WO2023151312A1

WO2023151312A1 - Betacoronavirus broad-spectrum neutralizing antibody and use thereof

Info

Publication number: WO2023151312A1
Application number: PCT/CN2022/126628
Authority: WO
Inventors: 谢晓亮; 曹云龙
Original assignee: 北京昌平实验室
Priority date: 2022-02-14
Filing date: 2022-10-21
Publication date: 2023-08-17
Also published as: CN116621972A; TW202342513A

Abstract

The present invention relates to the field of immunology, in particular to a betacoronavirus broad-spectrum neutralizing antibody and a use thereof. Specifically, the present invention relates to the betacoronavirus broad-spectrum neutralizing antibody or an antigen-binding fragment thereof, and a use of the antibody or the fragment for diagnosis, prevention and treatment of various diseases caused by betacoronavirus infection.

Description

B-coronavirus broad-spectrum neutralizing antibody and its application

technical field

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

Background technique

Coronaviruses (CoVs) are the largest RNA viruses identified so far, which can infect mammalian humans, mice, pigs, cats, dogs, bats, cattle, and avian vertebrates, causing respiratory, intestinal, liver, and nervous system damage in hosts. disease. Coronaviridae is divided into 4 genera, namely α, β, δ and γ coronaviruses, among which β is the most harmful to humans. Betacoronavirus is divided into βcoronavirus genus and five subgenus, namely Embecovirus, Sarbecovirus, Merbecovirus, Nobecovirus and Hibecovirus. The subgenus of beta coronavirus (Sarbecovirus) includes the new coronavirus (SARS-CoV-2) and its variants, severe acute respiratory syndrome coronavirus (SARS-CoV) and its variants, SARS-related Coronavirus (SARSr-CoV), subdivided as shown in Figure 1 (DOI: 10.1038/s41586-020-2294-9).

Betacoronavirus, as a single-stranded RNA virus, translates and replicates directly in cells, and is more prone to mutation than DNA viruses. Since the outbreak of SARS-CoV-2 at the end of 2019, a variety of mutant strains have been produced, among which the World Health Organization has listed as Variant of concern (VOC), which has reached 5 types: Alpha (Alpha, B. 1.1.7), Beta (Beta, B.1.351), Gamma (Gamma, P.1), Delta (Delta, B.1.617.2) and Omicron (B.1.1.529). Due to changes in the genetic sequence of these mutant strains, the structure of the spike protein on the surface of the virus changes, thereby affecting the ability of the virus to be recognized by antibodies or the affinity of the virus to the receptor, resulting in a reduction or failure of the neutralizing ability of antibodies produced after vaccination. Risks such as enhanced virus transmission ability and changes in virus pathogenic mechanism have added many difficulties to epidemic prevention and fighting. Continuous mutations may bring greater risks of epidemics in the future.

Therefore, it is necessary to find more effective broad-spectrum neutralizing antibodies against B-coronavirus including the above-mentioned variant strains, and provide effective means for diagnosing, preventing and/or treating variant strain infections, so as to help stabilize and eliminate the epidemic.

The present invention applies techniques in many fields such as immunology, molecular biology, virology, protein structure analysis, etc., develops a set of unique technical routes, and obtains a series of B-coronavirus broad-spectrum neutralizing antibodies. These antibodies come from the memory B cells of SARS-CoV-infected recovered patients who have been vaccinated with SARS-CoV-2 vaccines. They have the characteristics of broad-spectrum high-binding activity and broad-spectrum high neutralization of B-coronaviruses, and can efficiently neutralize novel coronaviruses, including novel coronaviruses. Viruses, atypical coronaviruses and other pathogenic viruses are especially suitable for the diagnosis, prevention and treatment of various diseases caused by B-coronavirus infection, and have important clinical value.

Through the optimized high-throughput yeast display technology, the present invention successfully predicts the binding epitope and escape map of these neutralizing antibodies on B-coronavirus, and finds antibody pairs based on this information, which is verified by cryo-electron microscopy. Thus the sequences of these antibodies were reported for the first time.

Summary of the invention

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

In some embodiments, the heavy and light chain variable regions comprise specific amino acid sequences.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a constant region derived from a human immunoglobulin, eg, a heavy chain constant region and a light chain constant region comprising specific amino acid sequences.

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

In a second aspect, the invention also provides an isolated nucleic acid molecule encoding an antibody of the 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 nucleic acid molecule is operably linked to an expression control sequence.

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

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

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

(1) cultivating 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

(2) Isolating and purifying the antibody or antigen-binding fragment thereof expressed from the nucleic acid molecule or expression vector.

In the sixth aspect, the present invention also provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof of the present invention, and a pharmaceutically acceptable carrier and/or excipient.

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

In the eighth aspect, the present invention also provides the use of the antibody or antigen-binding fragment thereof of the present invention for preparing a medicament for preventing and/or treating diseases caused by B-coronavirus infection.

In some embodiments, the beta coronavirus includes novel coronavirus (SARS-CoV-2) and variants thereof, severe acute respiratory syndrome coronavirus (SARS-CoV) and variants thereof, SARS-related coronaviruses (SARSr-CoV).

