CN112250763B

CN112250763B - Antibody targeting SARS-CoV-2 coronavirus and its diagnosis and detection use

Info

Publication number: CN112250763B
Application number: CN202011523501.XA
Authority: CN
Inventors: 胡宇豪; 郞国竣; 邵俊斌; 闫鑫甜
Original assignee: Shanghai ZJ Bio Tech Co Ltd; Sanyou Biopharmaceuticals Co Ltd
Current assignee: Shanghai ZJ Bio Tech Co Ltd; Sanyou Biopharmaceuticals Co Ltd
Priority date: 2020-12-21
Filing date: 2020-12-21
Publication date: 2021-03-26
Anticipated expiration: 2040-12-21
Also published as: CN112250763A

Abstract

The present invention relates to antibody targeting SARS-CoV-2 coronavirus and its diagnosis and detection use. In particular to an antibody or an antigen-binding fragment thereof and an antibody pair which specifically bind to coronavirus S protein and a detection product containing the antibody or the antigen-binding fragment thereof and the antibody pair. The invention also relates to nucleic acids encoding the antibodies or antigen-binding fragments and host cells comprising the same, as well as methods of making the antibodies or antigen-binding fragments. Furthermore, the invention relates to the prophylactic, therapeutic or diagnostic use of said antibody or antigen-binding fragment thereof, antibody pair. Compared with the conventional IgG/IgM detection, the detection method directly detects the RBD protein of the virus in the sample, can effectively avoid the interference of irrelevant IgG/IgM in the sample on the detection, and effectively improves the detection sensitivity. The antibodies or antibody pairs are useful for diagnosing and/or detecting coronaviruses.

Description

Antibody targeting SARS-CoV-2 coronavirus and its diagnosis and detection use

Technical Field

The present disclosure relates generally to antibodies and uses thereof. More specifically, the disclosure relates to antibodies that specifically recognize the spike protein of coronavirus SARS-CoV-2, methods of making, and uses thereof.

Background

The current clinical practice for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is mainly symptomatic support therapy. Considering that such coronaviruses continuously endanger human health and have the characteristics of high transmission and fatality rate and the like, more public health problems are likely to be caused, so that the development of preventive and therapeutic antiviral agents capable of inhibiting or blocking viral infection and the development of diagnostic kits for rapidly screening viral infection are needed for the pathogenic mechanism of such coronaviruses, and prevention and early diagnosis and treatment for the coronaviruses are provided for the future when the coronaviruses are epidemic or even widely appear in the population, and particularly, detection products capable of rapidly, highly sensitively and highly specifically detecting SARS-CoV-2 virus are needed.

Disclosure of Invention

In one aspect, the present disclosure provides an antibody pair for use in detecting SARS-CoV-2 virus, the antibody pair comprising a part I and a part II, the part I comprising one or more antibodies or antigen-binding fragments as described in (a), (b), and (c); part II comprises one or more antibodies or antigen-binding fragments as described in (d) and (e);

wherein (a) an antibody comprising the heavy chain CDR1 (HCDR 1) comprising the amino acid sequence of SEQ ID NO. 5 or a variant thereof having NO more than 2 amino acid changes; a heavy chain CDR2 (HCDR 2) comprising the amino acid sequence of SEQ ID NO 6 or a variant thereof having NO more than 2 amino acid changes; a heavy chain CDR3 (HCDR 3) comprising the amino acid sequence of SEQ ID NO. 7 or a variant thereof having NO more than 2 amino acid changes; a light chain CDR1 (LCDR 1) comprising the amino acid sequence of SEQ ID NO. 8 or a variant thereof having NO more than 2 amino acid changes; a light chain CDR2 (LCDR 2) comprising the amino acid sequence of SEQ ID NO. 9 or a variant thereof having NO more than 2 amino acid changes; and a light chain CDR3 (LCDR 3) comprising the amino acid sequence of SEQ ID NO. 10 or a variant thereof having NO more than 2 amino acid changes;

(b) an antibody comprising HCDR1 or a variant thereof having NO more than 2 amino acid changes in the heavy chain variable region amino acid sequence set forth in SEQ ID NO. 3; 3 or a variant thereof having NO more than 2 amino acid changes to HCDR2 in the heavy chain variable region amino acid sequence set forth in SEQ ID NO; 3 or a variant thereof having NO more than 2 amino acid changes to HCDR3 in the heavy chain variable region amino acid sequence set forth in SEQ ID NO; LCDR1 or a variant thereof having NO more than 2 amino acid changes in the light chain variable region amino acid sequence set forth in SEQ ID NO. 4; LCDR2 or a variant thereof having NO more than 2 amino acid changes in the light chain variable region amino acid sequence set forth in SEQ ID NO. 4; and LCDR3 or a variant thereof having NO more than 2 amino acid changes in the light chain variable region amino acid sequence set forth in SEQ ID NO. 4;

(c) an antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO 3 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 4 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

(d) an antibody comprising HCDR1 or a variant thereof having NO more than 2 amino acid changes in the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 11; 11 or a variant thereof having NO more than 2 amino acid changes of HCDR2 in the heavy chain variable region amino acid sequence set forth in SEQ ID NO; 11 or a variant thereof having NO more than 2 amino acid changes of HCDR3 in the heavy chain variable region amino acid sequence set forth in SEQ ID NO; LCDR1 or a variant thereof having NO more than 2 amino acid changes in the light chain variable region amino acid sequence set forth in SEQ ID NO. 12; LCDR2 or a variant thereof having NO more than 2 amino acid changes in the light chain variable region amino acid sequence set forth in SEQ ID NO. 12; and LCDR3 or a variant thereof having NO more than 2 amino acid changes in the light chain variable region amino acid sequence set forth in SEQ ID NO. 12;

(e) an antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO 11 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 12 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

In some embodiments, the antibody of group (a), (b) or (c) further comprises an IgG1 heavy chain and/or a Kappa light chain.

In some embodiments, the antibody of group (a), (b), or (c) further comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO:1 or a sequence at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 2 or a sequence at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; preferably, the heavy chain constant region comprises the nucleotide sequence set forth in SEQ ID No. 13 or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and the light chain constant region comprises the nucleotide sequence set forth in SEQ ID No. 14 or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In one aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to a coronavirus S protein, wherein the antibody is at least one antibody selected from the group consisting of:

(a) an antibody comprising the heavy chain CDR1 (HCDR 1) comprising the amino acid sequence of SEQ ID NO. 5 or a variant thereof having NO more than 2 amino acid changes; a heavy chain CDR2 (HCDR 2) comprising the amino acid sequence of SEQ ID NO 6 or a variant thereof having NO more than 2 amino acid changes; a heavy chain CDR3 (HCDR 3) comprising the amino acid sequence of SEQ ID NO. 7 or a variant thereof having NO more than 2 amino acid changes; a light chain CDR1 (LCDR 1) comprising the amino acid sequence of SEQ ID NO. 8 or a variant thereof having NO more than 2 amino acid changes; a light chain CDR2 (LCDR 2) comprising the amino acid sequence of SEQ ID NO. 9 or a variant thereof having NO more than 2 amino acid changes; and a light chain CDR3 (LCDR 3) comprising the amino acid sequence of SEQ ID NO. 10 or a variant thereof having NO more than 2 amino acid changes;

(d) an antibody comprising HCDR1 or a variant thereof having NO more than 2 amino acid changes in the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 11; 11 or a variant thereof having NO more than 2 amino acid changes of HCDR2 in the heavy chain variable region amino acid sequence set forth in SEQ ID NO; 11 or a variant thereof having NO more than 2 amino acid changes of HCDR3 in the heavy chain variable region amino acid sequence set forth in SEQ ID NO; LCDR1 or a variant thereof having NO more than 2 amino acid changes in the light chain variable region amino acid sequence set forth in SEQ ID NO. 12; LCDR2 or a variant thereof having NO more than 2 amino acid changes in the light chain variable region amino acid sequence set forth in SEQ ID NO. 12; and LCDR3 or a variant thereof having NO more than 2 amino acid changes in the light chain variable region amino acid sequence set forth in SEQ ID NO. 12; or

In some embodiments, the antibodies of the present disclosure exhibit at least one of the following biological activities:

(a) specifically binds to the S protein RBD domain of SARS-CoV-2 coronavirus, but does not cross-bind to SARS-CoV and MERS coronavirus; (b) EC50 that specifically binds to the S protein RBD domain of SARS-CoV-2 coronavirus is less than about 0.05 μ g/mL; or (c) the sensitivity for detecting SARS-CoV-2 coronavirus S protein can reach 1 ng/mL at most.

In one aspect, the present disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof according to the present disclosure.

In one aspect, the present disclosure provides an isolated vector comprising a polynucleotide according to the present disclosure.

In one aspect, the present disclosure provides a host cell comprising a polynucleotide or vector according to the present disclosure.

In one aspect, the present disclosure provides a method of expressing an antibody or antigen-binding fragment thereof of the present disclosure, the method comprising culturing a host cell according to the present disclosure under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and optionally recovering the antibody or antigen-binding fragment thereof of the present disclosure from the host cell or from the culture medium.

In one aspect, the present disclosure provides a product for detecting SARS-CoV-2 virus comprising an antibody pair according to the present disclosure, preferably the product is a test strip or a test kit.

In some embodiments, one or more antibodies or antigen-binding fragments thereof described in groups (a), (b), and (c) have a chromogenic label and are used as the labeled antibody; and one or more antibodies or antigen-binding fragments thereof described in groups (d) and (e) are immobilized and used as capture antibodies.

In some embodiments, the product comprises a substrate, a sample region, a binding region, and a reaction region, wherein the sample region, the binding region, and the reaction region are disposed on the substrate in that order along the flow direction of a sample to be tested; the reaction zone is provided with a detection zone loaded with one or more antibodies or antigen-binding fragments thereof described in groups (d) and (e); the binding region is loaded with one or more antibodies or antigen-binding fragments thereof described in groups (a), (b) and (c) having a chromogenic label.

In some embodiments, the reaction zone is further provided with a quality control region loaded with a secondary antibody, e.g., an anti-human Fc secondary antibody, for one or more of the antibodies described for groups (a), (b), and (c), or antigen-binding fragments thereof.

In some embodiments, the product further comprises an absorption zone on a side of the reaction zone remote from the sample zone.

In some embodiments, the chromogenic label is a colloidal gold label or a fluorescent label.

In one aspect, the disclosure provides the use of an antibody pair according to the disclosure in the preparation of a product for the detection of SARS-CoV-2 virus.

In one aspect, the disclosure provides use according to the description herein for the manufacture of a diagnostic agent for diagnosing SARS-CoV-2 virus infection.

In one aspect, the disclosure provides a method for detecting SARS-CoV-2 virus for non-diagnostic purposes comprising providing a sample and detecting the presence of SARS-CoV-2 virus in the sample using a product according to the disclosure, preferably by double antibody sandwich method. In some embodiments, the sample is an environmental sample. In some further embodiments, the sample is a biological sample.

The method for detecting SARS-CoV-2 virus includes a detection method for diagnostic purposes and a detection method for non-diagnostic purposes. The detection method for diagnosis can detect the change of the number and the activity of the new coronavirus before and after treatment of a patient. Methods for non-diagnostic purposes include, for example:

1. preventive medical research and institutional health regulations: as known to those skilled in the art, modern medicine is divided into two parts: preventive medicine and clinical medicine. The invention relates to a non-diagnosis detection … … method, which can detect samples (including human secretion) collected in the environment in preventive medicine, judge whether the environment is polluted, decide whether to block, suspend business, kill and other treatment modes to the environment, and decide whether to upgrade infectious disease prevention and control measures to the region, city and even higher level. The infection is prevented from the public health point of view, and the virus remained in the environment is prevented from becoming the infection source.

2. The field of scientific research: (1) basic medicine and ecology: for example, one skilled in the art can test water samples to determine the time and breadth of transmission of the virus. (2) Research and development of virology products: it is known to those skilled in the art that when multiple viruses are infected simultaneously in the same host, viral mutations may occur, including recombination of the viral nucleic acids themselves, or recombination of nucleic acids from different viruses. This is even more so if a mutation of the virus occurs, especially when it occurs in the autumn and winter flu season. The antibody of the invention is used for detection, can be used for virology research, detects whether the integration of virus nucleic acid is generated, and can monitor and timely warn from the aspect of preventive medicine whether the integration of virus is generated.

In one aspect, the disclosure provides the use of an antibody or antigen-binding fragment thereof according to the disclosure in a product for the detection, diagnosis, prevention and/or treatment of SARS-CoV-2 infection.

Drawings

FIG. 1 shows the process of fully human antibody production targeting SARS-CoV-2 coronavirus S protein.

FIG. 2A shows the determination of the binding activity of the recombinant protein Spike protein (Spike) RBD-mFc to human ACE 2-huFc.

FIG. 2B shows the determination of the binding activity of the recombinant proteins Spike protein (Spike) RBD-His and Spike RBD-mFc to human ACE 2-His.

FIG. 3A shows the enrichment effect of each round of the haining output, which shows the binding capacity of phage output from each round of the antibody haining process to the Spike RBD-mFc protein at ELISA level.

FIG. 3B shows the enrichment effect of each round of the hain selection output, which shows the binding ability of phage output from each round of the antibody hain selection process to the Spike RBD-mFc protein at the ELISA level.

FIG. 4 shows the detection of affinity activity of candidate antibodies binding to Spike RBD mFc based on ELISA.

FIG. 5A shows grouping of epitopes of candidate antibodies using the double antibody sandwich method of ELISA, which is grouping of epitopes of candidate antibodies when R16-F10 is in the liquid phase.

FIG. 5B shows the grouping of epitopes for candidate antibodies using the double antibody sandwich of ELISA, which is the grouping of epitopes for candidate antibodies when P16-A3 is in the liquid phase.

