Broadly neutralizing antibodies against betacoronavirus and uses thereof

Abstract

The present application relates to the field of immunology, in particular, to broad-spectrum neutralizing antibodies against betacoronavirus and uses thereof. Specifically, the present application relates to broad-spectrum neutralizing antibodies against betacoronavirus or antigen-binding fragments thereof, and uses of the antibodies or fragments in diagnosis, prevention and treatment of various diseases caused by betacoronavirus infection.

Description

Technical Field

[0001] This invention relates to the field of immunology, and in particular to broad-spectrum neutralizing antibodies against coronavirus beta and their applications. Specifically, this invention relates to broad-spectrum neutralizing antibodies against coronavirus beta or antigen-binding fragments thereof, and the application of said antibodies or fragments in the diagnosis, prevention, and treatment of various diseases caused by coronavirus beta infection. Background Technology

[0002] Coronaviruses (CoVs) are the largest RNA viruses identified to date, infecting mammals (humans, mice, pigs, cats, dogs, bats, cattle, and birds), causing respiratory, intestinal, hepatic, and nervous system diseases in their hosts. The Coronaviridae family is divided into four genera: α, β, δ, and γ coronaviruses, with β coronaviruses posing the most serious threat to humans. β coronaviruses are further divided into the genus βcoronavirus, which contains five subgenera: Embecovirus, Sarbecovirus, Merbecovirus, Nobecovirus, and Hibecovirus. The Sarbecovirus subgenus includes the novel coronavirus (SARS-CoV-2) and its variants, which have ravaged the world in recent years; Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its variants; and SARS-related coronaviruses (SARSr-CoV), which are further subdivided as follows: Figure 1 (DOI: 10.1038 / s41586-020-2294-9).

[0003] As a single-stranded RNA virus, beta-coronaviruses translate and replicate directly within cells, making them more prone to mutation than DNA viruses. Since the outbreak of SARS-CoV-2 in late 2019, multiple variants have emerged, among which five have been designated as Variant of Concern (VOC) by the World Health Organization: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529). These variants, due to alterations in their genetic sequences, have changed the structure of the viral spike protein, affecting the virus's ability to be recognized by antibodies or its affinity for receptors. This leads to risks such as reduced or ineffective neutralizing antibodies after vaccination, increased viral transmissibility, and altered pathogenic mechanisms, significantly increasing the challenges of epidemic prevention and control. Continuous mutations may bring even greater epidemic risks in the future.

[0004] Therefore, it is necessary to find more effective broad-spectrum neutralizing antibodies against beta coronaviruses, including the aforementioned variants, to provide effective means of diagnosing, preventing, and / or treating variant infections, thereby contributing to the stabilization and elimination of the epidemic.

[0005] This invention utilizes techniques from immunology, molecular biology, virology, protein structure analysis, and other fields to develop a unique technical approach, resulting in a series of broad-spectrum neutralizing antibodies against coronavirus beta. These antibodies are derived from memory B cells of SARS-CoV-2 recovered patients who have received the SARS-CoV-2 vaccine. They possess broad-spectrum, high-binding activity and broad-spectrum, high-neutralizing properties characteristic of coronavirus beta, enabling them to efficiently neutralize pathogenic viruses including the novel coronavirus and atypical coronaviruses. They are particularly suitable for the diagnosis, prevention, and treatment of various diseases caused by coronavirus beta infection, and have significant clinical value.

[0006] This invention successfully predicted the binding epitopes and escape patterns of these neutralizing antibodies on beta coronavirus using optimized high-throughput yeast display technology, and identified antibody pairs based on this information, which were then verified by cryo-electron microscopy, thus reporting the sequences of these antibodies for the first time. Invention Overview

[0008] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising a complementarity-determining region of a specific amino acid sequence.

[0009] In some implementations, the heavy chain variable region and the light chain variable region contain specific amino acid sequences.

[0010] In some embodiments, the antibody or its antigen-binding fragment includes constant regions derived from human immunoglobulins, such as heavy chain constant regions and light chain constant regions containing specific amino acid sequences.

[0011] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody, sdAb, chimeric antibody, bispecific antibody, or multispecific antibody.

[0012] In a second aspect, the present invention also provides isolated nucleic acid molecules that encode the antibody of the present invention or an antigen-binding fragment thereof, or a variable region of the heavy chain and / or a variable region of the light chain thereof.

[0013] In some implementations, the nucleic acid molecule is operatively linked to an expression regulatory sequence.

[0014] In a third aspect, the present invention also provides an expression vector comprising the nucleic acid molecule of the present invention.

[0015] In a fourth aspect, the present invention also provides a host cell transformed by the nucleic acid molecule of the present invention or the expression vector of the present invention.

[0016] In a fifth aspect, the present invention also provides a method for preparing an antibody or an antigen-binding fragment thereof, comprising:

[0017] (1) The host cells of the present invention are cultured under conditions suitable for expression of the nucleic acid molecules or expression vectors of the present invention, and

[0018] (2) Isolate and purify the antibody or its antigen-binding fragment expressed by the nucleic acid molecule or expression vector.

[0019] In a sixth aspect, the present invention also provides pharmaceutical compositions comprising the antibody of the present invention or an antigen-binding fragment thereof, and pharmaceutically acceptable carriers and / or excipients.

[0020] In a seventh aspect, the present invention also provides a method for preventing and / or treating disease caused by coronavirus beta infection, the method comprising administering to a subject an effective amount of an antibody of the present invention or an antigen-binding fragment thereof or a pharmaceutical composition of the present invention; the subject is preferably a mammal, more preferably a human.

[0021] In an eighth aspect, the present invention also provides the use of the antibody of the present invention or an antigen-binding fragment thereof for the preparation of a medicament for the prevention and / or treatment of diseases caused by coronavirus beta infection.

[0022] In some implementations, the beta-coronavirus includes SARS-CoV-2 and its variants, SARS-CoV and its variants, and SARS-related coronavirus (SARSr-CoV).

[0023] In some embodiments, the SARS-CoV-2 variant is selected from Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron (B.1.1.529), or any combination thereof.

[0024] In a ninth aspect, the present invention also provides conjugates comprising the antibody of the present invention or an antigen-binding fragment thereof, and a detectable label linked to said antibody or antigen-binding fragment thereof.

[0025] In some embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acrid esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (e.g., fluorescein or fluorescent protein), radionuclides, or biotin.

[0026] In a tenth aspect, the present invention also provides a kit comprising the antibody of the present invention or an antigen-binding fragment thereof or a conjugate thereof.

[0027] In some embodiments, the kit comprises the conjugate of the present invention.

[0028] In some embodiments, the kit comprises the antibody of the present invention or its antigen-binding fragment, and a second antibody that specifically recognizes the antibody or its antigen-binding fragment.

[0029] In some embodiments, the second antibody further includes a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acrid esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0030] In an eleventh aspect, the present invention also provides a method for detecting the presence or level of beta-coronavirus in a sample, comprising:

[0031] (1) Contact the sample with the antibody or its antigen-binding fragment of the present invention or the conjugate of the present invention;

[0032] (2) Detect the binding of the antibody or its antigen-binding fragment or conjugate to the target antigen in the sample.

[0033] In some implementations, detecting the binding indicates the presence of beta-coronavirus in the sample.

[0034] In some implementations, the strength of the binding is used to represent the level of coronavirus beta in the sample.

[0035] In some implementations, the sample is a blood sample (e.g., whole blood, plasma, or serum), excrement, oral or nasal secretions, or bronchoalveolar lavage fluid from the subject.

[0036] In some implementations, the subject is a mammal, such as a human.

[0037] In some implementations, the sample is not from the subject; for example, the sample is from a vaccine sample.

[0038] In an eleventh aspect, the present invention also provides the use of the antibody of the present invention or its antigen-binding fragment or conjugate in the preparation of a kit for detecting the presence or level of beta coronavirus in a sample.

[0039] The antibodies or antigen-binding fragments thereof in this invention should be understood to be more than one type, for example, combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 types. Furthermore, the antibodies or antigen-binding fragments thereof in this invention can also be combined with other antibodies or antigen-binding fragments thereof.

[0040] In some embodiments, the antibody or its antigen-binding fragment is a combination of two specific antibodies or their antigen-binding fragments, such as a combination of BD55-5840 and BD55-5514. Attached Figure Description

[0041] Figure 1 This shows the subgenus breakdown of Sarbecovirus.

[0042] Figure 2 The curve of mouse body weight change during SARS-CoV-2 (Omicron BA.1) challenge experiment is shown.

[0043] Figure 3 The viral load in mouse tissues and organs during the SARS-CoV-2 (Omicron BA.1) challenge experiment is shown.

[0044] Figure 4 The curve of mouse body weight change during SARS-CoV-2 (Omicron BA.5) challenge experiment is shown.

[0045] Figure 5 The viral load in mouse tissues and organs during the SARS-CoV-2 (Omicron BA.5) challenge experiment is shown.

[0046] Figure 6 The cryo-electron microscopy structure of BD55-5514+BD55-5840 combined with BA.1 Spike is shown.

[0047] Figure 7 The combined epitopes of BD55-5514 on BA.1 RBD are shown.