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

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

In some embodiments, the detectable label is selected from enzymes (such as horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (such as acridinium esters, luminol and derivatives thereof, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or biotin.

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

In some embodiments, the kit comprises a conjugate of the invention.

In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof of the invention, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof.

In some embodiments, the second antibody further includes a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium esters, luminol, and derivatives thereof, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or biotin.

In the eleventh aspect, the present invention also provides a method for detecting the presence or level of B-coronavirus in a sample, comprising:

(1) contacting the sample with an antibody or antigen-binding fragment thereof of the invention or a conjugate of the invention;

(2) detecting the binding of the antibody or antigen-binding fragment or conjugate thereof to the target antigen in the sample.

In some embodiments, detection of the binding represents the presence of B-coronavirus in the sample.

In some embodiments, the detection of the strength of the binding represents the level of the B-coronavirus in the sample.

In some embodiments, the sample is a blood sample (eg, whole blood, plasma, or serum), feces, oral or nasal secretions, or alveolar lavage fluid from the subject.

In some embodiments, the subject is a mammal, such as a human.

In some embodiments, the sample is not a sample from a subject, eg, the sample is from a vaccine sample.

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

Antibodies or antigen-binding fragments thereof in the present invention should be understood as more than one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 kinds, 13 kinds, 14 kinds, 15 kinds, 16 kinds, 17 kinds, 18 kinds, 19 kinds, 20 kinds, 21 kinds of combinations. In addition, the antibodies or antigen-binding fragments thereof of the present invention can also be combined with other antibodies or antigen-binding fragments thereof.

In some embodiments, the antibody or antigen-binding fragment thereof is a combination of 2 specific antibodies or antigen-binding fragments thereof, such as a combination of BD55-5840 and BD55-5514.

Description of drawings

Figure 1 shows the sub-genre breakdown of Sarbecovirus.

Figure 2 shows the body weight change curve of mice in the SARS-CoV-2 (Omicron BA.1) challenge experiment.

Figure 3 shows the viral load of mouse tissues and organs in the SARS-CoV-2 (Omicron BA.1) challenge experiment.

Figure 4 shows the body weight change curve of mice in the SARS-CoV-2 (Omicron BA.5) challenge experiment.

Figure 5 shows the virus load of mouse tissues and organs in the SARS-CoV-2 (Omicron BA.5) challenge experiment.

Figure 6 shows the cryo-EM structure of BD55-5514+BD55-5840 combined with BA.1 Spike.

Figure 7 shows the binding epitope of BD55-5514 on the BA.1 RBD.

Figure 8 shows the binding epitope of BD55-5840 on the BA.1 RBD.

Detailed description of the invention

1. Definition

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation steps used herein are all terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether native or partially or wholly synthetically (e.g., recombinantly) produced, including retained full-length immunoglobulins comprising at least part of the variable region of an immunoglobulin molecule. Any fragment of the binding specificity of a globulin. Thus, an antibody includes any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding domain (antibody combining site). Antibodies include antibody fragments. As used herein, the term antibody includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments, For example, 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), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the foregoing. Antibodies provided herein include any immunoglobulin class (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) members.

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 includes at least a portion of the variable region (e.g., one or more CDRs and/or one or more antibody combining sites), and thus retain binding specificity and at least part of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that comprises an antigen-binding portion that binds the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as derivatives produced synthetically, eg, recombinantly. 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, diabodies, 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 fragments may comprise multiple chains linked together, for example by disulfide bonds and/or by peptide linkers. Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any fragment of an antibody which, when inserted into an antibody framework (e.g., by replacing the corresponding region), results in an antibody that immunospecifically binds (i.e. exhibits a Ka of at least or at least about 10 ⁷ -10 ⁸ M ⁻¹ ) an antigen .

As used herein, "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in the population of monoclonal antibodies is identical to the other antibody molecules. This property is in contrast to that of polyclonal populations of antibodies, which comprise antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by many 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 by immortalizing B cells, eg, by fusing with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies in vitro from clonal populations of host cells by transforming the host cells with plasmids carrying artificial sequences of nucleotides encoding the antibodies.

As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (typically a myeloma or lymphoma cell) resulting from the fusion of antibody-producing lymphocytes and non-antibody-producing cancer cells. As is known to those of ordinary skill in the art, hybridomas can proliferate and sustain production of specific monoclonal antibodies. Methods for producing hybridomas are known in the art (see, eg, Harlow & Lane, 1988). When the term "hybridoma" or "hybridoma cell" is referred to, it also includes subclones and progeny cells of the hybridoma.

As used herein, "conventional antibody" refers to an antibody comprising two heavy chains (which may be designated as H and H') and two light chains (which may be designated as L and L') and two antigen binding sites, wherein Each heavy chain may be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen binding ability (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 can be a full-length light chain or any functional region (eg, 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).

As used herein, a full-length antibody is one that has 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. Antibodies, such as those produced naturally by antibody-secreting B cells, as well as antibodies produced synthetically with the same domain.

As used herein, dsFv refers to a Fv with an engineered intermolecular disulfide bond that stabilizes the VH-VL pair.

