1 antibody can bind to the S proteins of various novel coronavirus mutant strains and resist viral mutations. As shown in Figure 2I, the 76E1 antibody binds with high affinity to the S proteins of various novel coronavirus mutant strains.

[0151] (3) 76E1 antibody broadly neutralizes various coronavirus pseudoviruses To further confirm the function of 76E1, pseudoviruses of SARS-CoV, MERS-CoV, SARS-CoV-2, and various mutant strains of SARS-CoV-2 were packaged, and neutralization experiments of SARS-CoV and SARS-CoV-2 pseudoviruses were performed in 293T cells stably transfected with human ACE2, and neutralization experiments of MERS-CoV and HCoV-229E pseudoviruses were performed in huh7 cells. The experiments showed that 76E1 neutralized various strains of pseudoviruses with different abilities, had the highest neutralizing activity against SARS-CoV-2 and mutant strains, and had the highest IC 50 The IgG antibody titer was 397 ng / ml (Figure 3, AF), followed by SARS-CoV, MARS-CoV, and HCoV-229E (Figure 3, GI).

[0152] (4) 76E1 antibody, which can neutralize the SARS-CoV-2 virus To further confirm the neutralizing activity of 76E1 against SARS-CoV-2 virus, we performed a neutralizing activity test for SARS-CoV-2 virus in a BSL-3 laboratory. The results showed that 76E1 could effectively inhibit SARS-CoV-2 virus infection in three sensitive cell lines (Figure 4).

[0153] (5) 76E1 antibody, which recognizes linear epitopes at S2 809-833 and 809-823 First, ELISA experiments revealed that 76E1 maintained a higher binding activity to the denatured S protein, suggesting that 76E1 primarily recognizes a linear epitope on the S protein. Comparison of the sequences of seven coronavirus S proteins revealed that six sequences in the S2 region are highly conserved. Because 76E1 can bind broadly to the S proteins of these seven virus strains, the epitope of 76E1 is most likely located on these six polypeptides. ELISA experiments demonstrated that 76E1 can bind to the 809-833 polypeptide (Figure 5, A-C). By synthesizing a polypeptide of amino acids 809-823 of the truncated S protein and a series of polypeptides in which each site was individually mutated to alanine, it was found that 815(R), 819(E), and 823(F) are the most important epitopes acting on the antibody 76E1 of the present invention, 820(D) and 822(L) are the second most important epitopes, and that 76E1 can bind to the 809-823 polypeptide (Figure 5D).

[0154] Therefore, the main epitope of 76E1 is located on this polypeptide, which is a fusion peptide and plays an important role in mediating the fusion of the viral envelope with the host cell membrane. This polypeptide is also highly conserved among all coronaviruses (Fig. 5E), and 76E1 can distinguish the corresponding polypeptide sequences of all four coronavirus subfamilies (corresponding to amino acids 809-823 of the SARS-CoV-2 Spike protein) (Fig. 5F).

[0155] (6) 76E1 antibody inhibits viral infection by blocking fusion between the virus and cell membranes Because 76E1 binds to the S protein fusion peptide, we speculate that 76E1 inhibits viral infection by inhibiting fusion between the virus and cell membranes. We simulated the fusion process between the viral envelope and the host envelope by transfecting Hela-S cells with Hela-ACE2 cells and then mixing and fusing them with Hela-S and Hela-ACE2 cells, respectively. The results show that 76E1 effectively inhibits membrane fusion in SARS-CoV-2 and SARS-CoV, whereas the control antibody 28-12 cannot (Figure 6). Although membrane fusion mediated by MERS-CoV is less frequent than that of SARS-CoV-2 and SARS-CoV, 76E1 still effectively inhibits membrane fusion, whereas the control antibody 28-12 cannot (Figure 6). These experiments confirm that 76E1 inhibits viral infection by inhibiting fusion between the virus and cell membranes.