[0048] Figure 8 The combined epitopes of BD55-5840 on BA.1 RBD are shown. Invention Details

[0050] I. Definition

[0051] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0052] As used herein, “antibody” means immunoglobulin and immunoglobulin fragments, whether natural or partially or wholly synthetic (e.g., recombinant), including any fragment that retains the binding specificity of the full-length immunoglobulin molecule, containing at least a portion of the variable region of the immunoglobulin molecule. Therefore, an antibody includes any protein having a binding domain homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). Antibodies include antibody fragments. As used herein, the term antibody includes synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab′ fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), biantibodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The antibodies described herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0053] As used herein, an “antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the antibody’s variable region (e.g., one or more CDRs and / or one or more antibody-binding sites) that binds to an antigen, and thus retains binding specificity as well as at least a portion of the full-length antibody’s specific binding capacity. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen as an antibody fragment derived from the antibody fragment. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinant derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab')2, single-chain Fv(scFv), Fv, dsFv, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragment may comprise multiple chains linked together, for example by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, upon insertion into an antibody framework (e.g., by replacing the corresponding region), acquires immune-specific binding (i.e., exhibits at least or at least about 10 amino acids). 7 -10 8 M -1 Antibodies against the Ka antigen.

[0054] As used herein, “monoclonal antibody” refers to a population of identical antibodies, meaning that each individual antibody molecule in a population of monoclonal antibodies is identical to the others. This characteristic contrasts with that of a polyclonal population of antibodies, which contains antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by a number of well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared from immortalized B cells, for instance, by fusing with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant technologies can also be used to prepare antibodies in vitro from a clonal population of host cells by transforming host cells with plasmids carrying artificial sequences of nucleotides encoding the antibody.

[0055] As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (typically myeloma or lymphoma cells) resulting from the fusion of antibody-producing lymphocytes and non-antibody-producing cancer cells. As is known to those skilled in the art, a hybridoma can proliferate and continuously supply cells that produce a specific monoclonal antibody. Methods for generating hybridomas are known in the art (see, for example, Harlow & Lane, 1988). When referring to the term "hybridoma" or "hybridoma cell," it also includes subclones and progeny cells of the hybridoma.

[0056] As used herein, a “conventional antibody” refers to an antibody comprising two heavy chains (which may be labeled H and H’) and two light chains (which may be labeled L and L’) and two antigen-binding sites. Each heavy chain may be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen-binding capability (e.g., heavy chains include, but are not limited to, VH chains, VH-CH1 chains, and VH-CH1-CH2-CH3 chains), and each light chain may be a full-length light chain or any functional region thereof (e.g., light chains include, but are not limited to, VL chains and VL-CL chains). Each heavy chain (H and H’) is paired with one light chain (L and L’, respectively).

[0057] As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as antibodies naturally produced by antibody-secreting B cells and synthetically produced antibodies with the same domains.

[0058] As used in this article, dsFv refers to an engineered intermolecular disulfide bond with a stable VH-VL pair.

[0059] As used herein, the Fab fragment is an antibody fragment obtained by digesting a full-length immunoglobulin with papain, or a fragment with the same structure synthesized, for example, by a recombinant method. The Fab fragment comprises a light chain (containing VL and CL) and another chain containing a variable domain (VH) of the heavy chain and a constant region domain (CH1) of the heavy chain.

[0060] As used herein, the F(ab')2 fragment is an antibody fragment resulting from the digestion of immunoglobulins with pepsin at pH 4.0–4.5, or a fragment with the same structure synthesized, for example, by a recombinant method. The F(ab')2 fragment essentially comprises two Fab fragments, each heavy chain containing several additional amino acids, including cysteine ​​residues that form the disulfide bond connecting the two fragments.

[0061] As used in this article, the Fab' fragment is a fragment that contains half of the F(ab')2 fragment (one heavy chain and one light chain).

[0062] As used herein, scFv fragments refer to variable light chains (V) covalently linked in any order via peptide linkers. L ) and variable heavy chain (V H The antibody fragment has a linker length that allows the two variable domains to bridge with minimal interference. An exemplary linker is (Gly-Ser) with some Glu or Lys residues dispersed to increase solubility. n Residues.

[0063] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0064] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2, or other antigen-binding subsequences of antibodies) containing minimal sequences derived from non-human immunoglobulins. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the complementarity-determining region (CDR) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) with the desired specificity, affinity, and capability, such as mouse, rat, or rabbit.

[0065] Furthermore, in humanization, amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may be mutated to improve one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, through PCR-mediated mutations, and their effects on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conserved mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, mutations within the CDRs typically do not exceed one or two. Therefore, the humanized antibodies described in this invention also cover antibodies containing one or two amino acid mutations within the CDRs.

[0066] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which an antibody binds at its complementary site. Epitope determinants typically comprise chemically active surface subtypes of a molecule, such as amino acid or sugar side chains, and often possess specific three-dimensional structural features as well as specific charge characteristics.

[0067] As used herein, a variable domain or variable region is a specific Ig domain of an antibody heavy or light chain, containing an amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has one variable region domain, VL and VH, respectively. Variable domains provide antigen specificity and are therefore responsible for antigen recognition. Each variable region contains a CDR and a frame region (FR), the CDR being part of the antigen-binding site domain.

[0068] As used herein, "antigen-binding domain" and "antigen-binding site" are used synonymously to refer to a domain within an antibody that recognizes and physically interacts with a cognate antigen. Natural, conventional full-length antibody molecules have two conventional antigen-binding sites, each containing a variable region portion of the heavy chain and a variable region portion of the light chain. The conventional antigen-binding site contains a loop connecting antiparallel β-chains within the variable region domain. The antigen-binding site may contain other portions of the variable region domain. Each conventional antigen-binding site contains three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. The hypervariable regions are also called complementarity-determining regions (CDRs).

[0069] As used herein, the terms “hypervariant region,” “HV,” “complementarity-determining region,” “CDR,” and “antibody CDR” can be used interchangeably to refer to one of the multiple portions within each variable region that together form the antigen-binding site of the antibody. Each variable region domain contains three CDRs, named CDR1, CDR2, and CDR3. For example, the light chain variable region domain contains three CDRs, named VL CDR1, VL CDR2, and VL CDR3; the heavy chain variable region domain contains three CDRs, named VH CDR1, VH CDR2, and VH CDR3. The three CDRs in the variable region are discontinuous along the linear amino acid sequence but are close together in the folded polypeptide. The CDRs are located within the loop of the parallel chain connecting the β-sheet of the variable region. As described herein, those skilled in the art know and can identify CDRs based on Kabat or Chothia numbers (see, for example, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196: 901-917).

[0070] As used in this article, the frame region (FR) is a domain located within the antibody variable region domain within the β-sheet; in terms of amino acid sequence, the FR region is relatively more conserved than the hypervariable region.

[0071] As used herein, a "constant region" domain is a domain in the antibody heavy or light chain that contains a more conserved amino acid sequence than the variable region domain. In a typical full-length antibody molecule, each light chain has a single light chain constant region (CL) domain, while each heavy chain contains one or more heavy chain constant region (CH) domains, including CH1, CH2, CH3, and CH4. Full-length IgA, IgD, and IgG isoforms contain CH1, CH2, CH3, and hinge regions, while IgE and IgM contain CH1, CH2, CH3, and CH4. The CH1 and CL domains extend the Fab arms of the antibody molecule, thus facilitating interaction with antigens and the rotation of the antibody arms. Antibody constant regions can serve effector functions, such as, but not limited to, clearing antigens, pathogens, and toxins specifically bound to the antibody, for example, through interactions with various cells, biomolecules, and tissues.

[0072] As used herein, the functional region of an antibody is an antibody portion that contains at least VH, VL, CH (e.g., CH1, CH2, or CH3), CL, or hinge region domains of the antibody or at least its functional region.

[0073] As used herein, a functional region of a VH domain is at least a portion of a complete VH domain that retains at least a portion of the binding specificity of the complete VH domain (e.g., by retaining one or more CDRs of the complete VH domain), such that the functional region of the VH domain binds the antigen alone or in combination with another antibody domain (e.g., a VL domain) or a region thereof. An exemplary functional region of a VH domain is a region containing CDR1, CDR2, and / or CDR3 of the VH domain.

[0074] As used herein, a functional region of a VL domain is at least a portion of an intact VL domain that retains at least a portion of the binding specificity of the intact VL domain (e.g., by retaining one or more CDRs of the intact VL domain), such that the functional region of the VL domain binds the antigen alone or in combination with another antibody domain (e.g., a VH domain) or a region thereof. An exemplary functional region of a VL domain is a region comprising CDR1, CDR2, and / or CDR3 of the VL domain.

[0075] As used herein, the terms “specific binding” or “immune-specific binding” for antibodies or their antigen-binding fragments are used interchangeably and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with the same antigen through a non-covalent interaction between the antibody and the antigen’s antibody-binding site. The antigen may be an isolated antigen or present in a biological sample. Typically, antibodies that immune-specifically bind (or specifically bind) antigens are present in quantities of approximately 1 × 10⁻⁶. 7 M -1 Or 1x 10 8 M-1 Or a larger affinity constant Ka (or 1 x 10⁻⁶) -7 M or 1×10 -8 M or a lower dissociation constant (K) d The affinity constant can be determined by standard kinetic methods of antibody reactions, such as immunoassay, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); also see U.S. Patent No. 7,229,619, which describes exemplary SPR and ITC methods for calculating the binding affinity of antibodies). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare LifeSciences; Malmqvist (2000) Biochem. Soc. Trans. 27: 335).