As used herein, a Fab fragment is an antibody fragment obtained by papain digestion of a full-length immunoglobulin, or a fragment having the same structure produced synthetically, for example, by recombinant methods. The Fab fragment comprises a light chain (comprising VL and CL) and another chain comprising the variable domain (VH) of the heavy chain and one constant region domain (CH1) of the heavy chain.

As used herein, an F(ab') ₂ fragment is an antibody fragment resulting from pepsin digestion of an immunoglobulin at pH 4.0-4.5, or a fragment having the same structure produced synthetically, eg, by recombinant methods. An F(ab')2 fragment essentially consists of two Fab fragments, where each heavy chain portion contains an additional few amino acids, including a cysteine that forms a disulfide bond linking the two fragments.

As used herein, a Fab' fragment is a fragment comprising half (one heavy chain and one light chain) of an F(ab') ₂ fragment.

As used herein, a scFv fragment refers to an antibody fragment comprising a variable light chain ( _VL ) and a variable heavy chain ( _VH ) covalently linked by a polypeptide linker in any order. The length of the linker is such that the two variable domains are bridged substantially without interference. An exemplary linker is (Gly-Ser) _n residues interspersed with some Glu or Lys residues to increase solubility.

The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody antibodies.

"Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab') ₂ or Antigen-binding subsequences of antibodies), containing minimal sequence derived from non-human immunoglobulins. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the complementarity determining regions (CDRs) of the recipient antibody are derived from a non-human species having the desired specificity, affinity and capacity ( Donor antibody) such as mouse, rat or rabbit CDR residue substitutions.

In addition, in humanization, it is also possible to mutate amino acid residues within the CDR1, CDR2 and/or CDR3 regions of the VH and/or VL, thereby improving one or more binding properties (e.g., affinity) of the antibody . Mutations can be introduced, for example by PCR-mediated mutagenesis, whose effect on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations may be amino acid substitutions, additions or deletions. In addition, there are usually no more than one or two mutations within a CDR. Therefore, the humanized antibody of the present invention also covers an antibody containing 1 or 2 two amino acid mutations in the CDR.

As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually comprise chemically active surface types of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.

As used herein, a variable domain or variable region is a specific Ig domain of an antibody heavy or light chain comprising an amino acid sequence that varies from antibody to antibody. Each light chain and each heavy chain has one variable region domain, VL and VH, respectively. The variable domains provide antigen specificity and are thus responsible for antigen recognition. Each variable region comprises CDRs and framework regions (FRs), the CDRs being part of the antigen binding site domain.

As used herein, "antigen-binding domain" and "antigen-binding site" are used synonymously to refer to the domain within an antibody that recognizes and physically interacts with a cognate antigen. A natural conventional full-length antibody molecule has two conventional antigen binding sites, each comprising a heavy chain variable region portion and a light chain variable region portion. The conventional antigen binding site comprises loops linking antiparallel beta strands within the variable region domains. The antigen binding site may comprise other portions of the variable region domain. Each conventional antigen binding site contains 3 hypervariable regions from the heavy chain and 3 hypervariable regions from the light chain. Hypervariable regions are also known as complementarity determining regions (CDRs).

As used herein, "hypervariable region", "HV", "complementarity determining region", "CDR" and "antibody CDR" are used interchangeably to refer to the regions within each variable region that together form the antigen binding site of an antibody One of many parts. Each variable region domain contains 3 CDRs, named CDR1, CDR2 and CDR3. For example, the light chain variable region domain contains 3 CDRs, named VL CDR1, VL CDR2, and VL CDR3; the heavy chain variable region domain contains 3 CDRs, named VH CDR1, VH CDR2, and VH CDR3. The three CDRs in the variable region are not contiguous along the linear amino acid sequence, but are close together in the folded polypeptide. The CDRs are located within loops connecting the parallel strands of the beta sheets of the variable domains. As described herein, those skilled in the art know and can identify CDRs based on Kabat or Chothia numbering (see, e.g., Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).

As used herein, the framework regions (FRs) are the domains within the antibody variable region domains located within the beta sheet; the FR regions are relatively more conserved than the hypervariable regions in terms of amino acid sequence.

As used herein, a "constant region" domain is a domain in an antibody heavy or light chain that comprises a relatively more conserved amino acid sequence than that of a variable region domain. In conventional full-length antibody molecules, 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 isotypes comprise CH1, CH2, CH3 and the hinge region, while IgE and IgM comprise CH1, CH2, CH3 and CH4. The CH1 and CL domains extend the Fab arms of the antibody molecule, thus facilitating interaction with the antigen as well as turning the antibody arms. Antibody constant regions may serve effector functions such as, but not limited to, clearance of antigens, pathogens and toxins to which the antibody specifically binds, such as by interacting with various cells, biomolecules and tissues.

As used herein, a functional region of an antibody is a portion of an antibody comprising at least the VH, VL, CH (eg, CH1, CH2 or CH3), CL or hinge domain of the antibody, or at least a functional region thereof.

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 VH A functional region of a domain binds antigen alone or in combination with another antibody domain (eg a VL domain) or region thereof. Exemplary functional regions of the VH domain are regions comprising CDR1, CDR2 and/or CDR3 of the VH domain.