FIG. 6 shows the sensitivity of detection of S protein standard in colloidal gold assay cards prepared using R16-F10 antibody binding to epitope one and P16-A3 antibody binding to epitope two, which are 2. mu.g/mL, 1. mu.g/mL, 500 ng/mL, 250 ng/mL, 125 ng/mL, 62.5 ng/mL, 31 ng/mL, 15.6 ng/mL, 7.8 ng/mL, 3.9 ng/mL, 1.95 ng/mL, 650 pg/mL, 0 pg/mL (blank 1), 0 pg/mL (blank 2), 0 pg/mL (blank 3), respectively, in each row from top to bottom in FIG. 6.

FIG. 7 shows the detection of cross-binding of antibody R16-F10 to SARS-CoV-2 coronavirus S protein, SARS-CoV (SARS) recombinant protein, MERS recombinant protein using ELISA; wherein the binding of CR3022 (Monoclonal Antibody capable of binding to SARS-CoV, see Jan ter Meulen, Human Monoclonal Antibody Combination against SARS Coronavir: Synergy and Coverage of Escape variants, PLoS Med.2006 Jul; 3(7) e237, GenBank accession numbers DQ168569 and DQ168570, respectively, for the variable region of CR3022 Antibody) to the SARS-CoV recombinant protein serves as a positive control.

Detailed Description

The following provides a description of antibodies or antigen-binding portions thereof, antibody pairs, detection products, methods, and uses that can be used to detect, diagnose, prevent, and treat SARS-CoV-2 coronavirus infection. These antibodies, antibody pairs, products, methods and uses meet at least to some extent at least one of the needs existing in the art.

While this disclosure may be embodied in many different forms, there are disclosed herein specific illustrative embodiments thereof which are indicative of the principles of the disclosure. It should be emphasized that this disclosure is not limited to the particular embodiments illustrated. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this disclosure, the following terms are defined below.

The term "about," when used in conjunction with a numerical value, is intended to encompass a numerical value within a range having a lower limit that is 10% less than the stated numerical value and an upper limit that is 10% greater than the stated numerical value.

The term "and/or" when used to connect two or more selectable items should be understood to mean any one of the selectable items or any two or more of the selectable items.

As used herein, the term "comprising" or "comprises" is intended to mean including the stated elements, integers or steps, but not excluding any other elements, integers or steps. When the term "comprising" or "includes" is used herein, unless otherwise specified, it also encompasses the presence of stated elements, integers or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.

The term "Coronaviruses (CoV)" herein refers to viruses belonging to the genus beta coronavirus (Coronaviridae) of the family Coronaviridae, the virus particles being spherical or elliptical and having a diameter of about 60 to 220 nm. The virus is a single-stranded positive-stranded RNA (+ ssRNA) virus. Among several human pathogenic coronaviruses, most are associated with mild clinical symptoms (SuS, Wong G, Shi W et al, epidemic, genetic organization, and pathogenesis of coronaviruses. Trends Microbiol 2016; 24: 490-502), but two coronaviruses are notable exceptions: SARS-CoV; the other is a 2019 novel coronavirus (2019-nCoV) which causes a novel human coronavirus Disease (Corona Virus Disease 2019, COVID-19), has strong capability of spreading in the population, most infected patients suffer from high fever, some suffer from dyspnea, and chest X-ray films show that both lungs have invasive lesions (Huang C, Wang Y, Li X et al, Lancet, 24 th day on 1/2020). The World Health Organization (WHO) named the 2019-nCoV as SARS-CoV-2. "2019-nCoV" and "SARS-CoV-2" are used interchangeably herein.

The term "antibody" is used herein in the broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) so long as they exhibit the desired antigen binding activity. The antibody can be a whole antibody (e.g., having two full-length light chains and two full-length heavy chains) of any type and subtype (e.g., IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, IgA1, and IgA 2). A monomer of a complete antibody is a tetrapeptide chain molecule formed by disulfide bonding two full-length light chains and two full-length heavy chains, also known as a monomer of an Ig molecule. Antibody monomers are the basic structure that constitutes an antibody.

As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (abs) having different antigen specificities (e.g., an isolated antibody or antigen-binding fragment thereof that specifically binds a coronavirus S protein is substantially free of abs that specifically binds an antigen other than a coronavirus S protein). In certain embodiments, the antibody is purified to greater than 95% or 99% purity as determined, for example, by electrophoresis (e.g., SDS-PAGE, electrofocusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC).

As used herein, "blocking antibody," "neutralizing antibody," "antibody having neutralizing activity," or "neutralizing antibody" are used interchangeably herein and refer to an antibody that binds to or interacts with a target antigen and prevents the target antigen from binding to a ligand or receptor, thereby inhibiting or blocking a biological response that would otherwise result from the interaction of the target antigen with a binding partner, such as a receptor. In the context of the present disclosure, it is meant that binding of the antibody to the coronavirus S protein results in inhibition of at least one biological activity of the coronavirus. For example, the neutralizing antibodies of the present disclosure may prevent or block binding of coronavirus S protein to ACE 2.

An "epitope" or "antigenic determinant" refers to an antigenic determinant that interacts with a specific antigen-binding site, called a paratope, in the variable region of an antibody molecule. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and may have different biological effects. Epitopes can be formed from contiguous amino acids or noncontiguous amino acids joined in parallel via tertiary folding of a protein. Epitopes formed by consecutive amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally absent upon treatment with denaturing solvents. Epitopes typically comprise at least 3, and more typically at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.

The term "antigen-binding fragment" is a portion or fragment of an intact or complete antibody having fewer amino acid residues than the intact or complete antibody, which is capable of binding to an antigen or competing with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived) for binding to an antigen. Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen binding fragments include, but are not limited to, Fab ', F (ab ') 2, Fv, single chain Fv (scFv), single chain Fab, diabodies (diabodies), single domain or single domain antibodies (sdabs, nanobodies), camelid Ig, Ig NAR, F (ab) '3 fragments, bis-scFv, (scFv) 2, minibody, diabody, trifunctional antibody, tetrafunctional antibody, disulfide stabilized Fv protein ("dsFv"). The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof. For a more detailed description, see, for example, the Pierce Catalog and Handbook (Pierce Catalog and Handbook), 1994-1995 (Pierce chemical Co., Rockford, Ill.); Kuby, J. Immunol, 3rd edition, W.H. Flliman corporation (W.H.Freeman & Co., N.Y.), 1997.

The terms "whole antibody", "full-length antibody", "whole antibody" and "intact antibody" are used interchangeably herein to refer to a glycoprotein comprising at least two Heavy Chains (HC) and two Light Chains (LC) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of 3 domains, CH1, CH2, and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region consists of one domain CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as lambda or kappa. Immunoglobulins comprising alpha, delta, epsilon, gamma and mu heavy chains are classified as immunoglobulins (Ig) a, IgD, IgE, IgG and IgM. The complete antibody formed a "Y" shape. The stem of Y consists of the second and third constant regions of the two heavy chains (and for IgE and IgM, the fourth constant region) joined together, and disulfide bonds (interchain) are formed in the hinge. Heavy chains γ, α, and δ have a constant region consisting of three tandem (in-line) Ig domains, and a hinge region for increased flexibility; heavy chains mu and epsilon have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH 2 domain" and the "CH 3 domain", respectively. Each arm of Y comprises the variable region and the first constant region of a single heavy chain joined to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

The light and heavy chain variable regions each comprise a "framework" region interspersed with three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs"). "complementarity determining regions" or "CDR" or "hypervariable region" (used interchangeably herein with hypervariable region "HVR") are regions in an antibody variable domain which are mutated in sequence and form structurally defined loops ("hypervariable loops") and/or which contain antigen-contacting residues ("antigen-contacting points"). The CDRs are primarily responsible for binding to an epitope of the antigen. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR 3. The precise amino acid sequence boundaries of each CDR in a given light or heavy chain variable region amino acid sequence may be determined using any one or combination of a number of well known antibody CDR assignment systems, including, for example, Chothia (Chothia et Al (1989) Nature 342: 877-883, Al-Lazikani et Al, "Standard constraints for the structural organization of immunology", Journal of Molecular Biology,273, 927-948 (1997), Kabat (Kabat et Al, Sequences of Proteins of Immunological Interest, 4 th edition), U.S. Deutment of Health and Services, Nature of Health (1987)), or the CDR of Molecular Biology (Biotech) (Biotech ) (Biotech) GT www.imgt.cines.fr), or combinations thereof, based on the three-dimensional structure of antibodies, And North CDR definition based on affinity propagation clustering with a large number of crystal structures. The specific CDR sequences listed in the present disclosure are generally defined by AbM, but those skilled in the art should understand that the amino acid sequences corresponding to other CDR definition rules should also fall within the scope of the present invention.

However, it should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may differ. I.e., the CDR sequences of the same antibody variable region defined under different assignment systems differ. For example, the residue ranges for CDR regions numbered using Kabat and Chothia under different assignment systems are shown in table a below.

Table a. CDR residue ranges under different assignment system definitions

Thus, where reference is made to an antibody defined with a particular CDR sequence as defined in the present disclosure, the scope of the antibody also encompasses an antibody whose variable region sequences comprise the particular CDR sequence but whose claimed CDR boundaries differ from the particular CDR boundaries as defined in the present disclosure due to the application of different protocols (e.g., different assignment system rules or combinations).

The CDRs of the antibodies of the disclosure can be assessed manually to determine boundaries according to any protocol or combination thereof in the art. Unless otherwise indicated, in the present disclosure, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the ways described above.

The sequences of the framework regions of different light or heavy chains have relative preservability within a species (e.g., human). The framework regions of the antibody, which are the combined framework regions of the component light and heavy chains, are used to locate and align the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to the epitope of the antigen. Antibodies with different specificities (i.e., different combinatorial sites for different antigens) have different CDRs. Despite the CDR differences from antibody to antibody, only a limited number of amino acid positions within the CDR are directly involved in antigen binding. These positions within the CDRs are called Specificity Determining Residues (SDRs).

A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by cells into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by preparing hybrid antibody-forming cells from fusions of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

"Fv" is the smallest antibody fragment containing the complete antigen binding site. In one example, a two-chain Fv species consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. In the single-chain Fv (scFv) species, one heavy chain variable domain and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure similar to that of a two-chain Fv species. In this configuration, the three hypervariable regions (HVRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. The six HVRs collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, but with a lower affinity than the entire binding site.

Fab fragments contain a heavy chain variable domain and a light chain variable domain and also contain the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the name given herein for Fab', in which the cysteine residues of the constant domains carry free thiol groups. F (ab ') 2 antibody fragments were originally produced as Fab' fragment pairs with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The term "specifically binds" or "binds" when used in reference to an antigen/epitope and an antibody means that the antibody forms a complex with the antigen which is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen/epitope are well known in the art and include, for example, surface plasmon resonance assay, MSD assay (Estep, P. et al, High throughput solution-based measurement of antibody-antibody affinity and epitope binding, MAbs, 2013.5 (2): P. 270) -278), ForteBio affinity assay (Estep, P et al, High throughput solution-based measurement of antibody-antibody affinity and epitope binding. MAbs, 2013.5 (2): P. 270-8), and the like.

In one embodiment, an antibody of the present disclosure that "specifically binds" to a coronavirus S protein, e.g., as measured in a ForteBio affinity assay, is expressed in a molar ratio of at least about 10 "8M, preferably 10" 9M; more preferably 10-10M, even more preferably 10-11M, more preferably 10-12M, whereby the KD of the S protein binds to the S protein, thereby blocking or inhibiting the binding of the coronavirus S protein to its receptor ACE2 and subsequent membrane fusion, and thereby for the detection and diagnosis of coronavirus infections.

"affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the binding dissociation equilibrium constant (KD). Affinity can be measured by common methods known in the art, including those known in the art and described herein.

The term "variant" as used herein refers to a polypeptide or fragment, such as a heavy chain variable region or a light chain variable region, having at least one, e.g., 1, 2, or 3 amino acid changes, e.g., amino acid substitutions, deletions, or additions. A modified antibody or antigen-binding fragment thereof comprising a heavy chain or light chain variant substantially retains the biological characteristics of the pre-modified antibody or antigen-binding fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprising a variable heavy chain variable region or light chain variable region sequence retains 60%, 70%, 80%, 90%, 100% or more of the biological characteristics of the pre-modified antibody or antigen-binding fragment thereof. It will be appreciated that each heavy chain variable region or light chain variable region may be modified alone or in combination with another heavy chain variable region or light chain variable region. In one embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain variable region amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to a heavy chain variable region amino acid sequence described herein. In one embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises a light chain variable region amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to a light chain variable region amino acid sequence described herein. The percent homology may be across the entire heavy chain variable region and/or the entire light chain variable region, or the percent homology may be limited to framework regions, while the sequences corresponding to the CDRs are 100% identical to the CDRs disclosed herein within the heavy chain variable region and/or the light chain variable region.

As used herein, the term "CDR variant" refers to a CDR having at least one, e.g., 1, 2, or 3 amino acid changes, e.g., substitutions, deletions, or additions, wherein the modified antibody or antigen-binding fragment thereof comprising the CDR variant substantially retains the biological characteristics of the pre-modified antibody or antigen-binding fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof containing the variant CDRs retains at least 60%, 70%, 80%, 90%, 100% or more of the biological characteristics of the pre-modified antibody or antigen-binding fragment thereof. It is understood that each CDR that can be modified alone or in combination with another CDR. In one embodiment, the modification or change is a substitution, in particular a conservative substitution.

"humanized antibody" refers to a class of engineered antibodies having CDRs derived from a non-human donor immunoglobulin, with the remaining immunoglobulin portion of the humanized antibody being derived from one (or more) human immunoglobulin(s). Furthermore, framework support residues may be altered to preserve binding affinity (see, e.g., Queen et al, Proc. Natl. Acad. Sci. USA, 86:10029-10032 (1989), Hodgson et al, Bio/Technology, 9:421 (1991)). Suitable human acceptor antibodies may be antibodies selected from conventional databases, such as the Los Alamos database and the Swiss protein database, by homology to the nucleotide and amino acid sequences of the donor antibody. Human antibodies characterized by homology (based on amino acids) to the framework regions of the donor antibody may be suitable for providing heavy chain constant regions and/or heavy chain variable framework regions for insertion of the donor CDRs. Suitable acceptor antibodies that provide light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains need not be derived from the same acceptor antibody.