[0076] As used herein, the term "competition" with respect to antibodies refers to a first antibody or its antigen-binding fragment binding to an epitope in a manner sufficiently similar to that of a second antibody or its antigen-binding fragment, such that the binding of the first antibody to its associated epitope is detectably reduced in the presence of the second antibody compared to the absence of the second antibody. Alternatively, the binding of the second antibody to its epitope may also be detectably reduced in the presence of the first antibody; this is possible but not necessary. That is, the first antibody may inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, in cases where each antibody detectably inhibits the binding of another antibody to its associated epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to "cross-compete" with each other for binding to their respective epitopes. Both competing and cross-competing antibodies are covered in this invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or fragment thereof), those skilled in the art will recognize, based on the teachings provided in this invention, that such competing and / or cross-competing antibodies are covered in this invention and can be used in the methods disclosed herein.

[0077] As used herein, a polypeptide refers to two or more amino acids covalently linked together. The terms "polypeptide" and "protein" are used interchangeably herein.

[0078] "Separated protein," "separated polypeptide," or "separated antibody" means that the protein, polypeptide, or antibody: (1) is not associated with its natural associated component in its native state, (2) does not contain other proteins from the same species, (3) is expressed by cells from a different species, or (4) does not occur naturally. Therefore, a chemically synthesized polypeptide or a polypeptide synthesized in a cellular system of a different natural source cell will be "separated" from its natural associated component. Separation can also render a protein substantially free of its natural associated component, i.e., using protein purification techniques well known in the art.

[0079] In peptides or proteins, suitable conserved amino acid substitutions are known to those skilled in the art and can generally be performed without altering the biological activity of the resulting molecule. Typically, those skilled in the art recognize that single amino acid substitutions in non-essential regions of polypeptides do not substantially alter biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0080] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved substitutions of amino acids are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0081] The twenty common amino acids discussed herein are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0082] As used herein, the terms “polynucleotide” and “nucleic acid molecule” refer to oligomers or polymers containing at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which are typically linked together by phosphodiester bonds.

[0083] As used herein, isolated nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in natural sources of nucleic acid molecules. “Isolated” nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding provided antibody or antigen-binding fragments.

[0084] Sequence “identity” has a generally accepted meaning in the art, and the percentage of sequence similarity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence similarity can be measured along the full length of a polynucleotide or polypeptide or along a region of that molecule. (See, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991). Although there are many methods for measuring the similarity between two polynucleotides or polypeptides, the term “similarity” is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0085] As used herein, “operably linked” in relation to a nucleic acid sequence, region, element, or domain indicates that the nucleic acid regions are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide, thereby regulating or mediating the transcription of that nucleic acid.

[0086] As used herein, a "vector" is a reproducible nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can typically be introduced via restriction enzyme digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplification of nucleic acids or for expression / display of the polypeptide encoded by the nucleic acid. Vectors are typically kept free but can be designed to integrate genes or portions thereof into the chromosome of the genome. Vectors for artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes, are also considered. The selection and use of such vectors are well known to those skilled in the art.

[0087] As used in this article, vectors also include “viral vectors” or “vectors of viruses.” Viral vectors are engineered viruses that are operatively linked to a foreign gene to transfer (as a medium or shuttle) the foreign gene into cells.

[0088] As used herein, “expression” refers to the process by which a polypeptide is produced through the transcription and translation of polynucleotides. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from host cells. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assays, and Bradford protein assays.

[0089] As used herein, "expression vector" includes a vector capable of expressing DNA operatively linked to regulatory sequences, such as promoter regions, that influence the expression of such DNA fragments. These additional fragments may include promoter and terminator sequences and optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into a suitable host cell, results in the expression of clonal DNA. Suitable expression vectors are well known to those skilled in the art and include reproducible expression vectors in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain free or are integrated into the host cell genome.

[0090] "Codon optimization" refers to methods of modifying nucleic acid sequences to enhance expression in host cells of interest by replacing at least one codon of the natural sequence with codons that are used more frequently or most frequently in the gene in the host cell (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) while maintaining the natural amino acid sequence. Different species exhibit specific preferences for certain codons of specific amino acids. Codon preference (differences in codon use between organisms) is often associated with the translation efficiency of messenger RNA (mRNA), which is thought to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell generally reflects the codons most frequently used for peptide synthesis. Therefore, genes can be customized to achieve optimal gene expression in a given organism based on codon optimization. Codon utilization tables are readily available, for example in... www.kazusa.orjp / codon / The codon usage databases available online (“Codon Usage Database”) are available, and these tables can be adapted in different ways. See, Nakamura Y. et al., “Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0091] As used herein, a “host cell” is a cell used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When a host cell divides, the nucleic acids contained in the vector replicate, thereby amplifying the nucleic acids. Host cells can be eukaryotic or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK 293 cells.

[0092] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Absorption delaying agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art as being capable of stabilizing the desired activity of the active ingredient in the pharmaceutical product, including, but not limited to, monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such sterile injectable fluids are selected from water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0093] As used herein, the term "prevention" refers to methods implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., SARS-CoV-2 infection) in a subject. As used herein, the term "treatment" refers to methods implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include (but are not limited to) alleviating symptoms, reducing the extent of disease, stabilizing (i.e., no longer worsening) the state of disease, delaying or slowing the progression of disease, improving or alleviating the state of disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to the expected survival (if no treatment was received).

[0094] As used in this article, “therapeutic effect” refers to the effect resulting from treatment of an individual, which alters, usually improves or enhances the symptoms of a disease or condition, or cures a disease or condition.

[0095] As used herein, "preventive effective dose" or "preventive effective amount" refers to the amount of a substance, compound, material, or composition containing a compound that, when applied to a subject, would have the intended preventive effect, such as preventing or delaying the onset or recurrence of a disease or symptom, or reducing the likelihood of the onset or recurrence of a disease or symptom. A fully preventive effective dose does not necessarily occur through the administration of a single dose and can occur only after a series of doses have been administered. Therefore, a preventive effective dose can be administered in one or more applications.

[0096] As used herein, "therapeutic effective amount" or "therapeutic effective dose" refers to an amount of substance, compound, material, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition.

[0097] As used herein, the term "subject" preferably refers to a mammal, such as a human. In some embodiments, the subject (e.g., a human) has SARS-CoV-2 infection or a disease associated with SARS-CoV-2 infection (e.g., COVID-19), or is at risk of having such a disease.

[0098] As used in this article, "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)," formerly known as "novel coronavirus" or "2019-nCov," belongs to the β-coronavirus genus and is an enveloped, single-stranded, positive-sense RNA virus. SARS-CoV-2 contains at least three membrane proteins, including a surface spike protein (S), an integrated membrane protein (M), and an epithelial protein (E). Like SARS-CoV, SARS-CoV-2's receptor specifically binds to angiotensin-converting enzyme 2 (ACE2) on the host cell via the receptor-binding domain (RBD) on the S protein, leading to viral membrane fusion and cell entry. This receptor plays a crucial role in viral infection of cells.

[0099] In this document, the term "SARS-CoV-2" encompasses a variety of known isolates, including both the original strain (e.g., the first sequenced isolate, GenBank: MN908947.3) and subsequently discovered variants. In some embodiments, the term "SARS-CoV-2" includes both isolates whose S protein does not contain mutations (e.g., compared to the reference strain MN908947.3) and isolates whose S protein contains mutations (e.g., amino acid substitutions compared to the reference strain MN908947.3, such as K417N, E484K, N501Y, L452R, T478K, or any combination thereof). In some embodiments, the variants are preferably selected from isolates whose S protein contains mutations (e.g., amino acid substitutions, such as K417N, E484K, N501Y, L452R, T478K, or any combination thereof). In some exemplary embodiments, the mutant strain is selected from Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529).

[0100] As used in this article, the terms “novel coronavirus pneumonia” and “COVID-19” refer to pneumonia caused by SARS-CoV-2 infection. They have the same meaning and can be used interchangeably.

[0101] As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment to bind to antigenic proteins on a virus, thereby preventing the virus from infecting cells and / or maturing and / or releasing viral progeny. Antibodies or antibody fragments with neutralizing activity can prevent viral amplification, thereby inhibiting or eliminating viral infection.

[0102] II. Antibody or its antigen-binding fragment

[0103] The present invention provides an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise a complementarity-determining region (CDR) of a specific amino acid sequence, or an amino acid sequence having one or two amino acid residues substituted, deleted, or added relative to a specific amino acid sequence.

[0104] In some embodiments, the heavy chain variable region and the light chain variable region comprise a specific amino acid sequence or a variant thereof. The variant has one or more amino acid substitutions, deletions, or additions compared to the sequence from which it originates, such as substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; preferably, the substitution is a conservative substitution.

[0105] In some embodiments, the antibody or its antigen-binding fragment includes constant regions derived from human immunoglobulins, such as heavy chain constant regions and light chain constant regions containing specific amino acid sequences.