As used herein, a functional region of a VL domain is at least a portion of a complete VL domain that retains at least a portion of the binding specificity of the complete VL domain (e.g., by retaining one or more CDRs of the complete VL domain), such that the VL domain A functional region of a domain binds antigen alone or in combination with another antibody domain (eg a VH domain) or region thereof. Exemplary functional regions of the VL domain are regions comprising CDR1, CDR2 and/or CDR3 of the VL domain.

As used herein, "specifically binds" or "immunospecifically binds" with respect to an antibody or antigen-binding fragment thereof is used interchangeably herein and refers to the passage of the antibody or antigen-binding fragment between the antibody-binding site of the antibody and the antigen. The ability of non-covalent interactions to form one or more non-covalent bonds with the same antigen. The antigen may be an isolated antigen or present in a biological sample. Typically ^, ^an antibody that immunospecifically binds (or specifically binds ⁾ an antigen has an affinity constant Ka (or 1 x ^{10 -7} ^M or A dissociation constant (K _d ) of 1×10 ^-8 M or lower) binds the antigen. Affinity constants can be determined by standard kinetic methods of antibody reactions, for example, immunoassays, 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); see also US Patent No. 7,229,619 describing exemplary SPR and ITC methods for calculating the binding affinity of antibodies). Instruments and methods for real-time detection and monitoring of association rates are known and commercially available (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem.Soc.Trans.27 :335).

As used herein, the term "compete" with respect to antibodies means that a first antibody or antigen-binding fragment thereof binds an epitope in a manner sufficiently similar to a second antibody or antigen-binding fragment thereof such that the first antibody binds to its cognate epitope. Binding results are detectably reduced in the presence of the second antibody compared to the absence of the second antibody. Alternatively, this may, but need not be the case, where the binding of the second antibody to its epitope is also detectably reduced in the presence of the primary antibody. That is, a first antibody can inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody to its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to "cross-compete" binding with each other their respective epitopes. Both competing and cross-competing antibodies are encompassed by the 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 appreciate that such competing and/or cross-competing antibodies are based on the teachings provided herein. are contemplated by the present invention and may be used in the methods disclosed herein.

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

"Isolated protein," "isolated polypeptide," or "isolated antibody" means a protein, polypeptide, or antibody that: (1) is not associated with its naturally associated components in its native state, (2) is free from the same Other proteins of the species, (3) are expressed by cells from a different species, or (4) do not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which the polypeptide is naturally derived will be "isolated" from its naturally associated components. Proteins can also be rendered substantially free of naturally associated components by isolation, ie, using protein purification techniques well known in the art.

In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Generally, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub .co., p. 224).

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions for amino acid residues with amino acid residues that have similar side chains, e.g., are physically or functionally similar (e.g., have similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc.) Families of amino acid residues having similar side chains have been defined in the art. These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acid, proline, phenylalanine, methionine), beta branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine) amino acids. Therefore, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, e.g., 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).

The writing of the twenty conventional amino acids referred to herein follows conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present 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.

As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including nucleotides linked together usually by phosphodiester bonds. Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

As used herein, an isolated nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules that exist in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when prepared by recombinant techniques, 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 antibodies or antigen-binding fragments.

Sequence "identity" has an art-recognized meaning, and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide, or along a region of the molecule. (See, for example: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., 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., M Stockton Press, New York, 1991). Although there are many methods of measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ).

As used herein, "operably linked" with reference to nucleic acid sequences, regions, elements or domains means 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 such that the promoter regulates or mediates transcription of the nucleic acid.

As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Reference to vectors includes those into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction digestion and ligation. Reference to vectors also includes those vectors comprising a nucleic acid encoding a polypeptide. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplifying nucleic acids or expressing/displaying polypeptides encoded by nucleic acids. Vectors usually remain episomal, but can be designed to allow integration of a gene, or part thereof, into the chromosome of the genome. Also contemplated are vectors for artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art.

As used herein, vector also includes "viral vector" or "viral vector". Viral vectors are engineered viruses that are operably linked to foreign genes to transfer (either as vehicles or shuttles) foreign genes into cells.

As used herein, "expression" refers to the process by which a polypeptide is produced by the transcription and translation of a polynucleotide. Expression levels of a polypeptide can be assessed by any method known in the art, including, for example, methods for determining the amount of polypeptide produced by a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

As used herein, "expression vector" includes a vector capable of expressing DNA operably linked to regulatory sequences, such as a promoter region, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which, when introduced into an appropriate host cell, results in the expression of cloned DNA. Appropriate expression vectors are well known to those skilled in the art and include expression vectors replicable in eukaryotic cells and/or prokaryotic cells as well as expression vectors that remain episomal or integrate into the host cell genome.