"human antibody" refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human and/or made using any of the techniques for making human antibodies as disclosed herein. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be made using a variety of techniques known in the art, including phage display libraries.

As known in the art, "polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a chain of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate capable of being incorporated into a strand by a DNA or RNA polymerase.

The calculation of sequence identity between sequences is performed as follows. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, the length of the aligned reference sequences is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. Sequence comparisons between two sequences and calculation of percent identity can be accomplished using mathematical algorithms. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. mol.biol. 48: 444-. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http:// www.gcg.com), using the NWSgapdna. CMP matrix and GAP weights 40, 50, 60, 70 or 80 and

length weights

1, 2, 3, 4, 5 or 6. A particularly preferred set of parameters (and one that should be used unless otherwise specified) is the Blossum 62 scoring matrix using a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The PAM120 weighted residue table, gap length penalty 12, gap penalty 4) can also be used to determine the percent identity between two amino acid or nucleotide sequences using the E.Meyers and W.Miller algorithms that have been incorporated into the ALIGN program (version 2.0) ((1989) CABIOS, 4: 11-17). Additionally or alternatively, the nucleic acid sequences and protein sequences described herein may be further used as "query sequences" to perform searches against public databases, for example, to identify other family member sequences or related sequences.

As used herein, "vector" means a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the gene or sequence in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably in this disclosure and can include cells into which an exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as the cell screened or selected for in the originally transformed cell.

As used herein, a "subject", "individual" or "subject" refers to an animal, preferably a mammal, more preferably a human, in need of detection, diagnosis, amelioration, prevention and/or treatment of a disease or disorder, such as a viral infection. Mammals also include, but are not limited to, farm animals, racing animals, pets, primates, horses, dogs, cats, mice, and rats. The term includes human subjects having or at risk of having a coronavirus infection. The antibodies, or antigen-binding portions thereof, and antibody pairs described in the present disclosure are useful for detecting or diagnosing coronavirus infection, particularly SARS-CoV-2 virus infection, in a human subject. In the present disclosure, administering an antibody or antigen-binding portion thereof described in the present disclosure to a subject in need thereof refers to administering an effective amount of the antibody or antigen-binding portion thereof, and the like.

As used in this disclosure, the term "effective amount" means the amount of a drug or pharmaceutical agent that elicits the biological or pharmacological response in a tissue, system, animal or human that is being sought, for example, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount that causes improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or a decrease in the rate of progression of a disease or condition, as compared to a corresponding subject not receiving that amount. The term also includes within its scope an amount effective to enhance normal physiological function.

Coronavirus II

Coronaviruses are spherical single-stranded positive-stranded RNA viruses characterized by having spike proteins protruding from the virion surface (Barcena, M. et al, Cryo-electron biology of mouse epitopes virus: insight into the syndrome of the coronaviridation, Proc. Natl. Acad. Sci. USA2009, 106, 582-. The spherical morphology of the virus particles and the spike projections make coronaviruses look like coronaries under an electron microscope and are named coronavirus.

Coronaviruses are enveloped viruses (the Envelope is derived from the lipid bilayer of the host cell Membrane), having a viral structure formed mainly of viral structural proteins such as Spike protein (S), Membrane protein (M), Envelope protein (E) and Nucleocapsid protein (N), wherein the S, M and E proteins are all embedded in the viral Envelope, and the N protein interacts with the viral RNA, is localized at the core of the viral particle, forming the Nucleocapsid (Fehr, a.r. et al, Coronaviruses: An overview of the same reproduction and pathogenesis. Methods mol. biol.2015, 1282, 1-23). The S protein is a highly glycosylated protein that forms homotrimeric spikes on the surface of viral particles and mediates viral entry into host cells. SARS-CoV-2 is a single-stranded positive-strand RNA virus having a membrane structure and a size of 80-120 nm, and has a genome length of about 29.9 kb and a homology of 80% with the genome sequence of SARS-CoV belonging to the genus β coronavirus belonging to the family Coronaviridae. Open Reading Frame (ORF) ORF1a and ORF1b of the viral genome account for 2/3 of the genome, expressing hydrolases as well as replication, transcription related enzymes, e.g., cysteine protease (PLpro) and serine protease (3 CLpro), RNA-dependent RNA polymerase (RdRp) and helicase (Hel); the latter 1/3 region is responsible for encoding major structural proteins, including spike (S), envelope (E), membrane (M), nucleocapsid (N), etc., where the N protein envelops the viral genome to form a nucleoprotein complex, the E and M proteins are involved in the viral assembly process, and the S protein mediates viral entry and determines the host specificity of the virus, mainly through binding to host cell receptors. Sequence alignment revealed that SARS-CoV-2 virus and SARS-CoV virus S protein have 75% similarity, and it was reported that amino acid residues at the 442, 472, 479, 487 and 491 sites of the S protein and ACE2 receptor complex interface (distributed mainly in respiratory epithelial cells, lung, heart, kidney and digestive tract in humans) of multiple SARS-CoV coronavirus isolates are highly conserved. Compared with the S protein of SARS-CoV, the SARS-CoV-2 protein has only the 491 th amino acid identical at the 5 th position and has mutations at the other 4 th amino acids (Xu X et al, Sci China Life Sci., 3.2020; 63 (3): 457-. Nevertheless, it was found through protein 3D structure simulation prediction that although the S protein of SARS-CoV-2 is substituted with the 4 key amino acids bound to ACE2 receptor, the three-dimensional structure of the Receptor Binding Domain (RBD) in the S protein of SARS-CoV-2 is hardly changed relative to the S protein of SARS-CoV, and thus the S protein of SARS-CoV-2 still has high affinity to human ACE 2. A recent article (WrapPD et al, Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion formulation, Science, 19.2.2020, published on the web, pii: eabb2507. DOI: 10.1126/Science. abb 2507) and (Xiaoolong Tianan et al, tension binding of 2019 novel coronavir Spike protein by a SARS coronavir-specific human monoclonal antibody, engineering Microbes & innovations, 2020,9:1, p382-385, DOI: 10.1080/22221751.2020.1729069) found by Fortebio assay that the S protein of SARS-CoV-2 binds to human 2 with an affinity (SARS) of about 15 nM, and the S protein of human CoV-2 binds to human S protein, whereby the internal affinity of human CoV-3838 is also seen for the human CoV-2-intrinsic receptor infection. High affinity neutralizing antibodies directed against coronavirus S protein and blocking its binding to the ACE2 receptor are expected to be effective in preventing and treating coronavirus (e.g., SARS-CoV-2, SARS-CoV) infection.

Furthermore, given the sequence and structural similarity of SARS-CoV and the S protein of SARS-CoV-2, a detection antibody that is capable of distinguishing between the two would be particularly beneficial.

Antibodies against coronavirus S protein

The terms "antibody against coronavirus S protein," "anti-coronavirus S protein antibody," "anti-S protein antibody," "coronavirus S protein antibody," "S protein antibody," or "antibody that binds to S protein" are used interchangeably herein to refer to an antibody of the present disclosure that is capable of binding coronavirus S protein (e.g., SARS-CoV-2S protein) with sufficient affinity such that the antibody can be used as a detection, diagnostic, prophylactic and/or therapeutic agent that targets coronavirus S protein.

Antibodies and antigen binding portions thereof

In one aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to a coronavirus (particularly SARS-CoV-2) S protein.

In some embodiments, the antibodies provided by the present disclosure are directed against different epitopes of the SARS-CoV-2 coronavirus S protein. In some embodiments, the present disclosure provides the following antibodies against epitope 1 of the S protein of SARS-CoV-2 coronavirus:

group (a) -an antibody comprising HCDR1 comprising the amino acid sequence of SEQ ID NO:5 or a variant thereof having NO more than 2 amino acid changes; HCDR2 comprising the amino acid sequence of SEQ ID No. 6 or a variant thereof having NO more than 2 amino acid changes; HCDR3 comprising the amino acid sequence of SEQ ID No. 7 or a variant thereof having NO more than 2 amino acid changes; LCDR1 comprising the amino acid sequence of SEQ ID No. 8 or a variant thereof having NO more than 2 amino acid changes; LCDR2 comprising the amino acid sequence of SEQ ID No. 9 or a variant thereof having NO more than 2 amino acid changes; and/or LCDR3 comprising the amino acid sequence of SEQ ID No. 10 or a variant thereof having NO more than 2 amino acid changes;

group (b) -an antibody comprising HCDR1 or a variant thereof having NO more than 2 amino acid changes in the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 3; 3 or a variant thereof having NO more than 2 amino acid changes to HCDR2 in the heavy chain variable region amino acid sequence set forth in SEQ ID NO; 3 or a variant thereof having NO more than 2 amino acid changes to HCDR3 in the heavy chain variable region amino acid sequence set forth in SEQ ID NO; LCDR1 or a variant thereof having NO more than 2 amino acid changes in the light chain variable region amino acid sequence set forth in SEQ ID NO. 4; LCDR2 or a variant thereof having NO more than 2 amino acid changes in the light chain variable region amino acid sequence set forth in SEQ ID NO. 4; and/or LCDR3 or a variant thereof having NO more than 2 amino acid changes in the light chain variable region amino acid sequence set forth in SEQ ID NO. 4; or

Group (c) -an antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO 3 or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and/or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 4 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

In some embodiments, in the antibodies of group (a), (b), or (c), the HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID No. 5; HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO. 6; and/or HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO. 7.

In some embodiments, in the antibodies of group (a), (b), or (c), LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID No. 8; LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO. 9; and/or LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO. 10.

In some embodiments, the antibody of group (a), (b) or (c) comprises an IgG heavy chain (e.g. an IgG1, IgG2, IgG3 or IgG4 heavy chain, especially an IgG1 or IgG4 heavy chain) and/or a Kappa light chain. In some embodiments, the antibody of group (a), (b), or (c) further comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO. 1 or a sequence at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and/or a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO. 2 or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In some embodiments, the antibodies in group (a), (b), or (c) are fully human antibodies.

In some embodiments, the present disclosure provides the following antibodies against epitope 2 of the S protein of SARS-CoV-2 coronavirus:

group (d) -antibodies comprising HCDR1 or variants thereof having NO more than 2 amino acid changes in the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 11; 11 or a variant thereof having NO more than 2 amino acid changes of HCDR2 in the heavy chain variable region amino acid sequence set forth in SEQ ID NO; 11 or a variant thereof having NO more than 2 amino acid changes of HCDR3 in the heavy chain variable region amino acid sequence set forth in SEQ ID NO; LCDR1 or a variant thereof having NO more than 2 amino acid changes in the light chain variable region amino acid sequence set forth in SEQ ID NO. 12; LCDR2 or a variant thereof having NO more than 2 amino acid changes in the light chain variable region amino acid sequence set forth in SEQ ID NO. 12; and/or LCDR3 or a variant thereof having NO more than 2 amino acid changes in the light chain variable region amino acid sequence set forth in SEQ ID NO. 12; or

Group (e) -an antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and/or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 12 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In some embodiments, the antibody of group (d) or (e) comprises an IgG heavy chain (e.g. an IgG1, IgG2, IgG3 or IgG4 heavy chain, especially an IgG1 or IgG4 heavy chain) and/or a Kappa light chain. In some embodiments, the antibody in group (d) or (e) is a fully human antibody.

In some embodiments, epitope 1 and epitope 2 are located at different positions of the S protein RBD domain of SARS-CoV-2 coronavirus.

In some embodiments, the amino acid change can be an addition, deletion, or substitution of an amino acid, e.g., the amino acid change is a conservative amino acid substitution. In some embodiments, the amino acid change does not occur in a CDR region.

In some embodiments of the present disclosure, the amino acid changes described herein comprise substitutions, insertions, or deletions of amino acids. In some embodiments, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions. Conservative substitutions are those where one amino acid is substituted with another within the same class, for example where one acidic amino acid is substituted with another acidic amino acid, one basic amino acid is substituted with another basic amino acid, or one neutral amino acid is substituted with another neutral amino acid. Exemplary substitutions are shown in table B below;

table b. exemplary amino acid substitutions

In some embodiments, the amino acid changes described in the present disclosure occur in a region outside of the CDRs (e.g., in the FRs). In some embodiments, the amino acid changes described in the present disclosure occur in the Fc region. In some embodiments, an antibody or antigen-binding portion thereof comprising an Fc domain comprising one or more mutations that enhance or attenuate binding to the FcRn receptor at acidic pH, e.g., as compared to neutral pH, is provided. For example, the present disclosure includes an anti-coronavirus S protein antibody or antigen-binding portion that binds an epitope of a coronavirus S protein comprising a mutation in the CH2 or CH3 region of the Fc domain, wherein the one or more mutations increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome at a pH in the range of about 5.5 to about 6.0). Such mutations can result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or modification at position 428 and/or 433 (e.g.H/L/R/S/P/Q or K) and/or 434 (e.g. A, W, H, F or Y [ N434A, N434W, N434H, N434F or N434Y ]); or a modification at position 250 and/or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252. 254 and 256 (e.g., 252Y, 254T and 256E); 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and/or 308 modifications (e.g., 308F or 308P). In yet another embodiment, the modification comprises 265A (e.g., D265A) and/or 297A (e.g., N297A) modifications. For example, the present disclosure includes anti-coronavirus S protein antibodies or antigen-binding portions comprising an Fc domain comprising one or more pairs of mutations selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A, and 434A (e.g., T307A, E380A, and N434A); and 433K and 434F (e.g., H433K and N434F). In some embodiments, the present disclosure includes an anti-coronavirus S protein antibody or antigen-binding portion thereof comprising an Fc domain comprising an S108P mutation in the IgG4 hinge region to facilitate dimer stabilization. Any possible combination of the aforementioned Fc domain mutations and other mutations within the antibody variable domains disclosed herein are included within the scope of the present disclosure.