[0106] Information on some sequences involved in this invention is provided in Table 1 below, specifically relating to the VH CDR1, VHCDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of 21 antibodies (numbered BD55-1239, BD55-3372, BD55-3500, BD55-3546, BD55-4637, BD55-5242, BD55-5263, BD55-5300, BD55-5386, BD55-5477, BD55-5483, BD55-5484, BD55-5514, BD55-5549, BD55-5558, BD55-5585, BD55-5591, BD55-5640, BD55-5697, BD55-5700, and BD55-5840, respectively). The CDR3 sequence, as well as the VH and VL sequences, are shared by these 21 antibodies. These antibodies share the CH (SEQ ID NO: 169) and CL (SEQ ID NO: 170) sequences.

[0107] Table 1: Sequence Description

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] III. Antibody Preparation

[0118] The antibodies of the present invention can be prepared by various methods known in the art, such as through genetic engineering recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0119] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). Alternatively, these antigen-binding fragments can also be directly produced from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, the Fab' fragment can be obtained directly from host cells; the Fab' fragment can be chemically coupled to form the F(ab')2 fragment (Carter et al., Bio / Technology, 10: 163-167 (1992)). Additionally, Fv, Fab, or F(ab')2 fragments can also be directly isolated from recombinant host cell culture media. Other techniques for preparing these antigen-binding fragments are fully known to those skilled in the art.

[0120] Therefore, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof. In some embodiments, the isolated nucleic acid molecule encodes an antibody of the present invention or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof.

[0121] In some embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of the antibody or antigen-binding fragment of the present invention, and a second nucleotide sequence encoding a light chain or a light chain variable region of the antibody or antigen-binding fragment of the present invention, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecule of the present invention comprises a first nucleic acid molecule containing the first nucleotide sequence and a second nucleic acid molecule containing the second nucleotide sequence.

[0122] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising isolated nucleic acid molecules as described above. In some embodiments, the vector of the present invention is, for example, a plasmid, a granule, a bacteriophage, etc.

[0123] In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of the antibody or antigen-binding fragment of the present invention and a second nucleotide sequence encoding a light chain or a light chain variable region of the antibody or antigen-binding fragment of the present invention, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector containing the first nucleotide sequence and a second vector containing the second nucleotide sequence.

[0124] In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain variable region of an antibody or antigen-binding fragment of the present invention, and / or a second nucleotide sequence encoding a light chain variable region of an antibody or antigen-binding fragment of the present invention; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

[0125] In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain of an antibody of the present invention or an antigen-binding fragment thereof, and / or a second nucleotide sequence encoding a light chain of an antibody of the present invention or an antigen-binding fragment thereof; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

[0126] In another aspect, the present invention provides a host cell transformed by the nucleic acid molecule of the present invention or the expression vector of the present invention. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., *Escherichia coli* cells), eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). In some embodiments, the host cell of the present invention is a mammalian cell, such as CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK 293 cells.

[0127] In another aspect, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided, comprising culturing the host cell of the present invention under conditions suitable for expression of the nucleic acid molecule or expression vector of the present invention, and isolating and purifying the antibody or antigen-binding fragment thereof expressed by said nucleic acid molecule or expression vector.

[0128] IV. Pharmaceutical Compositions

[0129] As used herein, "pharmaceutically acceptable carriers and / or excipients" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. Preferably, the carrier and / or excipient is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, active compounds, such as antibody molecules or immunoconjugates, may be encapsulated in a material to protect the compound from acids and other natural conditions that could inactivate it.

[0130] The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as palmitic acid ascorbate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0131] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants.

[0132] The presence of microorganisms can be prevented through sterilization procedures or by including various antibacterial and antifungal agents such as parabens, chlorobutanol, and phenolic sorbic acid. In many cases, the composition preferably contains isotonic agents, such as sugars, polyols such as mannitol, sorbitol, or sodium oxide. Prolonged absorption of injectable drugs can be achieved by adding delayed absorption agents, such as monostearate and gelatin, to the composition.

[0133] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the provisional preparation of sterile injections or dispersions. The use of these media and reagents for pharmaceutically active substances is well known in the art. Conventional media or reagents, except for any ranges incompatible with the active compound, may be used in the pharmaceutical compositions of the present invention. Additional active compounds may also be incorporated into the compositions.

[0134] Therapeutic compositions must generally be sterile and stable under preparation and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersant containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. For example, by using coatings, such as lecithin, appropriate flowability can be maintained in the case of dispersions by maintaining the desired particle size, and by using surfactants.

[0135] Sterile injection solutions can be prepared by mixing the active compound in a suitable solvent in the required amount, and adding one or a combination of the components listed above as needed, followed by aseptic microfiltration. Dispersants are typically prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and other desired components listed above. For sterile powders used to prepare sterile injection solutions, preferred preparation methods include vacuum drying and freeze-drying (lyophilization), yielding a powder containing the active ingredient plus any additional desired components from a pre-sterile filtered solution.

[0136] The amount of active ingredient that can be combined with a carrier material to prepare a single-dose formulation varies depending on the target population and the specific route of administration. Generally, the amount of active ingredient that can be combined with a carrier material to prepare a single-dose formulation is the amount of the composition that produces the therapeutic effect. Typically, this amount, in 100% terms, ranges from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0137] Dosing regimens can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus can be administered, several separate doses can be administered over time, or the dose can be proportionally reduced or increased as needed for an emergency in the treatment situation. It is particularly advantageous to formulate the parenteral composition into easily administered and uniformly dosed unit forms. The term "dose unit form" as used herein refers to a physically discontinuous unit suitable as a unit dose for use on the subject being treated; each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect when combined with the desired drug carrier. Specific descriptions of the dose unit forms of the invention are limited to and directly dependent on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art for formulating such active compounds for treating individual sensitivities.

[0138] For the administration of antibody molecules, the dosage range is approximately 0.0001 to 100 mg / kg, more typically 0.01 to 20 mg / kg of recipient body weight. For example, the dosage may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight, or 20 mg / kg body weight, or in the range of 1–20 mg / kg. Exemplary treatment regimens may require weekly, bi-weekly, tri-weekly, quadri-weekly, monthly, 3-monthly, 3–6-monthly dosing, or slightly shorter initial dosing intervals (e.g., weekly to tri-weekly) followed by longer intervals (e.g., monthly to 3–6-monthly).

[0139] Alternatively, the antibody molecules of the present invention can also be administered as a sustained-release formulation, in which case a lower frequency of dosing is required. The dosage and frequency vary depending on the half-life of the antibody molecule in the patient. Typically, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency of administration vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic use, relatively low doses are administered at less frequent intervals over a longer period. Some patients continue treatment for the rest of their lives. In therapeutic use, sometimes higher doses are required at shorter intervals until disease progression is reduced or stopped, preferably until the patient shows partial or complete improvement in disease symptoms. Afterward, the patient can be given prophylactic medication.

[0140] The actual dose level of the active ingredient in the pharmaceutical compositions of this invention may be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and route of administration, without toxicity to the patient. The selected dose level depends on a variety of pharmacokinetic factors, including the activity of the particular composition of this invention applied, the route of administration, the time of administration, the excretion rate of the particular compound applied, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition applied, the age, sex, weight, condition, general health status, and medical history of the patient receiving treatment, and similar factors known in the medical field.

[0141] The antibodies or antigen-binding fragments thereof of the present invention, or the pharmaceutical compositions of the present invention, can be administered via one or more routes of administration using one or more methods known in the art. Those skilled in the art will understand that the route and / or manner of administration varies depending on the desired outcome. Preferred routes of administration for the antibodies of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, such as injection or infusion. The phrase “parenteral administration” as used herein refers to a mode of administration other than enteral and local administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions.

[0142] Alternatively, the antibodies or antigen-binding fragments of the present invention or the pharmaceutical compositions of the present invention may also be administered via non-parenteral routes, such as local, epidermal or mucosal routes, for example, intranasal, oral, vaginal, rectal, sublingual or local administration.

[0143] The active compound can be prepared with a carrier that protects the compound from rapid release, such as controlled-release formulations, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0144] The therapeutic compositions can be administered using medical devices known in the art. For example, in a preferred embodiment, the therapeutic compositions of the present invention can be administered using a needle-free subcutaneous injection device, such as those disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of known implants and modules that can be used in this invention include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispersing drugs at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233, which discloses a medical infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable-rate implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multiple compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0145] In some embodiments, the antibodies of the present invention may be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​blocks many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present invention can cross the BBB (if desired), they may be formulated in, for example, liposomes. For methods of preparing liposomes, see, for example, U.S. Patents 4,522,811; 5,374,548 and 5,399,331. Liposomes contain one or more targeting moieties that can be selectively transported into specific cells or organs, thereby enhancing targeted drug delivery (see, for example, VVRanade (1989) J. Clin. Pharmacol. 29:685). Examples of targeted components include folic acid or biotin (see, for example, U.S. Patent 5,416,016 to Low et al.); mannosides (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (PGBloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); p120 (Schreier et al. (1994) J. Biol. Chem. 269: 9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346: 123; JJ Killion; IJ Fidler (1994) Immunomethods 4: 273.

[0146] In some exemplary embodiments, the pharmaceutically acceptable carrier and / or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such a sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0147] V. Uses for disease prevention and / or treatment

[0148] The antibodies or antigen-binding fragments of the present invention can be used in vitro or in vivo to neutralize coronavirus beta, block or inhibit coronavirus beta infection of cells, thereby achieving the purpose of preventing and / or treating coronavirus beta infection or diseases related to coronavirus beta infection in subjects.