"Codon optimization" refers to the replacement of at least one codon of a native sequence (e.g. about or more than about 1, 2, 3, 4, 5 , 10, 15, 20, 25, 50 or more codons while maintaining the native amino acid sequence and modifying the nucleic acid sequence so as to enhance expression in the host cell of interest. Different species have certain codes for specific amino acids Codons exhibit specific preferences. Codon bias (differences in codon usage between organisms) is often associated with messenger RNA (mRNA) translation efficiency, which is thought to depend on the number of codons being translated The nature and availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs within a cell generally reflects the codons most frequently used for peptide synthesis. Thus, genes can be tailored based on codon optimization in a given organism Optimal gene expression for optimal gene expression. Codon usage tables are readily available, for example, in the Codon Usage Database ("Codon Usage Database") available at www.kazusa.orjp/codon/ , and these tables can be accessed through various Modal adjustments apply. 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).

As used herein, a "host cell" is a cell for receiving, maintaining, replicating and amplifying a vector. Host cells can also be used to express polypeptides encoded by vectors. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. Host cells can be eukaryotic or prokaryotic. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient compatible with the subject and the active ingredient pharmacologically and/or physiologically, These are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ions Strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents 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, and the like. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (eg, buffered saline), alcohols and polyols (eg, glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizer has the meaning generally understood by those skilled in the art, and it can stabilize the desired activity of the active ingredient in the medicine, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (eg, aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (eg 0.9% (w/v) NaCl), dextrose solutions (eg, 5% dextrose), solutions containing surfactants (eg, 0.01% polysorbate 20), pH buffered solutions (eg, phosphate buffered saline), Ringer's solutions, and any combination thereof.

As used herein, the term "prevention" refers to methods performed to prevent or delay the occurrence of a disease or disorder or symptom (eg, SARS-CoV-2 infection) in a subject. As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical result. For the purposes of this invention, a beneficial or desired clinical outcome includes, but is not limited to, relief of symptoms, reduction of the extent of the disease, stabilization (i.e., no longer worsening) of the disease state, delay or slowing of the progression of the disease, amelioration or palliation of the disease status, and relief of symptoms (whether partial or total), whether detectable or undetectable. In addition, "treating" can also refer to prolonging survival as compared to expected survival if not receiving treatment.

As used herein, "therapeutic effect" means the effect resulting from a treatment in an individual that alters, usually ameliorates or ameliorate the symptoms of, or cures a disease or condition.

As used herein, "prophylactically effective amount" or "prophylactically effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., prevent or delay a disease or symptom occurrence or recurrence of the disease or symptoms, and to reduce the likelihood of occurrence or recurrence of the disease or symptoms. A full prophylactically effective dose does not have to occur by administering one dose, and can only occur after administering a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect when administered to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest or partially arrest the symptoms of a disease or disorder.

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

As used herein, "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)", formerly known as "new coronavirus" or "2019-nCov", belongs to its beta coronavirus Viruses are enveloped, single-stranded, positive-sense RNA viruses. SARS-CoV-2 contains at least three membrane proteins, including surface spike protein (S), integral membrane protein (M) and membrane protein (E). The receptor of SARS-CoV-2 is the same as that of SARS-CoV, through the receptor binding domain (RBD) on the S protein to specifically bind to angiotensin transferase 2 (ACE2) on the host cell After receiving the membrane fusion and cell entry of the virus, it plays a vital role in the process of virus infection of cells.

In this paper, the term "SARS-CoV-2" includes various known isolates, such as both the original strain (such as the first sequenced isolate GenBank: MN908947.3), and the subsequently discovered mutant strain. In certain embodiments, the term "SARS-CoV-2" encompasses both isolates whose S protein does not contain a mutation (for example, compared to the reference strain MN908947.3) and includes a mutation in its S protein (for example, compared to Amino acid substitutions compared to the reference strain MN908947.3, eg K417N, E484K, N501Y, L452R, T478K or any combination thereof). In certain embodiments, the variant strain is preferably selected from isolates comprising mutations (eg amino acid substitutions such as K417N, E484K, N501Y, L452R, T478K or any combination thereof) in their S protein. In some exemplary embodiments, the mutant strain is selected from Alpha (Alpha, B.1.1.7), Beta (Beta, B.1.351), Gamma (Gamma, P.1), Delta (Delta, B.1.617.2) and Omicron (Omicron, B.1.1.529).

As used herein, the terms "new coronavirus pneumonia" and "COVID-19" refer to pneumonia caused by SARS-CoV-2 infection, both have the same meaning and can be used interchangeably.

As used herein, the term "neutralizing activity" means that the antibody or antibody fragment has the ability to bind to the antigenic protein on the virus, thereby preventing the virus from infecting the cells and/or the maturation of the virus progeny and/or the release of the virus progeny The functional activity of the antibody or antibody fragment with neutralizing activity can prevent the amplification of the virus, thereby inhibiting or eliminating the infection of the virus.

2. Antibodies or antigen-binding fragments thereof

The present invention provides an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region comprising complementarity determining regions (CDRs) of specific amino acid sequences, Or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions or additions relative to a specific amino acid sequence.

In some embodiments, the heavy and light chain variable regions comprise specific amino acid sequences or variants thereof. wherein said variant has a substitution, deletion or addition of one or several amino acids, such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions, compared to the sequence from which it is derived, or have 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% Sequences with sequence identity; preferably, said substitutions are conservative substitutions.