In some embodiments, a coronavirus S protein antibody or antigen-binding portion thereof provided herein is altered to increase or decrease the degree of glycosylation thereof. Addition or deletion of glycosylation sites to the coronavirus S protein antibody or antigen-binding portion thereof can be conveniently achieved by altering the amino acid sequence so as to create or remove one or more glycosylation sites. When the coronavirus S protein antibody or antigen-binding portion thereof comprises an Fc region, the carbohydrate attached to the Fc region may be altered. In some applications, modifications that remove unwanted glycosylation sites may be useful, for example, to remove fucose moieties to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al (2002) JBC277: 26733). In other applications, galactosylation modifications may be made to modulate Complement Dependent Cytotoxicity (CDC). In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of a coronavirus S protein antibody or antigen-binding portion thereof provided herein to generate an Fc region variant in order to enhance the effectiveness of a coronavirus S protein antibody, e.g., of the present disclosure, in preventing and/or treating a coronavirus infection. In some embodiments, native sequence Fc regions suitable for use with the antibodies described herein include human IgG1, IgG2 (IgG 2A, IgG 2B), IgG3, and IgG4, preferably human IgG 1. In some embodiments, ADCC and CDC effects are removed or reduced by mutating L234A and L235A of human IgG1 Fc.

In one aspect, the antibodies or antigen-binding fragments thereof provided by the present disclosure that specifically bind to the S protein of a coronavirus (particularly SARS-CoV-2) can be used alone for immunological detection or diagnosis of coronavirus infection and prevention or treatment of coronavirus infection, and can also be paired for detection and/or diagnosis of coronavirus (particularly SARS-CoV-2) infection by immunological methods such as a double antibody sandwich method.

Paired antibodies or antibody pairs

As used herein, the term "partner antibody" or "antibody pair," used interchangeably, refers to two antibodies that can bind to one antigenic molecule at the same time. The ligand antibody or antibody pair is generally applicable to a double antibody sandwich ELISA assay. In the present disclosure, a partner antibody or antibody pair can simultaneously bind to different epitopes of the S protein of a coronavirus, particularly SARS-CoV-2. In some embodiments, a partner antibody or antibody pair of the present disclosure can simultaneously bind to different epitopes on the RBD region of the S protein of a coronavirus, particularly SARS-CoV-2. A beneficial ligand antibody or antibody pair would be able to significantly improve the sensitivity and/or specificity of the detection.

As used herein, the term "double antibody sandwich" is a non-competitive binding assay that can be used to detect multivalent antigens having at least two antigenic determinants in a molecule. The detection method can utilize the capture antibody and the labeled antibody loaded on the solid phase carrier to be respectively combined with two antigenic determinants on the detected antigen molecules in a sample to form a solid phase antibody-antigen-labeled antibody immune complex. In some embodiments, since the amount of solid phase capture antibody and labeled antibody in the reaction system is excessive relative to the antigen to be detected, the amount of complex formed is directly proportional to the amount of antigen to be detected within the detectable range. General procedures and procedures for detecting antigens using the "double antibody sandwich" method are well known in the art, as reviewed in Methods in Enzymology, 1980, v.70, pages 166-198.

In some embodiments, the presence of a coronavirus (particularly SARS-CoV-2) S protein is determined using a pair of antibodies each specific for a coronavirus (particularly SARS-CoV-2) S protein. One of the pair of antibodies is referred to herein as a "labeled antibody" and the other of the pair of antibodies is referred to herein as a "capture antibody". The antibodies or binding fragments thereof of the present disclosure can be used as capture or label antibodies. The antibodies or binding fragments thereof of the present disclosure can also be used as both capture and label antibodies. Some embodiments of the present disclosure thus employ a double antibody sandwich method for the detection of coronavirus (particularly SARS-CoV-2) S protein in a biological or environmental sample. In this method, the analyte (coronavirus (especially SARS-CoV-2) S protein) is sandwiched between a labeled antibody and a capture antibody, wherein the capture antibody is irreversibly immobilized on a solid support. The labeled antibody comprises a detectable label to identify the presence of the antibody-analyte sandwich and thus the analyte.

In some embodiments, one or more antibodies or antigen-binding portions thereof in groups (a), (b), or (c) of the present disclosure are labeled and serve as detection antibodies, while one or more antibodies or antigen-binding portions thereof in groups (d) or (e) of the present disclosure are attached to a solid support or support as capture antibodies.

Methods and compositions for diagnosis and detection of coronaviruses

In some embodiments, any of the antibodies or antigen-binding portions thereof or counterpart antibodies provided herein can be used to detect the presence of a coronavirus in or an environmental biological sample. The term "detecting" as used herein includes quantitative or qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays.

In one embodiment, an antibody, or antigen-binding portion thereof, or companion antibody that specifically binds to coronavirus S protein is provided for use in a diagnostic or detection method. In another aspect, a method of detecting the presence of a coronavirus in a biological sample or a coronavirus contamination of an environment is provided. In certain embodiments, the method comprises detecting the presence of coronavirus S protein in a biological sample or environment. In certain embodiments, the method comprises contacting the biological or environmental sample with an antibody, or antigen-binding portion thereof, or a companion antibody, as described herein under conditions that allow binding of the antibody, or antigen-binding portion thereof, or companion antibody, to the coronavirus S protein, and detecting whether a complex is formed between the antibody, or antigen-binding portion thereof, or companion antibody, and the coronavirus S protein. The formation of a complex indicates the presence of coronavirus. The method may be an in vitro or in vivo method.

Exemplary diagnostic assays for coronaviruses include, for example, contacting a sample obtained from a patient with an antibody or antigen-binding portion thereof or a companion antibody of the present disclosure, wherein the antibody or antigen-binding portion thereof or the companion or capture ligand of the present disclosure is labeled with a detectable label or reporter molecule to selectively isolate coronaviruses from the patient sample. Alternatively, an unlabeled antibody of the present disclosure, or an antigen-binding portion thereof, can be used in diagnostic applications in combination with a second antibody that is itself detectably labeled. The detectable label or reporter may be a radioisotope, such as 3H, 14C, 32P, 35S or 125I; fluorescent or chemiluminescent moieties such as fluorescein isothiocyanate or rhodamine, or enzymes such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure coronavirus in a sample include enzyme linked immunosorbent assays (ELISAs), Radioimmunoassays (RIA), and Fluorescence Activated Cell Sorting (FACS).

Samples that can be used in a coronavirus diagnostic assay according to the present disclosure include any biological sample obtainable from a patient that comprises a coronavirus spike protein or fragment thereof in an amount detectable under normal or physiological conditions. In some embodiments, the biological sample is blood, serum, a pharyngeal swab, a lower respiratory tract sample (e.g., tracheal secretions, tracheal aspirates, alveolar lavage fluid), or other sample of biological origin. Generally, coronavirus spike protein levels in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with a coronavirus-related disease) will be measured to initially establish a baseline or standard coronavirus level. The baseline level of coronavirus may then be compared to a coronavirus level measured in a sample obtained from an individual suspected of having a coronavirus-related condition or a symptom associated with the condition. The antibody or antigen-binding portion thereof specific for the coronavirus spike protein may comprise no further label, or it may comprise an N-terminal or C-terminal label. In one embodiment, the label is biotin. In a binding assay, the location of the label (if present) can determine the orientation of the peptide relative to the bound surface. For example, if the surface is coated with avidin, a peptide comprising an N-terminal biotin will be oriented such that the C-terminal portion of the peptide is distal to the surface.

In one aspect, the present disclosure provides a method and product, such as a test or diagnostic strip or kit, for detecting a coronavirus (particularly SARS-CoV-2) and/or diagnosing an infection with a coronavirus (particularly SARS-CoV-2). These methods include well-known immunological methods commonly used to detect or quantify substances in biological or environmental samples. The terms "test or diagnostic strip", "test or diagnostic kit" refer to any test/diagnostic strip or kit known in the art, and may refer to a diagnostic kit as described in Zrein et al (1998), for example.

In some embodiments, a test kit can include a container holding one or more antibodies or binding portions thereof according to the present disclosure, and instructions for using the antibodies or antigen-binding portions thereof to bind to a coronavirus (particularly SARS-CoV-2) S protein to form an immune complex and detecting the formation of the immune complex, thereby correlating the presence or absence of the immune complex with the presence or absence of the coronavirus (particularly SARS-CoV-2) S protein.

Diagnosis and detection of coronaviruses (particularly SARS-CoV-2) can be accomplished by detecting immunospecific binding of an antibody or antigen-binding fragment thereof to an epitope of the S protein of a coronaviruse (particularly SARS-CoV-2) in a sample, this comprises contacting a sample or a specific body part or body area suspected to contain a coronavirus, in particular SARS-CoV-2, with an antibody that binds to an epitope of the coronavirus, in particular SARS-CoV-2, S protein, allowing the antibody to bind to the coronavirus, in particular SARS-CoV-2, S protein to form an immune complex, detecting the formation of the immune complex, and correlating the presence or absence of the immune complex with the presence or absence of coronavirus (particularly SARS-CoV-2) S protein in the sample or in a specific body part or region.

Biological samples that can be used for detection or diagnosis of coronaviruses (particularly SARS-CoV-2) can include, for example, bodily fluids such as pharyngeal swabs, nasal swabs, lower respiratory tract samples (e.g., tracheal secretions, tracheal aspirates, alveolar lavage fluid), and the like; or a tissue or cell sample obtained from an organism, such as neural, brain, cardiac or vascular tissue, and the like. To determine the presence or absence of coronavirus, particularly SARS-CoV-2, in a sample, any immunoassay known to those of ordinary skill in the art (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York 1988555-612)) may be used, for example, assays utilizing indirect detection methods by means of secondary reagents, ELISA, and immunoprecipitation and agglutination assays. A detailed description of these assays is given, for example, in WO96/13590, Zrein et al (1998) and WO96/29605 to Maertens and Stuyver.

Immunoassays for diagnostic applications typically rely on labeled antigens, antibodies, or secondary reagents for detection. These proteins or reagents may be labeled with compounds known to those skilled in the art, including enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances, including colored particles such as colloidal gold and latex beads. Among them, radiolabels can be applied to almost all types of assays and most variant forms. Enzyme-conjugated labels are particularly useful when radioactivity must be avoided or rapid results are required. While fluorescent dyes require expensive equipment in use, fluorescent dyes provide a very sensitive detection method. Antibodies used in these assays may include monoclonal antibodies, polyclonal antibodies, and affinity-purified polyclonal antibodies.

Alternatively, the antibody may be indirectly labeled by reacting with a labeled substance having affinity for immunoglobulin, such as protein a or protein G or a second antibody. The antibody may be conjugated to a second substance and detected with a third substance labeled with an affinity for the second substance conjugated to the antibody. For example, the antibody may be conjugated to biotin and the antibody-biotin conjugate detected with labeled avidin or streptavidin. Similarly, the antibody may be conjugated to a hapten and the antibody-hapten conjugate detected using a labeled anti-hapten antibody.

These and other suitable markers that may be employed in accordance with the present disclosure are known to those skilled in the art. Binding of these labels to the antibody or fragment thereof can be accomplished using standard techniques generally known to those of ordinary skill in the art. Typical techniques are described by Kennedy, J.H. et al, 1976 (Clin.Chim.acta 70: 1-31), and Schurs, A.H.W.M. et al, 1977 (Clin.Chim Acta 81: 1-40). The latter coupling techniques are mentioned as the glutaraldehyde method, the periodate method, the bismaleimide method and the like. All of these disclosures are incorporated herein by reference.

In some embodiments, the immunoassays of the present disclosure include detecting the presence of an analyte using a two-antibody approach. In some embodiments, the present disclosure provides a dual antibody system suitable for use in rapidly determining the presence of an analyte. Such rapid detection systems typically require a high affinity between the antibody and the analyte. In some embodiments, the presence of a coronavirus (particularly SARS-CoV-2) S protein is determined using a pair of antibodies each specific for a coronavirus (particularly SARS-CoV-2) S protein. The antibodies or binding fragments thereof of the present disclosure can be used as capture or label antibodies. The antibodies or binding fragments thereof of the present disclosure can also be used as both capture and label antibodies. Some embodiments of the present disclosure thus employ a double antibody sandwich method for the detection of coronavirus (particularly SARS-CoV-2) S protein in a biological or environmental sample. In this method, the analyte (coronavirus (especially SARS-CoV-2) S protein) is sandwiched between a labeled antibody and a capture antibody, wherein the capture antibody is irreversibly immobilized on a solid support. The labeled antibody comprises a detectable label to identify the presence of the antibody-analyte sandwich and thus the analyte. In some embodiments, one or more antibodies or antigen-binding portions thereof in groups (a), (b), or (c) of the present disclosure are labeled and serve as labeled antibodies, while one or more antibodies or antigen-binding portions thereof in groups (d) or (e) of the present disclosure are attached to a solid support or support as capture antibodies.

Exemplary solid phase materials include, but are not limited to, microtiter plates, polystyrene test tubes, magnetic, plastic or glass beads and slides as are well known in the immunoassay art. Methods for coupling antibodies to solid supports are also well known to those skilled in the art. Porous materials such as nylon, nitrocellulose, cellulose acetate, glass fiber, and other porous polymers may also be used as solid supports.

In some embodiments, the assay products of the present disclosure comprise a substrate, a sample region, a binding region, and a reaction region, wherein the sample region, binding region, and reaction region are disposed sequentially on the substrate along a flow direction of a sample to be tested. In some embodiments, the binding region is loaded with a labeled antibody. In some embodiments, the reaction zone is provided with a detection zone, and in further embodiments, the reaction zone is also provided with a quality control zone. In some embodiments, the detection product herein further comprises an absorbent zone on the side of the reaction zone remote from the sample zone.

In some embodiments, the assay products of the present disclosure comprise a substrate. The present disclosure is not particularly limited with respect to the material, size and source of the substrate, and any substrate known to those skilled in the art for immunoassay may be used. In some embodiments, the substrate is made of a plastic material such as PVC sheeting, which is commercially available.