[0149] In another aspect, the present invention provides a method for preventing and / or treating disease caused by coronavirus beta infection, the method comprising administering to a subject an effective amount of an antibody of the present invention or an antigen-binding fragment thereof, or a pharmaceutical composition of the present invention.

[0150] In some implementations, the beta-coronavirus includes SARS-CoV-2 and its variants, SARS-CoV and its variants, and SARS-related coronavirus (SARSr-CoV).

[0151] In some embodiments, the SARS-CoV-2 includes a variant strain. In some embodiments, the S protein of the variant strain contains a mutation, such as an amino acid substitution, deletion, or addition. In some embodiments, the S protein of the variant strain contains one or more amino acid substitutions selected from K417N, E484K, N501Y, L452R, and T478K. In some embodiments, the SARS-CoV-2 variant strain is selected from Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron (B.1.1.529), or any combination thereof.

[0152] In some implementations, the subject is preferably a mammal, and more preferably a human.

[0153] In some embodiments, the antibody or its antigen-binding fragment, or the pharmaceutical composition, is used alone or in combination with another pharmaceutically active agent (e.g., another antiviral agent). The antibody or its antigen-binding fragment of the present invention, or the pharmaceutical composition of the present invention, and the other pharmaceutically active agent can be administered simultaneously, separately, or sequentially.

[0154] In another aspect, the present invention provides methods for neutralizing beta-coronavirus, blocking or inhibiting the binding of beta-coronavirus to the ACE2 receptor, or blocking or inhibiting beta-coronavirus infection of cells, comprising using an antibody or antigen-binding fragment thereof or a pharmaceutical composition of the present invention. The methods can be used in vitro or in a subject (e.g., a human) to neutralize beta-coronavirus, block or inhibit the binding of beta-coronavirus to the ACE2 receptor, or block or inhibit beta-coronavirus infection of cells.

[0155] In some embodiments, the method is used to neutralize the virulence of beta-coronavirus in a sample, block or inhibit the binding of beta-coronavirus to the ACE2 receptor, or block or inhibit beta-coronavirus infection of cells. In some embodiments, the method includes contacting a sample containing beta-coronavirus with an antibody or antigen-binding fragment of the present invention or a pharmaceutical composition thereof.

[0156] In another aspect, the present invention relates to the use of the antibody or antigen-binding fragment thereof of the present invention in the preparation of a medicament for the prevention and / or treatment of diseases caused by coronavirus beta infection, said medicament being used for one or more of the following:

[0157] (1) Neutralizes coronavirus in vitro or in subjects (e.g., humans);

[0158] (2) Block or inhibit the binding of beta coronavirus to the ACE2 receptor;

[0159] (3) Blocking or inhibiting coronavirus beta infection of cells; and / or

[0160] (4) For the prevention and / or treatment of subjects with coronavirus beta infection or diseases associated with coronavirus beta infection (e.g., COVID-19).

[0161] The antibodies or antigen-binding fragments thereof of the present invention, or the pharmaceutical compositions thereof, can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The antibodies or antigen-binding fragments thereof or the pharmaceutical compositions thereof of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the antibody or antigen-binding fragment of the present invention, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0162] The antibodies or antigen-binding fragments thereof of the present invention, or the pharmaceutical compositions thereof, may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some embodiments, the antibodies or antigen-binding fragments thereof or the pharmaceutical compositions thereof of the present invention are administered by intravenous injection or bolus.

[0163] In this document, the dosing regimen may be adjusted to obtain the optimal target response (e.g., treatment or prevention). For example, it may be administered as a single dose, multiple times over a period of time, or the dose may be reduced or increased proportionally to the urgency of the treatment situation.

[0164] VI. Conjugates

[0165] The antibodies or antigen-binding fragments of the present invention can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of the antibody or antigen-binding fragment does not adversely affect its binding to beta-coronavirus. Therefore, the antibodies or antigen-binding fragments of the present invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide capable of mediating the binding of the antibody or antigen-binding fragment to another molecule (e.g., avidin or a multihistidine tag). Furthermore, the antibodies or antigen-binding fragments of the present invention can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups. These groups can be used to improve the biological properties of the antibody, such as increasing serum half-life.

[0166] Therefore, in some embodiments, the antibody or its antigen-binding fragment of the present invention carries a detectable marker.

[0167] In this document, the detectable markers described in this invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.) and radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent materials (e.g., chemiluminescent materials, such as acridine esters, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), thermal markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers.

[0168] In some embodiments, the detectable label is suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescent immunoassay, etc.). In some embodiments, the detectable label may be selected from enzymes (e.g., horseradish peroxidase, alkaline phosphatase, or β-galactosidase), chemiluminescent reagents (e.g., acrid esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (e.g., fluorescein or fluorescent proteins such as FITC, TRITC, or PE), radionuclides, or biotin.

[0169] In some implementations, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0170] VII. Reagent Kit and Detection Applications

[0171] The antibody or antigen-binding fragment of the present invention can specifically bind to the RBD of the S protein of beta coronavirus, thereby being used to detect beta coronavirus or its S protein or the RBD of the S protein, and optionally to diagnose whether a subject is infected with beta coronavirus based on the above detection results.

[0172] Therefore, in another aspect, the present invention provides a kit comprising the antibody of the present invention or an antigen-binding fragment thereof, or a conjugate of the present invention.

[0173] In some embodiments, the kit comprises the conjugate of the present invention.

[0174] In some embodiments, the kit comprises the antibody of the present invention or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment does not contain a detectable label. In some embodiments, the kit further comprises a second antibody that specifically recognizes the antibody of the present invention or its antigen-binding fragment; optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acrid esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0175] In some embodiments, the second antibody is specific to antibodies of the species (e.g., humans) from which the constant region contained in the antibody of the present invention or its antigen-binding fragment originates.

[0176] In some embodiments, the second antibody is an anti-immunoglobulin (e.g., human immunoglobulin) antibody, such as an anti-IgG antibody. In some embodiments, the second antibody is an anti-human IgG antibody.

[0177] In some embodiments, the kit of the present invention may further comprise reagents for detecting the corresponding detectable label. For example, when the detectable label is an enzyme, the kit may also comprise a chromogenic substrate for the corresponding enzyme, such as o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS, or luminol compounds for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase. For example, when the detectable label is a chemiluminescent reagent (e.g., acrid ester compounds), the kit may also comprise a pre-excitation solution and / or an excitation solution for chemiluminescence.

[0178] In another aspect, the present invention provides a method for detecting the presence or level of beta-coronavirus or its S protein or RBD of the S protein, or cells infected with beta-coronavirus, in a sample, comprising contacting the sample with an antibody or its antigen-binding fragment or conjugate of the present invention; detecting the binding of the antibody or its antigen-binding fragment or conjugate to a target antigen in the sample; wherein the detection of the binding represents the presence of beta-coronavirus in the sample, or the strength of the binding represents the level of beta-coronavirus in the sample.

[0179] In some implementations, the method is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0180] In some embodiments, the method includes using the conjugate of the present invention.

[0181] In other embodiments, the method includes using the antibody or antigen-binding fragment of the present invention. In some embodiments, the antibody or antigen-binding fragment does not contain a detectable label. In some embodiments, the method further includes using a second antibody with a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acrid esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin) to detect the antibody or antigen-binding fragment.

[0182] In some embodiments, the second antibody is specific to antibodies of the species (e.g., humans) from which the constant region contained in the antibody of the present invention or its antigen-binding fragment originates.

[0183] In some embodiments, the second antibody is an anti-immunoglobulin (e.g., human immunoglobulin) antibody, such as an anti-IgG antibody. In some embodiments, the second antibody is an anti-human IgG antibody.

[0184] In some embodiments, the method can be used for diagnostic purposes, such as diagnosing whether a subject is infected with beta-coronavirus based on the presence or level of beta-coronavirus in a sample. In such embodiments, the sample can be a blood sample (e.g., whole blood, plasma, or serum), excrement, oral or nasal secretions, or bronchoalveolar lavage fluid from the subject.

[0185] In some implementations, the subject is a mammal, such as a human.

[0186] In some implementations, the method can be used for non-diagnostic purposes, such as when the sample is not from a subject, for example, a vaccine sample.

[0187] In some implementations, the beta-coronavirus includes SARS-CoV-2 and its variants, SARS-CoV and its variants, and SARS-related coronavirus (SARSr-CoV).

[0188] In some embodiments, the SARS-CoV-2 includes a variant strain. In some embodiments, the S protein of the variant strain contains a mutation, such as an amino acid substitution, deletion, or addition. In some embodiments, the S protein of the variant strain contains one or more amino acid substitutions selected from K417N, E484K, N501Y, L452R, and T478K. In some embodiments, the SARS-CoV-2 variant strain is selected from Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron (B.1.1.529), or any combination thereof.

[0189] In another aspect, the use of the antibodies of the present invention or their antigen-binding fragments or conjugates of the present invention in the preparation of kits for detecting beta-coronavirus or its S protein or RBD of the S protein, or the presence or level of beta-coronavirus-infected cells in a sample, and / or for diagnosing whether a subject is infected with beta-coronavirus.