The information of the partial sequences involved in the present invention is provided in the following Table 1, specifically related to 21 kinds of antibodies (numbering respectively 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, BD55-5840) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, VL CDR3 sequences and VH, VL sequences. These 21 antibodies have common CH (SEQ ID NO: 169) and CL (SEQ ID NO: 170) sequences.

Table 1: Description of the sequences

3. Antibody preparation

The antibodies of the present invention can be prepared by various methods known in the art, for example, by genetic engineering and recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. Then, the transfected host cells are cultured under specific conditions, and express the antibody of the present invention.

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)) . In addition, these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000 )). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab') ₂ fragments (Carter et al., Bio/Technology, 10:163-167 (1992)). In addition, Fv, Fab or F(ab') ₂ fragments can also be directly isolated from the culture medium of recombinant host cells. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.

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

In some embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention and a first nucleotide sequence encoding the antibody or antigen-binding fragment thereof. The light chain or the second nucleotide sequence of the light chain variable region, wherein said first nucleotide sequence and said 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 second nucleotide sequence comprising the first nucleotide sequence. A nucleic acid molecule and a second nucleic acid molecule comprising the second nucleotide sequence.

In another aspect, the invention provides a vector (eg, a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above. In some embodiments, vectors of the invention are, for example, plasmids, cosmids, phage, and the like.

In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of an antibody of the invention or an antigen-binding fragment thereof and a light chain or a light chain encoding the antibody or an antigen-binding fragment thereof The second nucleotide sequence of the light chain variable region, 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 exist on different vectors, the vector of the present invention comprises a first vector containing the first nucleotide sequence and a vector containing the A second vector of the second nucleotide sequence.

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

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

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

In another aspect, there is provided a method for preparing the antibody of the present invention or an antigen-binding fragment thereof, comprising culturing the host cell of the present invention under the expression of a nucleic acid molecule or expression vector suitable for the present invention, and isolating and purifying the antibody obtained from the present invention. Antibodies or antigen-binding fragments thereof expressed by the nucleic acid molecules or expression vectors.

4. Pharmaceutical composition

As used herein, "pharmaceutically acceptable carrier and/or excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible . Preferably, the carrier and/or excipient are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg by injection or infusion). Depending on the route of administration, the active compound, ie, antibody molecule, immunoconjugate, may be encapsulated in a material to protect the compound from acids and other natural conditions that would render the compound inactive.

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 ascorbic acid palmitate esters, 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.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.

Prevention of the presence of microorganisms can be ensured by sterilization procedures or by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. In many cases it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium oxide in the composition. Prolonged absorption of injectable drugs can be brought about by including in the composition agents which delay absorption, for example, monostearate salts and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Conventional media or agents, to the extent they are incompatible with the active compounds, are possible in the pharmaceutical compositions of the present invention. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions generally must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by the use of surfactants.

Sterile injectable solutions can be prepared by mixing the active compound in the required amount in an appropriate solvent and, as required, adding one or a combination of ingredients enumerated above, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient which can be combined with carrier materials to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, based on 100%, this amount will range from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%, and pharmaceutically acceptable combination of carriers.

Dosage regimens can be adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to result in combination with the required pharmaceutical carrier. desired therapeutic effect. The particular specifications for the dosage unit forms of the invention are limited by and directly dependent upon (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such for the treatment of sensitivity in an individual. Active compound restrictions.

For administration of antibody molecules, dosages range from about 0.0001 to 100 mg/kg, more typically 0.01 to 20 mg/kg of recipient body weight. For example, dosages 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 within the range of 1-20 mg/kg. Exemplary treatment regimens entail weekly dosing, once every two weeks, once every three weeks, once every four weeks, once monthly, once every 3 months, once every 3-6 months, or an initial dosing interval Slightly shorter (eg once a week to once every three weeks) followed by longer dosing intervals (eg monthly to once every 3-6 months).

Alternatively, antibody molecules of the invention may also be administered as a sustained release formulation, in which case less frequent dosing is required. Dosage and frequency vary according to the half-life of the antibody molecule in the patient. In general, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. Dosage and frequency of administration will vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively lower doses are administered at infrequent intervals over a prolonged period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes desirable to administer higher doses at shorter intervals until the progression of the disease is reduced or stopped, preferably until the patient exhibits partial or complete amelioration of disease symptoms. Thereafter, the patient can be dosed in a prophylactic regimen.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, and the particular Other drugs, compounds and/or materials with which the composition is used in conjunction, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical field.

The antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention can be administered by one or more routes of administration using one or more methods known in the art. Those skilled in the art will appreciate that the route and/or manner of administration will vary depending on the desired result. Preferred routes of administration for antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intrasaccular, intraorbital, Intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

Alternatively, the antibody or antigen-binding fragment thereof of the invention or the pharmaceutical composition of the invention may also be administered by non-parenteral routes, such as topical, epidermal or mucosal routes, for example, intranasally, orally, vaginally, rectally, Sublingual or topical.

The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

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 may be administered with a needle-free hypodermic injection device, such as the devices disclosed in US Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; Examples of known implants and modules that can be used in the present 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 Patents disclose therapeutic devices for drug delivery through the skin; U.S. Patent No. 4,447,233, which discloses a medical infusion pump for delivering drugs at precise infusion rates; U.S. Patent No. 4,447,224, which discloses the use of Variable Flow Implantable Infusion Devices for Continuous Drug Delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with multiple chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems and modules are known to those skilled in the art.