In some embodiments, the assay products of the present disclosure comprise a sample zone disposed on the substrate. In some embodiments, the material of the sample region is glass fiber. The present disclosure is not limited in any way as to the source of the glass fibers, as would be known to one skilled in the art. In some embodiments, the sample absorbed by the sample area is a pharyngeal swab or nasal swab specimen.

In some embodiments, the detection products of the present disclosure comprise a binding region disposed on the substrate, the binding region being loaded with a labeled antibody. The binding region is contiguous with or may partially overlap the sample region, e.g. by 1-2 mm, such as about 1.5 mm. The present disclosure also does not have any limitation on the material of the bonding region, and may be glass fibers in some embodiments. In some embodiments, the binding region is loaded with one or more antibodies described in groups (a), (b), and (c) of the present disclosure or antigen-binding fragments thereof having a chromogenic label and used as a labeled antibody. In some embodiments, the chromogenic label is a colloidal gold label such as a colloidal gold particle or a fluorescent label.

In some embodiments, the assay products of the present disclosure comprise a reaction zone disposed on the substrate. The reaction zone is immediately adjacent to the binding zone and may overlap the binding zone, for example by 1-2 mm, such as about 1.5 mm. The material of the reaction zone is also not limited by the present disclosure, and may be a nitrocellulose membrane in some embodiments. In some embodiments, the reaction zone is provided with a detection zone or line T and a quality control zone or line C. In some embodiments, the detection zone or detection line T is located upstream of the quality control zone or line C along the direction of sample flow, the detection zone or detection line T being proximal to the binding zone and the quality control zone or line C being proximal to the absorption zone.

In some embodiments, the detection zone or detection line T is loaded with one or more antibodies or antigen-binding fragments thereof described in sets (d) and (e) of the present disclosure, immobilized at the detection zone and used as a capture antibody. In some embodiments, the control region or line C is loaded with a secondary antibody, e.g., an anti-human Fc secondary antibody, directed to one or more antibodies or binding fragments thereof described in groups (a), (b), and (C) of the present disclosure. In some embodiments, the color development in the detection zone or line T is determined visually to provide a qualitative or quantitative result based on the data obtained from the reader, or compared to the intensity of the control zone control line C to provide a semi-quantitative result.

In some embodiments, the present disclosure provides an antibody pair comprising antibodies R16-F10 and P16-A3 herein. In a further embodiment, the antibody P16-A3 is a capture antibody and the antibody R16-F10 is a labeled antibody, e.g., a colloidal gold labeled antibody. In some embodiments, the coating concentration of the antibodies of the present disclosure may be about 1-10 mg/ml, such as 1.5-5 mg/ml or 2-3 mg/ml.

In some embodiments, the detection products of the present disclosure include an absorbent region disposed on the substrate. In some embodiments, the absorption zone is immediately adjacent to the reaction zone and is disposed downstream of the quality control zone in the direction of sample flow. The present disclosure does not have any limitation on the source and material of the absorbent region, as long as a material capable of absorbing aqueous liquid to allow the sample to flow, such as absorbent paper, which is well known to those skilled in the art, can be used in the present disclosure.

In some embodiments, the test products of the present disclosure can further comprise a housing, such as a plastic housing, that houses the test strip, stick, or test strip. In some embodiments employing a plastic shell, the plastic shell can include a bottom cover and a top cover mated with the bottom cover, the bottom cover having a groove with a width and length corresponding to a test strip, stick, or test paper. The top cover can be provided with holes with the same size as the sample area and the reaction area, so that an operator can add samples and observe detection results.

Without being bound by theory, the detection product or device of the present disclosure for detecting coronaviruses (particularly SARS-CoV-2) based on the double antibody sandwich method can be based on the following principles:

the capture antibody of the present disclosure is first immobilized on a detection zone/line (T-line) and the labeled antibody of the present disclosure is loaded on the binding zone. The labeled antibody on the binding region is labeled with a chromogenic label (e.g., a fluorescent label or a colloidal gold label). After dropping a sample such as a respiratory tract or throat swab sample, the sample will flow toward the other end, and when flowing to the binding region, an antigen in the sample such as coronavirus (especially SARS-CoV-2) S protein will bind to the labeled antibody on the binding region, forming a "labeled antibody-antigen" complex; when the sample continues to flow to the detection line, the antigen will be captured by the capture antibody on the T-line, forming a "labeled antibody-antigen-capture antibody" complex. With increasing capture of antigen on the T-line, an increasingly pronounced color change of the T-line occurs. A secondary antibody (e.g., an anti-human Fc antibody) is immobilized on the C-line of the quality control region to ensure that the C-line will develop color regardless of whether the sample contains the antigen to be detected. And after the color development time reaches 15-20 min, if the color of the T line changes, the sample to be detected contains the antigen to be detected. The color intensity of the precipitation line will be directly proportional to the antigen concentration in the sample.

The assay products of the present disclosure can be prepared using general procedures known in the art. In one embodiment, the assay product of the present disclosure can be prepared according to the following exemplary method, which includes the following steps:

1) dissolving the labeled antibody in a buffer, coating, e.g., spraying, the diluted labeled antibody on a binding region such as a glass fiber paper, and then drying;

2) streaking a capture antibody solution onto a reaction region to form a detection line T, streaking a secondary antibody (e.g., an anti-human Fc secondary antibody) solution onto the reaction region to form a quality control line C, and drying the streaked reaction region, e.g., at 25-37 deg.C for about 10-20 min; and

3) the sample zone, the binding zone, the reaction zone and the absorption zone are immobilized on a substrate in sequence and optionally cut into strips to obtain the immunoassay strip.

The present invention is not limited to the method of scribing the reaction region, and the antibody spraying method known to those skilled in the art can be used. In one embodiment, the present disclosure may be streaked using a streaking instrument, such as Biodot ZX-1000CA, USA.

The method and the product of the present disclosure can be used for the rapid detection of the POCT of the new coronavirus. Based on the principle of a double-antibody sandwich method of complementary epitopes, the detection of the RBD structural domain of the Spike protein aiming at the high expression specificity of the surface of the new coronavirus can effectively distinguish the new coronavirus from SARS and MERS viruses. The antibody pair provided by the disclosure also has high sensitivity, and the detection sensitivity can reach 1 ng/mL, 0.9 ng/mL, 0.8 ng/mL, 0.5 ng/mL, 0.3 ng/mL, 0.1 ng/mL or lower. Compared with the conventional IgG/IgM detection, the detection method and the product disclosed by the invention can be used for directly detecting the RBD protein of the virus in the sample, so that the interference of possible unrelated cytokine substances in the sample on the detection can be effectively avoided, and the sensitivity and the accuracy of the detection are effectively improved. Moreover, since a human body requires a period of time to produce IgG/IgM, the double antibody sandwich assay for the novel coronavirus antigen of the present disclosure can bring the detection window significantly forward, and thus can be used to screen individuals in the latent phase.

In addition, the double antibody sandwich method-based product of the present disclosure, such as a colloidal gold test strip or kit, is convenient to carry and store, simple to operate, stable in method, capable of completing a test in a short time, such as 20 minutes, and has the characteristics of quantitative and semi-quantitative determination. Therefore, compared with nucleic acid detection, the method has the characteristics of shorter detection time and lower cost, and is particularly suitable for screening a large number of samples.

V. nucleic acids of the disclosure and host cells comprising the same

In one aspect, the disclosure provides nucleic acids encoding the antibodies or antigen-binding fragments thereof herein that specifically bind to coronavirus S protein. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising said nucleic acid or said vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., a CHO cell or 293 cell), or other cell suitable for production of an antibody or antigen-binding fragment thereof. In another embodiment, the host cell is prokaryotic. For example, a nucleic acid of the present disclosure comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs 1-4 and 11-12, or a nucleic acid encoding an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from any one of SEQ ID NOs 1-4 and 11-12. In some embodiments, a nucleic acid of the disclosure comprises a sequence set forth in any one of SEQ ID NOs 13-14 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

The present disclosure also encompasses nucleic acids that hybridize under stringent conditions to, or encode a polypeptide sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions, or insertions as compared to, a nucleic acid comprising a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of those set forth in any of SEQ ID NOs 1-4 and 11-12; or a nucleic acid comprising a nucleic acid sequence encoding an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from the group consisting of those set forth in any of SEQ ID NOs 1-4 and 11-12.

In one embodiment, one or more vectors comprising a nucleic acid or polynucleotide of the present disclosure are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or Yeast Artificial Chromosomes (YACs). In some embodiments, host cells comprising the expression vectors of the present disclosure are provided. In some embodiments, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for the production of antibodies. Suitable host cells include prokaryotic microorganisms such as E.coli. The host cell may also be a eukaryotic microorganism such as a filamentous fungus or yeast, or various eukaryotic cells, such as insect cells and the like. Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for growth in suspension may be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney (HEK 293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells (CV 1), Vero cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Bufaro rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOS cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63 and Sp2/0, and the like. For a review of mammalian host cell lines suitable for protein production see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (edited by B.K.C. Lo, Humana Press, Totowa, NJ), pp.255-268 (2003). In a preferred embodiment, the host cell is a CHO cell or 293 cell. Once an expression vector or DNA sequence has been prepared for expression, the expression vector may be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, the cells are grown in culture and screened for appropriate activity. Methods and conditions for culturing the resulting transfected cells and for recovering the resulting antibody molecules are known to those skilled in the art and may be varied or optimized depending on the particular expression vector and mammalian host cell used based on the present specification and methods known in the art. Alternatively, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The marker may, for example, provide prototrophy, biocidal resistance (e.g., antibiotics), or heavy metal (e.g., copper) resistance, etc., to an auxotrophic host. The selectable marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.

Production and purification of antibodies of the disclosure

In one embodiment, the present disclosure provides a method of making an antibody or antigen-binding portion thereof of the present disclosure, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody or antigen-binding portion thereof or an expression vector comprising the nucleic acid under conditions suitable for expression of the antibody or antigen-binding portion thereof, and optionally isolating the antibody or antigen-binding portion thereof. In some embodiments, the method further comprises recovering the antibody, or antigen-binding portion thereof, from the host cell (or host cell culture medium).

To recombinantly produce an antibody or antigen-binding portion thereof of the present disclosure, a nucleic acid encoding the disclosed antibody or antigen-binding portion thereof is first obtained and inserted into a vector for further cloning and/or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that are capable of specifically binding to nucleic acids encoding the antibodies or antigen-binding portions thereof of the present disclosure.

Antibodies of the present disclosure, or antigen-binding portions thereof, prepared as described herein can be purified by known prior techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein also depend on net charge, hydrophobicity, hydrophilicity, and like factors, and will be apparent to those skilled in the art. The purity of the antibodies or antigen-binding fragments thereof of the present disclosure can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

Assays for Activity of antibodies or antigen-binding portions thereof of the disclosure

The antibodies, or antigen-binding portions thereof, provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities by a variety of assays known in the art. In one aspect, antibodies of the disclosure, or antigen-binding portions thereof, are tested for antigen-binding activity, e.g., by known methods such as ELISA, Western blot, and the like. Binding to coronavirus S protein can be determined using methods known in the art. In some embodiments, binding of an antibody or antigen-binding portion thereof of the present disclosure to coronavirus S protein is determined using SPR or biofilm layer interference. The present disclosure also provides assays for identifying antibodies, or antigen-binding portions thereof, having biological activity. Biological activity may include, for example, blocking binding to ACE2 on the surface of a cell.

Pharmaceutical combinations and pharmaceutical preparations

It will be understood by those skilled in the art that since the antibodies of the present disclosure can specifically bind to the RBD region of the S protein of coronavirus, they can also be used as blocking antibodies or neutralizing antibodies for the prevention or treatment of coronavirus infection, particularly SARS-CoV-2 infection.

In some embodiments, the present disclosure provides a composition comprising any of the antibodies or antigen-binding portions thereof described herein, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier, such as a pharmaceutical excipient. In one embodiment, a composition (e.g., a pharmaceutical composition) comprises a pharmaceutically acceptable carrier of the present disclosure in combination with one or more other therapeutic agents (e.g., anti-infective active agents, small molecule drugs). The anti-infective active agent, small molecule drug suitable for the present disclosure may be any anti-infective active agent, small molecule drug used to treat, prevent or ameliorate a coronavirus infection in a subject, including but not limited to, ridciclovir, ribavirin, oseltamivir, zanamivir, hydroxychloroquine, interferon- α 2b, analgesics, azithromycin, and corticosteroids. In the context of the present disclosure, a coronavirus infection includes an infection caused by a coronavirus (particularly SARS-CoV-2).

In some embodiments, the pharmaceutical composition or pharmaceutical formulation of the present disclosure comprises a suitable pharmaceutically acceptable carrier, e.g., a pharmaceutical excipient, such as a pharmaceutical carrier, pharmaceutical excipient, known in the art, including a buffer. As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents and the like that are physiologically compatible. Pharmaceutical carriers suitable for use in the present disclosure can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. For the use of excipients and their use, see also "Handbook of Pharmaceutical excipients", fifth edition, r.c. rowe, p.j.seskey and s.c. owen, Pharmaceutical Press, London, Chicago. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard pharmaceutical carriers and/or excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin. A Pharmaceutical formulation or Pharmaceutical composition comprising an antibody of the present disclosure or an antigen-binding fragment thereof having a desired purity can be prepared by mixing the antibody or antigen-binding fragment thereof with one or more optional Pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16 th edition, Osol, a. eds. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution. The pharmaceutical compositions or formulations of the present disclosure may also contain more than one active ingredient as required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other anti-infective active ingredients, such as other antibodies, anti-infective active agents, small molecule drugs or immunomodulators and the like. The active ingredients are suitably present in combination in an amount effective for the intended use. Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibodies, or antigen-binding fragments thereof, of the present disclosure, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

IX. combination product or kit

In some embodiments, the present disclosure also provides a combination product comprising at least one antibody or antigen-binding portion thereof of the present disclosure, or further comprising one or more additional therapeutic agents (e.g., an anti-infective active agent, a small molecule drug or an immunomodulatory agent, etc.).