[0190] In some embodiments, the method is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0191] In some embodiments, the kit detects beta-coronavirus or its S protein or RBD of the S protein, or the presence or level of beta-coronavirus-infected cells in a sample using the detection methods described above, and optionally diagnoses whether a subject is infected with beta-coronavirus based on the detection results.

[0192] In some embodiments, the sample is a blood sample (e.g., whole blood, plasma, or serum), excrement, oral or nasal secretions, or bronchoalveolar lavage fluid from a subject (e.g., a mammal, preferably a human).

[0193] In some implementations, the beta-coronavirus includes SARS-CoV-2 and its variants, SARS-CoV and its variants, and SARS-related coronavirus (SARSr-CoV).

[0194] In some embodiments, the SARS-CoV-2 includes a variant strain. In some embodiments, the S protein of the variant strain contains a mutation, such as an amino acid substitution, deletion, or addition. In some embodiments, the S protein of the variant strain contains one or more amino acid substitutions selected from K417N, E484K, N501Y, L452R, and T478K. In some embodiments, the SARS-CoV-2 variant strain is selected from Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron (B.1.1.529), or any combination thereof.

[0195] In the above-described technical solutions of the present invention, such as pharmaceutical compositions, conjugates, kits, methods or uses for disease prevention and / or treatment, detection methods, and uses for preparing pharmaceuticals or kits, the antibody or its antigen-binding fragment should be understood to be more than one, for example, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 types. The antibody or its antigen-binding fragment of the present invention can also be combined with other antibodies or their antigen-binding fragments. The antibody or its antigen-binding fragment can be a combination of two specific antibodies or their antigen-binding fragments, such as a combination of BD55-5840 and BD55-5514. Furthermore, the combination of antibodies or their antigen-binding fragments exhibits superior binding activity and neutralizing effect against beta-coronavirus.

[0196] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments.

[0197] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Susubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the manufacturer's recommendations. Those skilled in the art will appreciate that the examples illustrate the invention by way of illustration and are not intended to limit the scope of the invention as claimed.

[0198] Example 1: Isolation of memory B cells

[0199] Whole blood was collected from recovered SARS-CoV patients who had received the SARS-CoV-2 vaccine. The blood samples were first diluted with PBS (Invitrogen) containing 2% FBS (Gibco) and then centrifuged using a Ficoll (Cytiva) gradient centrifugation. After lysis and washing, the blood was resuspended in PBS (Invitrogen) containing 2% FBS (Gibco) for downstream B cell separation, or stored long-term in FBS containing 10% DMSO (Sigma-Aldrich) at -80°C. CD19 was used for... + B cell isolation kits (STEMCELLS) enriched B cells through positive selection. Enriched B cells were stained in FACS buffer (1×PBS, 2% FBS, 1mM EDTA) with the following human antigens and antibodies: FITC anti-CD19 antibody (Biolegend), FITC anti-CD20 antibody (Biolegend), Brilliant Violet 421 anti-CD27 Antibody (Biolegend), PE / Cyanine7 anti-IgM, and fluorophore-labeled RBDs (SARS340 CoV-2 and SARS-CoV RBDs, Sino Biological Inc.) and ovalbumin (Ova) on ice for 30 minutes. Cells were stained with 7-AAD for 10 minutes prior to sorting and then individually labeled with CD19 or CD20 on an Astrios EQ (BeckMan Coulter). + CD27 + IgM-Ova- RBD-PE + RBD-APC + B cells were sorted into PBS containing 30% FBS. After flow cytometry sorting, the obtained cells were used to prepare 5′-mRNA and single-cell V(D)J libraries, and then further sequenced by Illumina using a two-sided sequencing mode of 26bp (barcode) + 91bp (insertion sequence) on the HiSeq 2500 platform.

[0200] Example 2: Obtaining and Identifying Antibody Sequences

[0201] The raw FASTQ file was processed using Cell Ranger (version 6.1.1) software with reference to the human sequence GRCh38 database. Sequences were generated using either "cellranger multi" or "cellranger vdj" with default parameters. Protein sequences were then extracted and processed using IMGT / DomainGapAlign (version 4.10.2) to obtain annotations for V(D)J, CDR regions, and mutation frequencies. The V gene amino acid mutation rate was calculated as mutation count / V gene peptide length.

[0202] Example 3: Antibody preparation and purification

[0203] Paired immunoglobulin heavy and light chain genes obtained from 10X Genomics V(D)J sequencing and analysis were submitted for recombinant monoclonal antibody synthesis. The heavy and light chain sequences were cloned into expression vectors based on Gibson assembly, and then the two plasmids were co-transfected into HEK293F cells. The secreted monoclonal antibodies were then purified from the cell culture medium by Protein A affinity chromatography.

[0204] Example 4: Evaluation of broad-spectrum antibody binding activity

[0205] 4.1 ELISA

[0206] The specific binding ability of these antibodies or plasma to the assay was determined by ELISA. In short, after coating, blocking, and washing with RBDs of 22 currently known sarbecoviruses (see Table 2) at 0.03 μg / mL and 1 μg / mL, 1 μg / mL of antibody or serially diluted plasma samples were added to the ELISA plate.

[0207] Table 2: Gene names and NCBI accession numbers of the beta-coronavirus spike protein

[0208]

[0209]

[0210] After incubation and washing, the 96-well plates were incubated with diluted goat anti-human IgG 373(H+L) / HRP antibody (JACKSON). Tetramethylbenzidine (TMB, Solarbio) was then added for color development for 10 minutes, followed by the addition of 2M H2SO4 to stop the reaction. The absorbance was measured at 450 nm using a PerkinElmer Ensight HH3400 microplate reader, and the EC50 was calculated using GraphPad Prism 8.0 software (see Table 3).

[0211] 4.2 Dynamic Experiment of Interference Analysis of Biological Layer

[0212] use The protein analysis system (Fortebio) was used for biolayer interferometry kinetic experiments, employing a protein A biosensor (Fortebio 18-5010). First, the sensor was immersed in a buffer (ForteBio 18-1105 buffer) for 10 minutes to complete a self-test, followed by a 30-second baseline immersion. In the antibody capture step, the sensor was immersed in 2 μg / ml antibody for 300 seconds with a threshold set at 0.4 nm. The sensor was then immersed in the buffer for only 120 seconds to complete the baseline. Next, in the antigen binding step, the sensor was immersed in serially diluted RBD protein buffer for 60 seconds. In the subsequent dissociation step, the sensor was transferred to a buffer and immersed for 600 seconds. Finally, the sensor was immersed in regeneration buffer (10 mM glycine hydrochloride, pH 1.5) for 30 seconds, then in the buffer for 30 seconds, repeated twice to complete the regeneration step. Data was recorded using the software Data Acquisition 11.1 (Fortebio), and data analysis was performed using the software Data Analysis HT11.1 (Fortebio) (see Table 4).

[0213] Example 5: Evaluation of broad-spectrum neutralizing activity of antibodies

[0214] Antibodies with broad-spectrum binding were screened using a pseudovirus method for neutralization assays to evaluate their neutralizing capacity in plasma. A series of two-fold diluted antibodies were incubated with seven VsV pseudoviruses (labeled with luciferase) of the sarbecovirus spike protein for 1 hour, and the mixture was then incubated with Huh-7 cells. After culturing at 37°C for 24 hours, cells were collected and lysed with luciferase substrate (PerkinElmer), and the luminescence intensity was measured using a microplate reader. IC50 was determined using a four-parameter nonlinear regression model (see Table 5).

[0215] Example 6: Mouse challenge experiment - SARS-CoV-2 (Omicron BA.1) virus

[0216] 1. Weight

[0217] Using the combination of antibodies BD55-5514 and BD55-5840 as an example, the antiviral efficacy of the candidate drug was evaluated. Human ACE2 (hACE2) transgenic mice were divided into five groups of five mice each. SARS-CoV-2 (Omicron BA.1) virus was detected at a rate of 1*10-1. 5 TCID 50 Mice were infected via nasal instillation at a dose of 20 mg / kg. The prevention group received an intraperitoneal or intramuscular injection of the drug 24 hours before challenge. The treatment group received an intraperitoneal injection of the drug 20 mg / kg 2 hours after challenge. The control group received an intraperitoneal injection of an equal volume of PBS 2 hours after challenge. Mouse body weight was monitored on days 0, 1, 2, and 3 after challenge. Body weight change curves (…) Figure 2 Analysis showed that mice in all groups experienced a slight decrease in body weight one day after challenge, followed by a recovery trend two to three days later, with all mice returning to their original body weight by day three. There was no statistically significant difference in body weight changes between the experimental groups and the control group, which may be due to the low pathogenicity of BA.1 in hACE2 transgenic mice.