In some embodiments, antibodies of the 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 invention are able to cross the BBB (if desired), they can be formulated, eg, in liposomes. For methods of preparing liposomes, see, eg, US 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, e.g., V.V. Ranade (1989) J. Clin. Pharmacol. 29:685 ). Examples of targeting moieties include folic acid or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al); mannosides (Umezawa et al (1988) Biochem. 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; J.J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

In certain exemplary embodiments, the pharmaceutically acceptable carrier and/or excipient comprises a sterile injectable liquid (eg, aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (eg 0.9% (w/v) NaCl), dextrose solutions (eg, 5% dextrose), solutions containing surfactants (eg, 0.01% polysorbate 20), pH buffered solutions (eg, phosphate buffered saline), Ringer's solutions, and any combination thereof.

5. Disease prevention and/or treatment purposes

The antibody of the present invention or its antigen-binding fragment can be used to neutralize B-coronavirus in vitro or in a subject, block or inhibit the infection of cells by B-coronavirus, so as to prevent and/or treat B-coronavirus in a subject beta-coronavirus infection or diseases associated with beta-coronavirus infection.

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

In some embodiments, the SARS-CoV-2 includes variant strains. In some embodiments, the S protein of the mutant strain contains mutations, such as amino acid substitutions, deletions or additions. In some embodiments, the S protein of the mutant strain comprises one or more amino acid substitutions selected from K417N, E484K, N501Y, L452R, and T478K. In some embodiments, the mutant strain of SARS-CoV-2 is selected from Alpha (Alpha, B.1.1.7), Beta (Beta, B.1.351), Gamma (Gamma, P.1), Delta ( Delta, B.1.617.2), Omicron (B.1.1.529) or any combination thereof.

In some embodiments, the subject is preferably a mammal, more preferably a human.

In some embodiments, the antibody or antigen-binding fragment thereof, or the pharmaceutical composition is used alone, or in combination with another pharmaceutically active agent (eg, another antiviral agent). The antibody or antigen-binding fragment thereof of the invention, or the pharmaceutical composition of the invention and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.

In another aspect, the present invention provides a method for neutralizing B-coronavirus, blocking or inhibiting the binding of B-coronavirus to ACE2 receptor, or blocking or inhibiting the infection of B-coronavirus to cells, which Included are the use of antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the invention. The method can be used in vitro or in a subject (such as a human) to neutralize B-coronavirus, block or inhibit the binding of B-coronavirus to ACE2 receptors, or block or inhibit the infection of B-coronavirus to cells .

In some embodiments, the method is used to neutralize the virulence of B-coronavirus in the sample, block or inhibit the binding of B-coronavirus to ACE2 receptor, or block or inhibit the binding of B-coronavirus to cells Infect. In some embodiments, the method comprises: contacting a sample comprising a betacoronavirus with an antibody or antigen-binding fragment thereof or a pharmaceutical composition of the invention.

In another aspect, the present invention relates to the use of the antibody of the present invention or its antigen-binding fragment for the preparation of a medicament for the prevention and/or treatment of diseases caused by B-coronavirus infection, and the medicament is used for one or more of the following item:

(1) neutralizing B-coronavirus in vitro or in a subject (such as a human);

(2) Blocking or inhibiting the binding of B-coronavirus to ACE2 receptor;

(3) blocking or inhibiting the infection of cells by B-coronavirus; and/or

(4) For the prevention and/or treatment of B-coronavirus infection or diseases related to B-coronavirus infection (such as COVID-19) in subjects.

The antibody or antigen-binding fragment thereof of the present invention, or the pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, Powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injections and concentrated solutions for injections), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. Antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating in an appropriate solvent the necessary dose of an antibody or antigen-binding fragment thereof of the present invention and, optionally, concomitantly incorporating other desired ingredients including, but not limited to pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filter sterilization. In addition, sterile injectable solutions can be prepared as sterile lyophilized powder (eg, by vacuum drying or freeze-drying) for ease of storage and use. Such sterile lyophilized powders can be dispersed in suitable carriers before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w/v) NaCl), Dextrose solution (eg 5% glucose), surfactant containing solution (eg 0.01% polysorbate 20), pH buffer solution (eg phosphate buffer solution), Ringer's solution and any combination thereof.

The antibodies of the invention or antigen-binding fragments thereof, or the pharmaceutical compositions of the invention may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ophthalmic, topical, parenteral, Rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (eg, powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (eg, intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will understand that the route and/or manner of administration will vary depending on the intended purpose. In some embodiments, an antibody or antigen-binding fragment thereof or pharmaceutical composition of the invention is administered by intravenous injection or bolus injection.