In some embodiments, two or more of the ingredients in a combination product of the present disclosure may be administered to a subject in combination, sequentially, separately or simultaneously.

In some embodiments, the present disclosure also provides a kit comprising an antibody or antigen-binding portion thereof, a pharmaceutical composition, or a combination product of the present disclosure, and optionally a package insert directing administration. In some embodiments, the present disclosure also provides a pharmaceutical product comprising an antibody or antigen-binding portion thereof, a pharmaceutical composition, a combination product of the present disclosure, optionally further comprising a package insert directing administration.

Prophylactic and/or therapeutic use of an antibody or antigen-binding portion thereof of the present disclosure

The present disclosure provides a method for preventing a coronavirus-associated disease or disorder, such as a coronavirus infection (particularly COVID-19), in a subject, comprising administering to the subject an antibody, or antigen-binding portion thereof, of the present disclosure. Subjects at risk for a coronavirus-related disease include subjects in contact with an infected person or subjects exposed to a coronavirus in some other manner. The prophylactic agent can be administered prior to exhibiting the symptomatic characteristics of the coronavirus-associated disease, so as to arrest the disease, or alternatively delay the progression of the disease.

The disclosure also provides methods of treating a coronavirus associated disease, such as a coronavirus infection (preferably COVID-19), in a patient. In one embodiment, the method involves administering an antibody or antigen-binding portion thereof or combination of antibodies of the present disclosure that neutralizes coronaviruses to a patient suffering from the disease.

In some embodiments, there is provided a method of treating a coronavirus infection in a patient, the method comprising administering an antibody, or antigen-binding portion thereof, of the disclosure. In a preferred embodiment, two of the antibodies of the present disclosure, or antigen-binding portions thereof, are administered together to the patient. In some embodiments, the antibodies of the present disclosure, or antigen-binding portions thereof, can cross-neutralize both human and animal infectious coronavirus isolates. In some embodiments, the antibody or antigen-binding portion thereof of the present disclosure is administered within the first 24 hours after coronavirus infection.

The pharmaceutical compositions of the present disclosure may be administered in vivo to a subject in need thereof by various routes including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intratracheal, buccal, intraperitoneal, intradermal, topical, transdermal and intrathecal or by inhalation. The pharmaceutical compositions of the present disclosure may be formulated in solid, semi-solid, liquid or gaseous form; including but not limited to tablets, capsules, powders, granules, ointments, solutions, injections, inhalants and aerosols. The appropriate formulation and route of administration may be selected according to the intended application and treatment regimen. The frequency of administration can be determined and adjusted during the course of treatment. In some embodiments, the dose administered may be adjusted or reduced to control potential side effects and/or toxicity. Alternatively, sustained continuous release formulations of the pharmaceutical compositions of the present disclosure for use in therapy may be suitable. One skilled in the art will appreciate that the appropriate dosage may vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit with any risk or deleterious side effects. The selected dosage level will depend upon a variety of factors including, but not limited to, the activity of the particular antibody or antigen-binding portion thereof, the route of administration, the time of administration, the rate of clearance, the duration of treatment, other drugs used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health and prior medical history of the patient, and the like. The amount of antibody, or antigen-binding portion thereof, and the route of administration is ultimately at the discretion of the physician, veterinarian, or clinician, but the dosage is generally selected to achieve a local concentration at the site of action that achieves the desired effect, without causing substantial deleterious or adverse side effects. In general, the antibodies of the present disclosure, or antigen-binding portions thereof, can be administered in various dosage ranges. In some embodiments, an antibody, or antigen-binding portion thereof, provided herein can be administered at a dose of about 0.01 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg). In certain of these embodiments, the antibody, or antigen-binding portion thereof, is administered at a dose of about 50 mg/kg or less, and in certain of these embodiments, the dose is 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less. In certain embodiments, the dosage administered may vary over the course of treatment. For example, in certain embodiments, the initial administered dose may be higher than the subsequently administered dose. In certain embodiments, the dosage administered may vary over the course of treatment depending on the subject's response. The frequency of administration can be determined by one skilled in the art, for example, by the attending physician based on considerations of the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, and the like. In certain preferred embodiments, the course of treatment involving an antibody or antigen-binding portion thereof of the present disclosure will comprise multiple doses of the selected drug administered over a period of weeks or months. More specifically, the antibodies, or antigen-binding portions thereof, of the present disclosure can be administered daily, every second day, every fourth day, weekly, every tenth day, every second week, every third week, or longer intervals. In this regard, it is understood that the dosage may be varied or the interval adjusted based on patient response and clinical practice.

Examples

The present disclosure generally described herein will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to limit the scope of the disclosure. These examples are not intended to indicate that the experiments below are all or only experiments performed.

Wherein, the process of producing the fully human antibody targeting SARS-CoV-2 coronavirus S protein is shown in FIG. 1.

Example 1 preparation and detection of coronavirus S protein antigen preparation of ACE2

The following antigens and ACE2 proteins were used in the examples, including RBD-His S protein (319 Arg-532 Asn), human ACE2-huFc (18 Gln-740 Ser), human ACE2-His (18 Gln-740 Ser), and RBD-mFc S protein (purchased from Sinobio, 40592-V05H), the first three of which were specifically prepared as follows.

1.1 plasmid construction

Each protein sequence was obtained from NCBI, where the human ACE2 sequence was obtained from NCBI Gene ID: 59272 and the S protein sequence was obtained from NCBI Gene ID: 43740568. Each protein sequence was obtained according to the amino acid fragment position, and after conversion to a gene sequence, the target fragment gene synthesis was carried out by the Kinsley Biotechnology Ltd. PCR amplifies each target segment, and then constructs to eukaryotic expression vector pcDNA3.3-TOPO (Invitrogen) by a homologous recombination method for the expression of subsequent recombinant protein.

1.2 plasmid preparation

And respectively transforming the constructed recombinant protein expression vectors into escherichia coli SS320, culturing overnight at 37 ℃, and performing plasmid extraction by using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids for eukaryotic expression.

1.3 expression purification of Each protein

The S protein RBD-His (319 Arg-532 Asn), human ACE2-huFc (18 Gln-740 Ser) and human ACE2-His (18 Gln-740 Ser) were all expressed by Expi293 transient expression system (ThermoFisher, A14635) as follows:

on the day of transfection, cell density of about 4.5X 106 to 5.5X 106 viable cells per ml was confirmed, and cell survival rate was confirmed>95% at which time the cells were adjusted to a final concentration of 3X 106 cells per ml with fresh Expi293 expression medium pre-warmed at 37 ℃. Diluting the target plasmid with 4 deg.C pre-cooled Opti-MEMTM (1 μ g plasmid was added to 1 mL Opti-MEMTM), diluting Expifeactamine 293 reagent with Opti-MEMTM, mixing the two solutions in equal volume, gently blowing and mixing to obtain mixture of Expifeacylamine 293 reagent/plasmid DNA, incubating at room temperature for 10-20 minSlowly adding into the prepared cell suspension while gently shaking, and finally placing into a cell culture shaker at 37 deg.C and 8% CO₂Culturing under the condition.

Expifeactine TM293 Transfection Enhancer 1 and Expifeactine TM293 Transfection Enhancer 2 were added 18-22h after Transfection, and the flasks were placed in a 32 ℃ shaker and 5% CO₂The culture was continued under the conditions that, after 5 to 7 days of transfection, the cell expression supernatant was subjected to high-speed centrifugation at 15000 g for 10 min, and the resulting Fc tag protein expression supernatant was subjected to affinity purification using MabSelect SuRe LX (GE, 17547403), followed by elution of the objective protein with 100 mM sodium acetate (pH 3.0) and subsequent neutralization with 1M Tris-HCl; the resulting His-tagged protein-expressing supernatant was subjected to affinity purification using Ni Smart Beads 6FF (Changzhou Tiandi and Biotech Co., Ltd., SA 036050), followed by elution of the target protein using a gradient concentration of imidazole. The eluted proteins were individually replaced into PBS buffer by ultrafiltration concentration tubes (Millipore, UFC 901096). After being qualified by SDS-PAGE identification and activity identification, the frozen stock is preserved at minus 80 ℃ for standby.

1.4 quality testing of the proteins

Spike RBD-His prepared in 1.3 above and a commercial S protein RBD-mFc (also referred to as Spike-RBD-mFc; or RBD-mFc) were assayed for binding to each other by ELISA assay with human ACE2-huFc and human ACE 2-His. The results show that in FIGS. 2A and 2B, human ACE2-huFc and RBD-mFc have good binding activity; the S protein S1-huFc and the commercial S protein RBD-mFc have good binding activity with human ACE2-his respectively, and the activity is equivalent.

Example 2 construction and screening of Natural human antibody phage display libraries

In this example, an antibody gene phage display library was constructed and screened against recombinant SARS-CoV-2 coronavirus RBD protein (i.e., Spike-RBD-mFc, Sinobio, 40592-V05H) as a screening antigen to obtain a plurality of antibody molecules having a specificity of binding to SARS-CoV-2 coronavirus RBD protein.

2.1 construction of a Gene library of human antibodies

15 mL of Ficoll-Paque density gradient fraction (from GE, Cat: 17144003S) was slowly added to a 50 mL centrifuge tube. The centrifugal tube is inclined and 15 mL of collected normal human blood is slowly added along the tube wall in batches, so that the Ficoll-Paque density gradient separation solution and the normal human blood keep a clear separation interface. And (3) centrifuging the 50 mL centrifuge tube filled with the blood and the separating medium at about 15 ℃ for 20min, wherein the centrifuge is set to be 400 g, the acceleration is 3, and the deceleration is 0. After centrifugation, the whole liquid surface is divided into four layers, the upper layer is a plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll-Paque liquid. A narrow band of white cloud layer mainly containing PBMC, i.e. PBMC cell layer, is arranged at the junction of the upper layer and the middle layer. The supernatant plasma mixture was carefully aspirated with a sterile pasteur pipette and the PBMCs were then aspirated with a fresh sterile pasteur pipette to obtain isolated PBMCs. The separated PBMCs were rinsed twice with PBS, centrifuged at 1500 rpm for 10 min at 4 ℃ and finally resuspended in 1.5 mL PBS and counted by a cell counter (CountStar, CountStar Altair).

Total RNA was extracted from isolated PBMC cells by conventional methods. The extracted total RNA was reverse-transcribed into cDNA using a reverse transcription kit (purchased from TaKaRa, Cat: 6210A). Based on the sequence similarity of heavy chain and light chain germline genes, degenerate primers (li xialin, construction and preliminary screening of large-capacity non-immune human Fab phage antibody library, article by university of china and medical science, 6 months 2007) were designed at the front end of the V region and the rear end of the first constant region of the heavy chain and light chain, respectively, and heavy chain variable region gene fragments and light chain variable region gene fragments of the antibodies were obtained after PCR. After recovering the heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody, a fragment containing the light chain variable region of the antibody is obtained by amplification by the fusion PCR method, then the PCR product and a phage display vector are subjected to enzyme digestion, recovery and ligation, and the ligation product is recovered by a recovery kit (Omega, catalog No.: D6492-02), and specific materials and methods are described in the above article of Lixialin. Finally, the strain was transformed into competent E.coli SS320 (Lucigen, MC 1061F) by an electrotransfer apparatus (Bio-Rad, MicroPulser), and the transformed E.coli SS320 strain solution was spread on ampicillin-resistant 2-YT solid plates (solid plates prepared from 1.5% tryptone, 1% yeast extract, 0.5% NaCl, 1.5% agar in terms of mass volume g/mL).

2.2 calculation of antibody Gene library volume

Inoculating transformed Escherichia coli SS320 bacterial liquid with antibiotic-free 2YT culture solution at a volume of 1:50, culturing at 37 deg.C and 220 rpm for 1.5-2 hr until OD600 reaches 0.5-0.6, and taking out to room temperature. The bacterial suspension was added to a 96-well round bottom dilution plate at 90. mu.L/well. Each bacterial fluid sample was diluted by 10-fold gradient, and 12 dilution gradients were obtained. The diluted sample was applied to a 2YT (hereinafter also referred to as C +/T + 2 YT) plate having a concentration of carbenicillin and tetracycline of 50. mu.g/mL and 50. mu.g/mL, respectively, in the order of dilution gradient from low to high by using an 8-channel 10. mu.L range pipette, and was allowed to stand upright for 5 minutes and then was allowed to stand upside down and incubated at 37 ℃ overnight. The next day, the growth of the clones was observed, and the stock capacity was calculated. The library capacity is calculated as follows, starting from line a, labeled in sequence as 1, 2, 3, 4, 5, 6, 7, 8 through line X. Firstly, selecting counting holes, firstly selecting the number of clones in the counting holes of 3-20 clones to obtain the number X of lines, counting the number n of clones in the corresponding holes, wherein the calculation formula is 5 multiplied by 100 multiplied by 10 multiplied by n, and obtaining the bacterial liquid storage capacity of 3 multiplied by 10 per milliliter through calculation¹¹cfu, i.e. 3X 10¹¹Antibody gene library of individual antibody genes.

2.3 preparation of antibody Gene phage display library

Based on the antibody gene pool capacity, 50 ODs (1 OD 5X 10) were aspirated⁸cfu) was added to a fresh 2-YT broth so that the initial OD value was 0.1. The resultant was cultured at 37 ℃ in a shaker at 220 rpm until logarithmic growth phase (OD 600= about 0.6), VSCM13 helper phage (purchased from Stratagene) was added in an amount 50 times as many as the number of bacteria (i.e., multiplicity of infection (MOI) was about 50), well mixed, and cultured for an additional 1 h in a shaker at 220 rpm after standing for 30 min. Subsequently, after the culture was centrifuged at 10000 rpm for 5 min, the supernatant was discarded, and the culture solution was replaced with carbenicillin 50. mu.g/mL/kanamycin 40. mu.g/mL double-resistant 2-YT medium (hereinafter also referred to as "kanamycin")C +/K + 2-YT medium) and incubation is continued overnight at 220 rpm at 30 ℃. The next day, the bacterial solution was centrifuged at 13000g for 10 min, the supernatant was collected and added with 20% PEG/NaCl (prepared from 20% PEG6000 and 2.5M NaCl) to give a final concentration of PEG/NaCl of 4%, mixed, placed on ice for 1 h, centrifuged at 13000g for 10 min, and the precipitated phage was rinsed with PBS and stored for subsequent phage selection.