[0218] 2. Viral load

[0219] Lung tissue and trachea of ​​mice were collected by dissection on day 3 after challenge. After homogenization, the viral ribonucleic acid (RNA) load in the samples was detected by qRT-PCR. Results ( Figure 3 The results showed that the viral load in the lung tissue and trachea of ​​the control group was high, with average values ​​of 4.91 (log10 copies / mL) and 3.82 (log10 copies / mL), respectively. Viral RNA copies were almost undetectable in the lung tissue and trachea of ​​the treatment group and the intraperitoneal prophylaxis group, showing a significant difference from the control group. The viral load in the lung tissue samples of the muscle prophylaxis group was low, and statistical results showed a significant difference from the control group (P < 0.0001). Viral RNA was almost undetectable in the tracheal samples of the muscle prophylaxis group. The viral load in the lung tissue and tracheal samples of the DXP-593 treatment group was slightly reduced in some cases, but there was no statistically significant difference compared to the control group. These experimental results indicate that, compared with the control group, the treatment group, the intraperitoneal prophylaxis group, and the muscle prophylaxis group can effectively reduce the viral load in the lung tissue and trachea of ​​mice.

[0220]

[0221]

[0222]

[0223] Example 7: Mouse challenge experiment - SARS-CoV-2 (Omicron BA.5) virus

[0224] 1. Weight

[0225] Using the combination of antibodies BD55-5514 and BD55-5840 as an example, the antiviral efficacy of the candidate drug was evaluated. Human ACE2 (hACE2) transgenic mice were divided into four groups of five mice each. SARS-CoV-2 (Omicron BA.5) virus was detected at a rate of 1*10-1... 5 TCID 50 Mice were infected via nasal instillation at a dose of 20 mg / kg. The prevention group received an intraperitoneal or intramuscular injection of the drug 24 hours before challenge. The treatment group received an intraperitoneal injection of the drug 20 mg / kg 2 hours after challenge. The control group received an intraperitoneal injection of an equal volume of PBS 2 hours after challenge. Mouse body weight was continuously monitored daily after challenge, and the body weight change curve was recorded. Figure 4 Analysis showed that mice in all groups experienced a slight decrease in body weight 1-2 days after challenge, followed by a recovery trend 3-4 days after challenge, with all groups essentially returning to their original body weight by day 4. There was no statistically significant difference in body weight changes between the experimental groups and the control group, which may be due to the low pathogenicity of BA.5 to hACE2 transgenic mice.

[0226] 2. Viral load

[0227] Lung tissue and trachea of ​​mice were collected by dissection on day 4 after challenge. After homogenization, the viral ribonucleic acid (RNA) load in the samples was detected by qRT-PCR. Results ( Figure 5 The results showed that the viral load in the lung tissue and trachea of ​​the control group was high, with average values ​​of 5.66 (log10 copies / mL) and 5.04 (log10 copies / mL), respectively. The viral load in the lung tissue and trachea of ​​the intraperitoneal prevention group was lower, with average reductions of 3.10 and 5.04 log10 values ​​compared to the control group, respectively, showing a significant difference (P < 0.0001). The viral load in the lung tissue and trachea of ​​the intraperitoneal treatment group was reduced by an average of 2.21 and 1.11 log10 values ​​compared to the control group, also showing a significant difference between the two groups (P < 0.05). The average viral load in the lung tissue of the muscle prevention group decreased by 2.89 log10 values ​​compared to the control group (P < 0.01), while the viral load in the trachea of ​​the muscle prevention group showed a slight decrease in some individuals, but overall there was no statistically significant difference compared to the control group. These experimental results indicate that, compared to the control group, both the intraperitoneal prevention and intraperitoneal treatment groups effectively reduced the viral load in the lung tissue and trachea of ​​mice, while the muscle prevention group had a certain effect on reducing the viral load in the lung tissue.

[0228] Example 8: Cryo-electron microscopy

[0229] We resolved the cryo-electron microscopy structure of antibodies BD55-5514+BD55-5840 co-binding with BA.1 Spike, demonstrating that the epitopes of BD55-5514 and BD55-5840 do not conflict and can simultaneously bind to the S protein (Spike). BD55-5840 can bind to both the "up" and "down" conformations of the receptor-binding domain (RBD), while BD55-5514 only binds to the "up" conformation of the RBD.

[0230] The binding epitope of BD55-5514 is similar to that of ADG20 (Adintrevimab). The sites on the BA.1 RBD involved in binding BD55-5514 mainly include Asp405, Asn437, Asn439, Gly502, Val503, Gly504, and Tyr508. Sites such as Pro373-Thr376, Val407-Arg408, Pro499, and Tyr501 are also close to the binding interface. However, the neutralization of BD55-5514 is not affected by Thr376Ala, Asp405Asn, and Arg408Ser contained in BA.2, but only by mutations in Val503 and Gly504. These two sites are highly conserved and almost never appear in nature.

[0231] The binding epitope of BD55-5840 on the RBD is similar to that of Ser309 (Sotrovimab), but the key binding sites are different. RBD residues appearing at the interface include Asp339, Glu340, Thr345, Arg346, Lys440, Leu441, Ser443, and Lys444, with mutations mainly affecting its binding concentrated on Asp339-Glu340 and Thr345-Arg346.

[0232] Neutralization experiments showed that the combination of BD55-5514 and BD55-5840 provides the most difficult-to-escape broad-spectrum Sarbecovirus lineage B neutralizing antibody drug, with extremely strong neutralizing ability against all current major SARS-CoV-2 variants, including all major lineages of Omicron.

[0233] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein, (1) The heavy chain variable region includes: VH CDR1, which consists of the amino acid sequence shown in SEQ ID NO:9 VH CDR2, which consists of the amino acid sequence shown in SEQ ID NO:10, and VH CDR3, which consists of the amino acid sequence shown in SEQ ID NO:11; The light chain variable region includes: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO:12, VL CDR2, which consists of the amino acid sequence shown in SEQ ID NO:13, and VL CDR3, which consists of the amino acid sequence shown in SEQ ID NO:14; or (2) The heavy chain variable region includes: VH CDR1, which consists of the amino acid sequence shown in SEQ ID NO:

33. VH CDR2, which consists of the amino acid sequence shown in SEQ ID NO:34, and VH CDR3, which consists of the amino acid sequence shown in SEQ ID NO:35; The light chain variable region includes: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO:

36. VL CDR2, which consists of the amino acid sequence shown in SEQ ID NO:37, and VL CDR3, which consists of the amino acid sequence shown in SEQ ID NO:38; or (3) The heavy chain variable region includes: VH CDR1, which consists of the amino acid sequence shown in SEQ ID NO:

57. VH CDR2, which consists of the amino acid sequence shown in SEQ ID NO:58, and VH CDR3, which consists of the amino acid sequence shown in SEQ ID NO:59; The light chain variable region includes: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO:60, VL CDR2, which consists of the amino acid sequence shown in SEQ ID NO:61, and VL CDR3, which consists of the amino acid sequence shown in SEQ ID NO:62; or (4) The heavy chain variable region includes: VH CDR1, which consists of the amino acid sequence shown in SEQ ID NO:81, VH CDR2, which consists of the amino acid sequence shown in SEQ ID NO:82, and VH CDR3, which consists of the amino acid sequence shown in SEQ ID NO:83; The light chain variable region includes: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO:

84. VL CDR2, which consists of the amino acid sequence shown in SEQ ID NO:85, and VL CDR3, which consists of the amino acid sequence shown in SEQ ID NO:86; or (5) The heavy chain variable region includes: VH CDR1, which consists of the amino acid sequence shown in SEQ ID NO:

97. VH CDR2, which consists of the amino acid sequence shown in SEQ ID NO:98, and VH CDR3, which consists of the amino acid sequence shown in SEQ ID NO:99; The light chain variable region includes: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO:100, VL CDR2, which consists of the amino acid sequence shown in SEQ ID NO:101, and VL CDR3, which consists of the amino acid sequence shown in SEQ ID NO:

102.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein... (1) The heavy chain variable region is composed of the sequence shown in SEQ ID NO: 15; the light chain variable region is composed of the sequence shown in SEQ ID NO: 16; or (2) The heavy chain variable region is composed of the sequence shown in SEQ ID NO: 39; the light chain variable region is composed of the sequence shown in SEQ ID NO: 40; or (3) The heavy chain variable region is composed of the sequence shown in SEQ ID NO: 63; the light chain variable region is composed of the sequence shown in SEQ ID NO: 64; or (4) The heavy chain variable region is composed of the sequence shown in SEQ ID NO: 87; the light chain variable region is composed of the sequence shown in SEQ ID NO: 88; or (5) The heavy chain variable region is composed of the sequence shown in SEQ ID NO: 103; the light chain variable region is composed of the sequence shown in SEQ ID NO:

104.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, further comprising a constant region derived from human immunoglobulin.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from human immunoglobulin selected from IgG1, IgG2, IgG3 or IgG4, and the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from human immunoglobulin selected from κ or λ.

5. The antibody or antigen-binding fragment thereof of claim 3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region as shown in SEQ ID NO: 169 and a light chain constant region as shown in SEQ ID NO:

170.

6. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein, The antigen-binding fragments are selected from Fab, Fab', (Fab')2, Fv, diabody, chimeric antibody, and multispecific antibody.

7. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein, The antigen-binding fragment is a bispecific antibody.

8. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein, The antigen-binding fragment is selected from disulfide-linked Fv or scFv.

9. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described in any one of claims 1-8, or a variable region of the heavy chain and a variable region of the light chain thereof.

10. The isolated nucleic acid molecule of claim 9, wherein the nucleic acid molecule is operatively linked to an expression regulatory sequence.

11. An expression vector comprising the nucleic acid molecule of claim 9 or 10.

12. A host cell, which is transformed by the nucleic acid molecule of claim 9 or 10 or the expression vector of claim 11.