[0156] (7) 76E1 inhibits membrane fusion by inhibiting enzymatic digestion of the S2' enzyme digestion site. The S protein is enzymatically digested by S1 / S2 and S2', exposing the fusion peptide and promoting viral fusion with the cell membrane. Because the identifying epitope of 76E1 is close to the S2' enzymatic digestion site, we speculate that 76E1 can inhibit the enzymatic digestion of S2'. Western blot experiments demonstrate that 76E1, but not the control antibody, can inhibit the enzymatic digestion of S2' of the S protein (Figure 7).

[0157] (8) 76E1 antibody protects ACE2-humanized mice from SARS-CoV-2 true virus infection Therapeutic and preventive animal experiments were designed (Figure 8). The results showed that the 76E1 antibody reduced weight loss in ACE2-humanized mice infected with the SARS-CoV-2 virus and reduced the viral load in the lungs of ACE2-humanized mice infected with the SARS-CoV-2 virus (Figure 9).

[0158] All documents mentioned in this application are incorporated by reference in this application as if each document were incorporated by reference individually. Furthermore, after reading the above teachings of the present invention, those skilled in the art will be able to make various changes or modifications to the present invention, and these equivalents will also fall within the scope defined by the appended claims of this application.

Claims

1. A specific anti-coronavirus antibody, said antibody comprising: (1) A heavy chain variable region comprising the following three complementarity-determining regions (CDRs): CDR1 as shown in SEQ ID NO.:3, CDR2 as shown in SEQ ID NO.:4, and CDR3 as shown in SEQ ID NO.:5, and (2) a light chain variable region comprising the following three complementarity-determining regions (CDRs): CDR1′ as shown in SEQ ID NO.:6, CDR2' having the amino acid sequence EVN, and CDR3' shown in SEQ ID NO.:7 wherein said antibody binds to a linear epitope of SARS-CoV-2 S2 protein 809-833.

2. The antibody of claim 1 , wherein the heavy chain variable region further comprises a human-derived FR region.

3. The antibody of claim 1 , wherein the light chain variable region further comprises a human-derived FR region.

4. The antibody of claim 1, wherein the heavy chain variable region sequence of the antibody is as shown in SEQ ID NO.:1, and the light chain variable region sequence of the antibody is as shown in SEQ ID NO.:

2.

5. The antibody of claim 1 , wherein the antibody is a monoclonal antibody.

6. A recombinant protein, the recombinant protein comprising: (i) an antibody according to any one of claims 1 to 5, and (ii) a tag sequence to aid in expression and / or purification; The recombinant protein having the following structure:

7. 10. A CAR construct, wherein the scFv segment of the antigen-binding region of the CAR construct is a binding region that specifically binds to a linear epitope of SARS-CoV-2 S2 protein 809-833, and the scFv has the heavy chain variable region and the light chain variable region of the antibody of claim 1.

8. 1. An antibody drug conjugate, comprising: (a) an antibody moiety selected from the group consisting of the antibodies of any one of claims 1 to 5; (b) a coupling moiety coupled to said antibody moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, and combinations thereof; The antibody-drug conjugate comprising:

9. A pharmaceutical composition, the pharmaceutical composition comprising: (i) an active ingredient selected from the group consisting of the antibody of any one of claims 1 to 5, the recombinant protein of claim 6, the antibody drug conjugate of claim 8, and combinations thereof; (ii) a pharmaceutically acceptable carrier; The pharmaceutical composition comprising:

10. A group consisting of: (1) The antibody according to any one of claims 1 to 5. (2) The recombinant protein according to claim 6, and (3) The CAR construct according to claim 7. A polynucleotide encoding a polypeptide selected from:

11. Use of an active ingredient selected from the group consisting of an antibody according to any one of claims 1 to 5, a recombinant protein according to claim 6, and combinations thereof, The use of the active ingredient, wherein the active ingredient is used for preparing a drug, a reagent, a detection plate, or a kit.

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