2.4 screening of antibody Gene phage display libraries

2.4.1 screening of antibody Gene phage display library by paramagnetic particle method

The magnetic bead method screening is based on labeling antigen protein (Spike-RBD-mFc, Sinobio, 40592-V05H) with biotin, binding to magnetic beads coupled with streptavidin, and subjecting the magnetic beads bound with antigen and an antibody gene phage display library to a panning process of incubation, washing and elution, usually 3-4 rounds of panning, whereby specific monoclonal antibodies against the antigen can be enriched in large quantities. In this example, the biotinylated SARS-CoV-2 coronavirus RBD protein was used for phage display library screening, and 3 rounds of panning were followed by primary screening with monoclonal antibodies against the RBD protein.

The specific implementation method of antibody screening is as follows:

first, streptavidin-coupled magnetic beads were incubated with biotin-labeled SARS-CoV-2 coronavirus RBD protein (Spike-RBD-mFc, Sinobio, 40592-V05H) to allow binding of the biotin-labeled RBD protein to the magnetic beads. The RBD protein-binding magnetic beads and the constructed phage library were incubated at room temperature for 2 h. After washing 6-8 times with PBST, the non-specifically adsorbed phage was removed, and Trypsin (Gibco, 25200072) was added to mix gently and react for 20min to elute the specifically bound antibody-displaying phage. Subsequently, SS320 thalli (Lucigen, MC 1061F) in the logarithmic phase are infected by the eluted phage and are kept still for 30 min, then are cultured for 1 h under the condition of 220 rpm, then VSCM13 auxiliary phage are added and are kept still for 30 min, the culture is continued for 1 h under the condition of 220 rpm, the phage are centrifuged and replaced into a C +/K + 2-YT culture medium, and finally the obtained phage are continuously used for the next round of panning.

2.4.2 screening of antibody Gene phage display libraries by the Immunity tube method

The purpose of the immune tube method and the magnetic bead method is to enrich specific antibodies aiming at antigens, and the method is two mutually complementary and verified experimental methods.

The principle of the screening by the immune tube method is that SARS-CoV-2 coronavirus RBD protein (Spike-RBD-mFc, Sinobio, 40592-V05H) is coated on the surface of the immune tube with high adsorption force, and through the panning process of adding phage display antibody library into the immune tube and incubating, washing and eluting with antigen protein adsorbed on the surface of the immune tube, 2-4 rounds of panning are carried out, and finally specific monoclonal antibody aiming at the antigen is enriched.

The specific implementation method comprises the following steps:

for the first round of selection, 1 mL of 100. mu.g/mL RBD-mFc was added to the tubes and coated overnight at 4 ℃. The coating solution is discarded the next day, 5% milk PBS is added for blocking for 2h, PBS is added for rinsing twice, then the constructed phage library with the total amount of 1014 whole human antibody genes is added, and incubation is carried out for 2 h. After rinsing 8 times with PBS and 2 times with PBST to remove non-specifically bound phage, 0.8 mL of pancreatin digest containing 0.05% EDTA was added to the immune tube for elution of phage specifically binding to the antigen of interest. Then the strain is infected with SS320 thallus (Lucigen, 60512-1) in the logarithmic phase, is kept stand for 30 min at 37 ℃, is cultured for 1 h under the condition of 220 rpm, is added with VSCM13 helper phage and is kept stand for 30 min. The cultivation was continued for 1 h at 220 rpm, centrifuged and replaced into C +/K + 2-YT medium and continued overnight at 30 ℃ in a 220 rpm environment. The next day phages were precipitated for subsequent 2-4 rounds of screening. The antigen coating concentrations commonly used for the second, third and fourth rounds of phage selection were decreasing sequentially, 30. mu.g/mL, 10. mu.g/mL and 3. mu.g/mL, respectively; in addition, the PBS rinsing intensity was gradually increased, and the PBS elution times were 12 times, 16 times and 20 times in this order.

The enrichment effect was evaluated by performing ELISA on phage pools eluted from each round, and 10 clones were randomly selected from phage pools screened in each round for sequence analysis, and the results are shown in FIGS. 3A and 3B, which show better enrichment in each round, with 3rd-1 being the most preferred enrichment.

The results showed that the antibody sequence was significantly enriched after the third round of screening, and therefore, the clones obtained in the third round were selected for positive clone screening by ELISA.

2.5 selection of the monoclonal

After a total of four rounds of screening, the resulting clones from the third round were selected for a positive clone ELISA screen by ELISA. Finally, a total of 88 positive clones capable of binding to the RBD protein were selected from 2304 clones, and after sequencing analysis, ELISA binding and FACS blocking detection at Fab level, the sequences of 10 clones were finally selected to construct full-length antibodies for further experiments. The specific implementation method is as follows.

2.5.1 sequencing and analysis of Positive clones

After the primary screening work is completed, 88 positive clones capable of combining with RBD protein are numbered, 2 mu L of bacterial liquid is sucked into 2 mL of 2YT culture medium, cultured overnight at 37 ℃ and 220 rpm, and plasmids are extracted for second-generation sequencing. And (3) integrating the original AB1 file, comparing and removing a non-antibody gene sequence through Seqman to generate a fasta file of an antibody gene integration version. The DNA sequence is then translated into an amino acid sequence by MEGA6, and a fasta file of the amino acid sequence is derived by finding out the amino acid sequence containing a terminator, an unconventional sequence, and the like.

2.5.2 ELISA assay for detecting the affinity Effect of Fab-form antibodies

First, the third round of selected clones was picked up and cultured overnight at 37 ℃ in a 96-well deep-well plate containing 300. mu.L of 2-YT medium, the supernatant containing the expressed Fab was taken, and the supernatant was diluted in a gradient and added to an ELISA plate coated with 2. mu.g/mL RBD-mFc, and then detected with HRP-labeled Goat anti-Human Fab as a secondary antibody (Goat anti-Human Fab-HRP, ThermoFisher, 31482, 1:6000 dilution), with higher signal values indicating stronger affinity. The results show that the Fab of 9 antibodies showed better affinity activity in ELISA assay.

EXAMPLE 3 construction, expression and purification of full-Length antibody

In this example, the 9 Fab antibodies with better activity for binding S-RBD protein obtained in example 2 were constructed into human IgG1 type, wherein the light chains are all kappa type and the antibody type is fully human, and the procedures are described by taking antibodies R14-F8 and R16-F10 as examples.

3.1 plasmid construction

From the screened strain containing the Fab antibody, the light chain variable region fragment and the heavy chain variable region fragment of the antibody are obtained by PCR amplification and are respectively constructed on an engineered eukaryotic expression vector plasmid pcDNA3.3-TOPO (Invitrogen) containing the light chain constant region fragment and the heavy chain constant region fragment by a homologous recombination method.

3.2 plasmid preparation

And respectively transforming the constructed vectors containing the full-length genes of the light and heavy chains of the antibody into escherichia coli SS320, culturing overnight at 37 ℃, and performing plasmid extraction by using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free light and heavy chain plasmids of the antibody for eukaryotic expression.

3.3 expression purification of antibodies

The candidate antibodies R14-F8 and R16-F10 were expressed by the ExpicHO transient expression system (Thermo Fisher, A29133) by the following specific method:

on the day of transfection, the cell density was confirmed to be 7X 10⁶To 1X 10⁷About one living cell/mL, cell survival rate>98 percent. At this time, the cells were adjusted to a final concentration of 6X 10 using fresh Expi CHO expression medium pre-warmed at 37 deg.C⁶Individual cells/mL. The plasmid of interest was diluted with 4 ℃ pre-cooled OptiPROTM SFM (1. mu.g of plasmid was added to 1 mL of the medium). Simultaneously, diluting Expifeactine (TM) CHO with OptiPROTM SFM, mixing the two in equal volume, gently blowing, beating and uniformly mixing to prepare Expifeactine (TM) CHO/plasmid DNA mixed solution, incubating at room temperature for 1-5 min, slowly adding into the prepared cell suspension, gently shaking, finally placing in a cell culture shaking table, and performing cell culture at 37 ℃ and 8% CO concentration₂Culturing under the condition.

At 18-22h after transfection, ExpichOTMenstationary and ExpichOTMFeed were added to the medium and the flasks were placed on a shaker at 32 ℃ and 5% CO₂The culture was continued under the conditions. On day 5 post-transfection, the same volume of ExpichOTMFeed was added, slowly while addingThe cell suspension was gently mixed. 7-15 days after transfection, the cell culture supernatant expressing the protein of interest was subjected to high-speed centrifugation at 15000 g for 10 min, and the resulting supernatant was subjected to affinity purification using MabSelect SuRe LX (GE, 17547403), followed by elution of the protein of interest with 100 mM sodium acetate (pH 3.0), followed by neutralization with 1M Tris-HCl, and finally replacement of the resulting protein into PBS buffer by an ultrafiltration concentration tube (Millipore, UFC 901096).

3.4 determination of antibody concentration

The concentration of the purified antibody protein is measured by an ultra-micro spectrophotometer (Nano-300, Osheng instruments Co., Ltd., Hangzhou), the value of A280 is divided by the theoretical extinction coefficient of the antibody to be used as the concentration value of the antibody for subsequent research, and the antibody is subpackaged and stored at-80 ℃ after quality inspection is qualified.

Example 4 affinity Activity of candidate antibodies at protein level

In this example, 3 candidate antibodies were selected and tested to verify the affinity activity of the full-length antibody for the SARS-CoV-2 coronavirus S protein.

4.1 detection of affinity Activity of candidate antibodies based on ELISA

Recombinant SARS-CoV-2 coronavirus Spike-RBD-His, 2. mu.g/mL, 30. mu.L/well, was coated on a 96-well ELISA plate overnight at 4 ℃. The following day, the well plates were washed 3 times with PBST and then blocked with 5% skim milk for 2h, after washing the plates 3 times with PBST, 3 candidate antibodies were added at a gradient dilution (gradient concentration see fig. 4) and incubated for 1 h. After 3 washes with PBST, HRP-labeled anti-human Fc secondary antibody (Abcam, ab 98624) was added at 1:5000 and incubated for 1 h. After incubation was complete, the plates were washed six times with PBST and developed with TMB (SurModics, TMBS-1000-01). According to the results of the color development, the reaction was terminated by adding 2M HCl, and the plate was read by a microplate reader (Molecular Devices, SpectreMax 190) at OD450, and the results are shown in FIG. 4, which shows that 3 candidate antibodies (designated as R14-F8 antibody, R16-F10 antibody, and P16-A3 antibody, respectively) had excellent affinity activity for the S protein. Indicating that the full-length constructed antibody has the same binding affinity for the RBD domain.

Example 5 grouping binding epitopes of candidate antibodies at the protein level

In this example, 9 candidate antibodies were selected to group the epitopes that bind to SARS-CoV-2 coronavirus S protein.

5.1 double antibody Sandwich method for grouping epitopes of candidate antibodies

In this example, the double antibody sandwich method includes a process of the previous method development, and parameters having both sensitivity and stability are determined for practical use based on the development result.

9 candidate antibodies were coated, 2. mu.g/mL, 30. mu.L/well, overnight at 4 ℃. The following day, the well plates were washed 3 times with PBST and then blocked with 5% skim milk for 2 h. Then, Spike RBD-His, 2. mu.g/mL, 30. mu.L/well was added and incubated for 1 h. After 3 washes with PBST, 9 biotinylated candidate antibodies were added in a gradient dilution (checkerboard) at 30. mu.L/well and incubated for 1 h. Thereafter, after 3 washes with PBST, a secondary antibody NeutrAvidin-HRP (Thermofisiher, 31001) was added and incubated for 1 h. After incubation was complete, the plates were washed six times with PBST and developed with TMB (SurModics, TMBS-1000-01). According to the color results, 2M HCl was added to stop the reaction, and the plate was read by a microplate reader (Molecular Devices, SpectreMax 190) at OD 450. The results of epitope grouping between antibodies are shown in the table below. The results show that 9 candidate antibodies can be divided into two groups of epitopes on the RBD domain that binds to the S protein of SARS-CoV-2 coronavirus, epitope 1 and epitope 2 in Table C.

Watch C

5.2 Effect of detecting Spike RBD protein under double antibody Sandwich method

In this example, since the same concentration of Spike RBD protein was used, the evaluation of its detection effect was mainly from OD450 and EC 50. The results of FIGS. 5A and 5B show that P16-A3 and R16-F10 are more excellent in RBD detection of SARS-CoV-2 coronavirus S protein by the paired antibodies in the double antibody sandwich method. In FIG. 5A, different candidate antibodies were coated on an ELISA plate, and the solution phase was biotinylated using R16-F10, which shows that the EC50 was 0.05. mu.g/mL when P16-A3 was coated on the ELISA plate and R16-F10 was biotinylated. In FIG. 5B, different candidate antibodies were coated on the ELISA plate, and the liquid phase was labeled with P16-A3, so that it can be seen that when R16-F10 was coated on the ELISA plate, P16-A3 was labeled with biotin, there was almost no upper platform, and the detection effect was poor. Thus, for these 2 candidate antibodies, the binding capacity to the S protein of SARS-CoV-2 coronavirus was higher when R16-F10 was in the liquid phase and P16-A3 was in the solid phase.

EXAMPLE 6 detection of SARS-CoV-2 coronavirus Spike RBD protein by colloidal gold assay card

In this example, antibody R16-F10 that binds epitope 1 and antibody P16-A3 that binds epitope 2 were used to prepare colloidal gold test cards that were tested for their sensitivity to detect SARS-CoV-2 coronavirus S protein.