13. A method for preparing an antibody or its antigen-binding fragment, including, (1) The host cells of claim 12 are cultured under conditions suitable for expression of the nucleic acid molecule or expression vector, and (2) Isolate and purify the antibody or its antigen-binding fragment expressed by the nucleic acid molecule or expression vector.

14. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof of any one of claims 1-8, and a pharmaceutically acceptable carrier and / or excipient.

15. The pharmaceutical composition of claim 14, wherein the antibody or its antigen-binding fragment is of two, three, four, or five types.

16. The pharmaceutical composition of claim 15, wherein the antibody or antigen-binding fragment thereof is of two types, wherein one antibody or antigen-binding fragment thereof is as described in claim (5) of any one of claims 1-8, and the heavy chain variable region of the other antibody or antigen-binding fragment thereof comprises: VH CDR1, which consists of the amino acid sequence shown in SEQ ID NO:161, VH CDR2, which consists of the amino acid sequence shown in SEQ ID NO: 162, and VH CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 163; The variable region of the light chain contains: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO: 164, VL CDR2, which consists of the amino acid sequence shown in SEQ ID NO: 165, and VL CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 166; or Another antibody or its antigen-binding fragment has a heavy chain variable region consisting of the sequence shown in SEQ ID NO: 167; and a light chain variable region consisting of the sequence shown in SEQ ID NO:

168.

17. The pharmaceutical composition of claim 16, wherein the other antibody or its antigen-binding fragment comprises a heavy chain constant region as shown in SEQ ID NO: 169 and a light chain constant region as shown in SEQ ID NO:

170.

18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-8 for the preparation of a medicament for the prevention and / or treatment of diseases caused by coronavirus beta infection.

19. The use of claim 18, wherein the antibody or its antigen-binding fragment is of two, three, four or five types.

20. The use of claim 19, wherein the antibody or antigen-binding fragment thereof is of two types, wherein one antibody or antigen-binding fragment thereof is as described in any one of claims 1-8 (5), and the heavy chain variable region of the other antibody or antigen-binding fragment thereof comprises: VH CDR1, which consists of the amino acid sequence shown in SEQ ID NO:161, VH CDR2, which consists of the amino acid sequence shown in SEQ ID NO: 162, and VH CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 163; The variable region of the light chain contains: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO: 164, VL CDR2, which consists of the amino acid sequence shown in SEQ ID NO: 165, and VL CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 166; or Another antibody or its antigen-binding fragment has a heavy chain variable region consisting of the sequence shown in SEQ ID NO: 167; and a light chain variable region consisting of the sequence shown in SEQ ID NO:

168.

21. The use of claim 20, wherein the other antibody or its antigen-binding fragment comprises a heavy chain constant region as shown in SEQ ID NO: 169 and a light chain constant region as shown in SEQ ID NO:

170.

22. The use of any one of claims 18-21, wherein the beta coronavirus includes novel coronavirus (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-related coronavirus (SARSr-CoV).

23. A conjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-8, and a detectable label linked to said antibody or antigen-binding fragment thereof.

24. The conjugate of claim 23, wherein the antibody or its antigen-binding fragment is of two, three, four or five types.

25. The conjugate of claim 24, wherein the antibody or antigen-binding fragment thereof is of two types, wherein one antibody or antigen-binding fragment thereof is as described in any one of claims 1-8 (5), and the heavy chain variable region of the other antibody or antigen-binding fragment thereof comprises: VH CDR1, which consists of the amino acid sequence shown in SEQ ID NO:161, VH CDR2, which consists of the amino acid sequence shown in SEQ ID NO: 162, and VH CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 163; The variable region of the light chain contains: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO: 164, VL CDR2, which consists of the amino acid sequence shown in SEQ ID NO: 165, and VL CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 166; or Another antibody or its antigen-binding fragment has a heavy chain variable region consisting of the sequence shown in SEQ ID NO: 167; and a light chain variable region consisting of the sequence shown in SEQ ID NO:

168.

26. The conjugate of claim 25, wherein the other antibody or its antigen-binding fragment comprises a heavy chain constant region as shown in SEQ ID NO: 169 and a light chain constant region as shown in SEQ ID NO:

170.

27. The conjugate of any one of claims 23-26, wherein the detectable marker is selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, or biotin.

28. The conjugate of claim 27, wherein the enzyme is selected from horseradish peroxidase or alkaline phosphatase, the chemiluminescent reagent is selected from acrid esters, luminol and its derivatives or ruthenium derivatives, and the fluorescent dye is selected from fluorescein or fluorescent protein.

29. A kit comprising the antibody or antigen-binding fragment thereof as claimed in any one of claims 1-8 or the conjugate as claimed in any one of claims 23-28.

30. The kit of claim 29, wherein the kit comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-8, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof.

31. The kit of claim 29 or 30, wherein the antibody or its antigen-binding fragment is of two, three, four or five types.

32. The kit of claim 31, wherein the antibody or antigen-binding fragment thereof is of two types, wherein one antibody or antigen-binding fragment thereof is as described in any one of claims 1-8 (5), and the heavy chain variable region of the other antibody or antigen-binding fragment thereof comprises: VH CDR1, which consists of the amino acid sequence shown in SEQ ID NO:161, VH CDR2, which consists of the amino acid sequence shown in SEQ ID NO: 162, and VH CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 163; The variable region of the light chain contains: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO: 164, VL CDR2, which consists of the amino acid sequence shown in SEQ ID NO: 165, and VL CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 166; or Another antibody or its antigen-binding fragment has a heavy chain variable region consisting of the sequence shown in SEQ ID NO: 167; and a light chain variable region consisting of the sequence shown in SEQ ID NO:

168.

33. The kit of claim 32, wherein the other antibody or its antigen-binding fragment comprises a heavy chain constant region as shown in SEQ ID NO: 169 and a light chain constant region as shown in SEQ ID NO:

170.

34. The kit of any one of claims 30-33, wherein the second antibody further comprises a detectable marker selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, or biotin.

35. The kit of claim 34, wherein the enzyme is selected from horseradish peroxidase or alkaline phosphatase, the chemiluminescent reagent is selected from acrid esters, luminol and its derivatives or ruthenium derivatives, and the fluorescent dye is selected from fluorescein or fluorescent protein.

36. A method for detecting the presence or level of beta-coronavirus in a sample for non-diagnostic purposes, comprising: (1) Contact the sample with the antibody or antigen-binding fragment of any one of claims 1-8 or the conjugate of any one of claims 23-28; (2) Detect the binding of the antibody or its antigen-binding fragment or the conjugate to the target antigen in the sample; The presence of coronavirus beta in the sample can be indicated by the detection of the binding, or the strength of the binding can be indicated by the level of coronavirus beta in the sample.

37. The method of claim 36, wherein the antibody or its antigen-binding fragment is of two, three, four, or five types.

38. The method of claim 37, wherein the antibody or antigen-binding fragment thereof is of two types, wherein one antibody or antigen-binding fragment thereof is as described in any one of claims 1-8 (5), and the heavy chain variable region of the other antibody or antigen-binding fragment thereof comprises: VH CDR1, which consists of the amino acid sequence shown in SEQ ID NO:161, VH CDR2, which consists of the amino acid sequence shown in SEQ ID NO: 162, and VH CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 163; The variable region of the light chain contains: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO: 164, VL CDR2, which consists of the amino acid sequence shown in SEQ ID NO: 165, and VL CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 166; or Another antibody or its antigen-binding fragment has a heavy chain variable region consisting of the sequence shown in SEQ ID NO: 167; and a light chain variable region consisting of the sequence shown in SEQ ID NO:

168.

39. The method of claim 38, wherein the other antibody or its antigen-binding fragment comprises a heavy chain constant region as shown in SEQ ID NO: 169 and a light chain constant region as shown in SEQ ID NO:

170.

40. The method of any one of claims 36-39, wherein the sample is not a sample from the subject.

41. The method of claim 40, wherein the sample is derived from a vaccine sample.

42. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-8 or the conjugate thereof of any one of claims 23-28 in the preparation of a kit for detecting the presence or level of beta coronavirus in a sample.

43. The use of claim 42, wherein the antibody or its antigen-binding fragment is of two, three, four or five types.

44. The use of claim 43, wherein the antibody or antigen-binding fragment thereof is of two types, wherein one antibody or antigen-binding fragment thereof is as described in claim (5) of any one of claims 1-8, and the heavy chain variable region of the other antibody or antigen-binding fragment thereof comprises: VH CDR1, which consists of the amino acid sequence shown in SEQ ID NO:161, VH CDR2, which consists of the amino acid sequence shown in SEQ ID NO: 162, and VH CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 163; The variable region of the light chain contains: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO: 164, VL CDR2, which consists of the amino acid sequence shown in SEQ ID NO: 165, and VL CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 166; or Another antibody or its antigen-binding fragment has a heavy chain variable region consisting of the sequence shown in SEQ ID NO: 167; and a light chain variable region consisting of the sequence shown in SEQ ID NO:

168.

45. The use of claim 44, wherein another antibody or antigen-binding fragment thereof comprises a heavy chain constant region as shown in SEQ ID NO: 169 and a light chain constant region as shown in SEQ ID NO: 170.

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