6.1 preparation of colloidal gold test cards

In this example, the colloidal gold test card was prepared by the method described in the example of chinese patent application CN102747040A, using the above-mentioned conjugated antibody, and passed various quality tests. In this test card, the antibody P16-A3 was used as a capture antibody, and the antibody R16-F10 was used as a labeled antibody.

6.2 sensitivity determination

In this example, the SARS-CoV-2 coronavirus Spike RBD protein was diluted from 2. mu.g/mL with a 2-fold gradient to 650 pg/mL. And adding the gradient diluted Spike RBD protein into the sample adding hole of the colloidal gold detection card (left lane of the card) by the volume of 120 mu L per hole respectively, and developing for 20min, wherein C line is a quality control line, T line is a detection line, 3 parallel groups are arranged, and 9 blank controls (only adding diluent) are arranged. As shown in FIG. 6, the detection sensitivity of the detection card for SARS-CoV-2 coronavirus Spike RBD protein can reach 1 ng/mL. Note that the right lane of the card is other schemes, and the disclosure is not set forth.

Example 7 detection of Cross-binding Properties of antibody R16-F10 with SARS and MERS recombinant proteins based on ELISA

In this example, the cross-binding of antibody R16-F10 to SARS-CoV-2 coronavirus S protein, SARS recombinant protein, MERS recombinant protein was evaluated.

ELISA-based detection of cross-binding Activity of candidate antibodies

Recombinant SARS-CoV-2 coronavirus Spike-RBD-His, SARS-His and MERS-His were coated separately on 96-well ELISA plates at 2. mu.g/mL, 30. mu.L/well and overnight at 4 ℃. The following day, the well plates were washed 3 times with PBST and then blocked with 5% skim milk for 2h, after washing the plates 3 times with PBST, a gradient dilution (gradient concentration see FIG. 7) of R16-F10 and CR3022 antibody was added, respectively, and incubated for 1 h. After 3 washes with PBST, HRP-labeled anti-human Fc secondary antibody (Abcam, ab 98624) was added at 1:5000 and incubated for 1 h. After incubation was complete, the plates were washed six times with PBST and developed with TMB (SurModics, TMBS-1000-01). According to the color results, the reaction was stopped by adding 2M HCl, and the plate was read by a microplate reader (Molecular Devices, SpectreMax 190) at OD450, and the results are shown in FIG. 7. The results show that R16-F10 has excellent affinity activity to Spike RBD protein, and R16-F10 has weak cross-binding with SARS recombinant protein and no cross-binding with MERS recombinant protein.

Summary of sequence listing

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Sequence listing

<110> Sanyou biomedical (Shanghai) Co., Ltd

SHANGHAI ZJ BIO-TECH Co.,Ltd.

<120> antibody targeting SARS-CoV-2 coronavirus and its diagnosis and detection use

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<211> 11

<212> PRT

<213> Artificial sequence

<400> 8

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 9

<211> 7

<212> PRT

<213> Artificial sequence

<400> 9

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 10

<211> 9

<212> PRT

<213> Artificial sequence

<400> 10

Gln Gln Ser Tyr Ser Thr Ser Leu Thr

1 5

<210> 11

<211> 127

<212> PRT

<213> Artificial sequence

<400> 11

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Tyr Ser Ser Ser Trp Leu Leu Gln Ser Phe Tyr Tyr Tyr

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 12

<211> 107

<212> PRT

<213> Artificial sequence

<400> 12

Asn Ile Gln Leu Thr Gln Ser Pro Val Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Leu Thr Cys Arg Ala Ser Gln Gly Ile Gly Tyr Ser

20 25 30

Leu Val Trp Tyr Gln Lys Lys Pro Gly Thr Ala Pro Lys Leu Leu Ile

35 40 45

Phe Asp Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Val Ile His Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 13

<211> 1353

<212> DNA

<213> Artificial sequence

<400> 13

caggtccagc tggtgcagtc tgggcctgag gtgaaggagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cctctggagg caccttcgac accgctgcta tcagctgggt gcgacaggcc 120

cctggacaag ggcctgagtg gatgggaggg atcatcccta tctttggtac agcaaactac 180

gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aagagaacat 300

acggtggctc ctgtgtacgg tttcgacgtg tggggccaag ggaccctggt caccgtctca 360

tcagcttcca ccaagggccc ctccgtgttc cccctggctc cctcttccaa gagcaccagc 420

ggcggcaccg ctgctctggg atgtctggtg aaggactact tccctgagcc tgtgaccgtg 480

tcctggaatt ccggcgccct gacctccggc gtgcacacat tccctgctgt gctgcagtcc 540

tccggcctgt atagcctgtc ctccgtggtg acagtgccta gctccagcct gggcacccag 600

acctatatct gcaacgtgaa ccacaagcct agcaatacca aggtggacaa gaaggtggag 660

cctaagagct gcgacaagac ccacacctgt cctccatgtc ctgctccaga actgctcggc 720

ggaccttccg tgttcctgtt tcctccaaag cctaaggaca ccctgatgat cagcagaacc 780

cctgaagtga cctgcgtggt ggtggatgtg tcccacgagg atcccgaagt gaagttcaat 840

tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 900

aacagcacct acagagtggt gtccgtgctg accgtgctgc accaggattg gctgaacggc 960

aaagagtaca agtgcaaggt gtccaacaag gccctgcctg ctcctatcga gaaaaccatc 1020

agcaaggcca agggccagcc tagggaaccc caggtttaca cactgcctcc aagcagggac 1080

gagctgacca agaatcaggt gtccctgacc tgcctggtca agggcttcta cccttccgat 1140

atcgccgtgg aatgggagag caatggccag cctgagaaca actacaagac aacccctcct 1200

gtgctggaca gcgacggctc attcttcctg tacagcaagc tgacagtgga caagagcaga 1260

tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320

acccagaagt ccctgagcct gtctcctggc aaa 1353

<210> 14

<211> 642

<212> DNA

<213> Artificial sequence

<400> 14

gacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cctcgctcac tttcggcgga 300

gggaccaagg tggagatcaa aaggaccgtg gctgccccca gcgtgttcat cttccctcct 360

agcgacgagc agctgaagag cggcaccgct agcgtggtgt gtctgctgaa taacttctat 420

cccagggagg ccaaggtgca gtggaaggtg gataacgccc tgcagagcgg caactcccag 480

gagtccgtga ccgagcagga ctccaaggac agcacctact ccctgagctc caccctgacc 540

ctgtccaagg ctgattatga gaagcacaag gtgtatgctt gcgaggtgac acaccagggc 600

ctgtccagcc ctgtgaccaa gagcttcaac cggggcgagt gc 642

Claims

1. An antibody pair for use in the detection of SARS-CoV-2 virus, said antibody pair comprising a part I and a part II, said part I comprising one or more antibodies or antigen-binding fragments as described in (a), (b) and (c); part II comprises one or more antibodies or antigen-binding fragments as described in (d) and (e);

wherein (a) an antibody comprising the heavy chain CDR1 of the amino acid sequence set forth in SEQ ID NO. 5; heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO. 6; the heavy chain CDR3 of the amino acid sequence set forth in SEQ ID NO. 7; a light chain CDR1 of the amino acid sequence set forth in SEQ ID NO. 8; a light chain CDR2 of the amino acid sequence set forth in SEQ ID NO. 9; and a light chain CDR3 of the amino acid sequence set forth in SEQ ID NO. 10;

(b) an antibody comprising heavy chain CDR1 in the heavy chain variable region amino acid sequence set forth in SEQ ID NO. 3; heavy chain CDR2 of the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 3; heavy chain CDR3 of the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 3; light chain CDR1 of the light chain variable region amino acid sequence set forth in SEQ ID NO. 4; light chain CDR2 of the light chain variable region amino acid sequence set forth in SEQ ID NO. 4; and light chain CDR3 from the light chain variable region amino acid sequence set forth in SEQ ID NO. 4;

(c) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO 3; and the variable region of the light chain of the amino acid sequence shown as SEQ ID NO. 4;

(d) an antibody comprising heavy chain CDR1 from the heavy chain variable region amino acid sequence set forth in SEQ ID NO. 11; heavy chain CDR2 of the heavy chain variable region amino acid sequence set forth in SEQ ID NO. 11; heavy chain CDR3 of the heavy chain variable region amino acid sequence set forth in SEQ ID NO. 11; light chain CDR1 of the light chain variable region amino acid sequence set forth in SEQ ID NO. 12; light chain CDR2 of the light chain variable region amino acid sequence set forth in SEQ ID NO. 12; and light chain CDR3 of the light chain variable region amino acid sequence set forth in SEQ ID NO. 12;

(e) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11; and the variable region of the light chain of the amino acid sequence shown as SEQ ID NO. 12.

2. The antibody pair of claim 1, wherein the antibody of (a), (b) or (c) further comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO. 1 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO. 2.

3. The antibody pair of claim 2, wherein the antibody of (a), (b) or (c) further comprises a heavy chain constant region comprising the nucleotide sequence set forth in SEQ ID NO. 13 and a light chain constant region comprising the nucleotide sequence set forth in SEQ ID NO. 14.

4. An isolated antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 coronavirus S protein, wherein the antibody is at least one antibody selected from the group consisting of:

(a) an antibody comprising the heavy chain CDR1 of the amino acid sequence set forth in SEQ ID NO. 5; heavy chain CDR2 of the amino acid sequence shown in SEQ ID NO. 6; the heavy chain CDR3 of the amino acid sequence set forth in SEQ ID NO. 7; a light chain CDR1 of the amino acid sequence set forth in SEQ ID NO. 8; a light chain CDR2 of the amino acid sequence set forth in SEQ ID NO. 9; and a light chain CDR3 of the amino acid sequence set forth in SEQ ID NO. 10;

(b) an antibody comprising heavy chain CDR1 in the heavy chain variable region amino acid sequence set forth in SEQ ID NO. 3; heavy chain CDR2 of the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 3; heavy chain CDR3 of the amino acid sequence of the heavy chain variable region shown in SEQ ID NO. 3; light chain CDR1 of the light chain variable region amino acid sequence set forth in SEQ ID NO. 4; light chain CDR2 of the light chain variable region amino acid sequence set forth in SEQ ID NO. 4; and light chain CDR3 from the light chain variable region amino acid sequence set forth in SEQ ID NO. 4; or

(c) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO 3; and the variable region of the light chain of the amino acid sequence shown as SEQ ID NO. 4.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the antibody of (a), (b), or (c) further comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO. 1 and a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO. 2.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the antibody of (a), (b), or (c) further comprises a heavy chain constant region comprising the nucleotide sequence set forth in SEQ ID NO. 13 and a light chain constant region comprising the nucleotide sequence set forth in SEQ ID NO. 14.

7. The antibody or antigen-binding fragment thereof of any one of claims 4-6, wherein the antibody exhibits at least one of the following biological activities:

(a) specifically binds to the S protein RBD domain of SARS-CoV-2 coronavirus, but does not cross-bind to SARS-CoV and MERS coronavirus;

(b) EC50 that specifically binds to the S protein RBD domain of SARS-CoV-2 coronavirus is less than about 0.05 μ g/mL; or

(c) The sensitivity for detecting SARS-CoV-2 coronavirus S protein can reach 1 ng/mL at most.

8. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 4-7.

9. An isolated vector comprising the polynucleotide of claim 8.

10. A host cell comprising a polynucleotide according to claim 8 or a vector according to claim 9.

11. A method of expressing the antibody or antigen-binding fragment thereof according to any one of claims 4-7, comprising culturing the host cell of claim 10 under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and optionally recovering the antibody or antigen-binding fragment thereof according to any one of claims 4-7 from the host cell or from the culture medium.

12. A product for detecting SARS-CoV-2 virus, comprising an antibody pair according to any one of claims 1 to 3.

13. The product of claim 12, wherein the product is a test strip or a test kit.

14. The product according to claim 12, wherein one or more antibodies or antigen-binding fragments thereof described in (a), (b) and (c) in any one of claims 1 to 3 have a chromogenic label and are used as labeled antibodies; and one or more antibodies or antigen-binding fragments thereof as described in any one of claims 1-3 (d) and (e) are immobilized and used as capture antibodies.

15. The product of claim 12, wherein the product comprises a substrate, a sample region, a binding region, and a reaction region, wherein the sample region, the binding region, and the reaction region are sequentially disposed on the substrate along a flow direction of a sample to be measured; the reaction zone is provided with a detection zone loaded with one or more antibodies or antigen-binding fragments thereof as described in any one of claims 1-3 (d) and (e); the binding region is loaded with one or more antibodies or antigen-binding fragments thereof as described in (a), (b) and (c) of any one of claims 1-3 having a chromogenic label.

16. The product of claim 15, wherein the reaction region is further provided with a quality control region loaded with a secondary antibody to one or more antibodies or antigen binding fragments thereof described in (a), (b) and (c) of any one of claims 1 to 3.

17. The product of claim 16, wherein the control region is loaded with an anti-human Fc secondary antibody to one or more antibodies or antigen-binding fragments thereof described in (a), (b), and (c) of any one of claims 1-3.

18. The product of any one of claims 15-17, wherein the product further comprises an absorbent region on a side of the reaction region remote from the sample region.

19. The product of claim 14 or 15, wherein the chromogenic label is a colloidal gold label or a fluorescent label.

20. Use of the pair of antibodies according to any one of claims 1-3 in the preparation of a product for the detection of SARS-CoV-2 virus.

21. Use of the pair of antibodies according to any one of claims 1-3 in the preparation of a diagnostic agent for the diagnosis of SARS-CoV-2 viral infection.

22. A method for detecting SARS-CoV-2 virus for non-diagnostic purposes, comprising: providing a sample, and detecting the presence of SARS-CoV-2 virus in the sample using the product of any one of claims 12-18.

23. The method of claim 22, wherein the method is performed by a double antibody sandwich method.

24. The method of claim 22 or 23, wherein the sample is an environmental sample.

25. The method of claim 22 or 23, wherein the sample is a biological sample.

26. Use of an antibody or antigen-binding fragment thereof according to any one of claims 4-7 in a product for the detection and/or diagnosis of SARS-CoV-2 infection.