Herein, dosing regimens may be adjusted to obtain the optimum desired response (eg, a therapeutic or prophylactic response). For example, a single dose can be administered, multiple doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

6. Conjugates

An antibody or antigen-binding fragment thereof of the invention can be derivatized, eg, linked to another molecule (eg, another polypeptide or protein). Typically, derivatization (eg, labeling) of an antibody or antigen-binding fragment thereof will not adversely affect its binding to B-coronavirus. Accordingly, the antibodies or antigen-binding fragments thereof of the invention are also intended to include such derivatized forms. For example, an antibody of the invention or an antigen-binding fragment thereof can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or otherwise) to one or more other molecular moieties, such as another antibody (e.g., forming bispecific antibodies), detection reagents, pharmaceutical reagents, and/or proteins or polypeptides capable of mediating the binding of an antibody or antigen-binding fragment to another molecule (eg, an avidin or polyhistidine tag). In addition, the antibodies or antigen-binding fragments thereof of the invention may also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl groups, or sugar groups. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life.

Accordingly, in some embodiments, an antibody or antigen-binding fragment thereof of the invention is detectably labeled.

Herein, the detectable label of the present invention can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, examples of which include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclide Chlorine (for example, ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C or ³² P), fluorescent dyes (for example, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC) , phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa 750)), luminescent substances (such as chemiluminescent substances, such as acridine ester compounds, Lu Minol and its derivatives, ruthenium derivatives such as ruthenium terpyridine), magnetic beads (for example,

), calorimetric markers such as colloidal gold or colored glass or plastic (eg, polystyrene, polypropylene, latex, etc.) beads, and avidin (eg, streptavidin ) of biotin.

In some embodiments, the detectable label can be adapted for immunological detection (eg, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable label may be selected from enzymes (such as horseradish peroxidase, alkaline phosphatase, or β-galactosidase), chemiluminescent reagents (such as acridinium esters, lucid Minol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent proteins, such as FITC, TRITC or PE), radionuclides or biotin.

In some embodiments, a detectable label as described above can be attached to an antibody or antigen-binding fragment thereof of the invention via linkers of various lengths to reduce potential steric hindrance.

7. Reagent kits and testing purposes

The antibody of the present invention or its antigen-binding fragment can specifically bind to the RBD of the S protein of the B-coronavirus, so that it can be used to detect the RBD of the B-coronavirus or its S protein or S protein, and optionally according to the above detection results Diagnose whether the subject is infected with B-coronavirus.

Thus, in another aspect, the invention provides a kit comprising an antibody of the invention or an antigen-binding fragment thereof, or a conjugate of the invention.

In some embodiments, the kit comprises a conjugate of the invention.

In other embodiments, the kit comprises an antibody or antigen-binding fragment thereof of the invention. In some embodiments, the antibody or antigen-binding fragment thereof does not comprise a detectable label. In some embodiments, the kit further comprises a second antibody that specifically recognizes the antibody of the invention or an antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., paprika root peroxidase or alkaline phosphatase), chemiluminescent reagents (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radioactive nuclei biotin or biotin.

In some embodiments, the second antibody is specific for an antibody of the species (eg, human) from which the constant region comprised by the antibody or antigen-binding fragment thereof of the invention is derived.

In some embodiments, the second antibody is an anti-immunoglobulin (eg, human immunoglobulin) antibody, eg, an anti-IgG antibody. In some embodiments, the second antibody is an anti-human IgG antibody.

In some embodiments, the kits of the invention may further comprise reagents for allowing the corresponding detectable label to be detected. 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), tetramethylbenzyldiamine (OPD) for horseradish peroxidase, Aniline (TMB), ABTS or luminol-like compounds, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase. For example, when the detectable label is a chemiluminescence reagent (such as an acridinium ester compound), the kit may also include a pre-excitation solution and/or an excitation solution for chemiluminescence.

In another aspect, the present invention provides a method for detecting the presence or level of a betacoronavirus or its S protein or the RBD of the S protein, or cells infected with a betacoronavirus in a sample, comprising making the sample contacting an antibody or antigen-binding fragment or conjugate thereof of the invention; detecting binding of said antibody or antigen-binding fragment or conjugate thereof to a target antigen in said sample; wherein detecting said binding represents said B-coronavirus exists in the sample, or the level of B-coronavirus in the sample is represented by detecting the strength of the combination.

In some embodiments, the method is an immunological assay, such as an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

In some embodiments, the method comprises using a conjugate of the invention.

In other embodiments, the method comprises the use of an antibody or antigen-binding fragment thereof of the invention. In some embodiments, the antibody or antigen-binding fragment thereof does not comprise a detectable label. In some embodiments, the method further comprises using a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters, luminol and derivatives thereof, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or biotin) to detect said antibodies or antigen-binding fragments thereof.

In some embodiments, the method can be used for diagnostic purposes, for example, whether a subject is infected with B-coronavirus can be diagnosed based on the presence or level of B-coronavirus in a sample. In such embodiments, the sample can be a blood sample (eg, whole blood, plasma, or serum), feces, oral or nasal secretions, or alveolar lavage fluid from the subject.

In some embodiments, the subject is a mammal, such as a human.

In some embodiments, the method may be used for non-diagnostic purposes, eg, the sample is not from a subject, eg, a vaccine sample.

In another aspect, the use of the antibody of the present invention or its antigen-binding fragment or the conjugate of the present invention in the preparation of a kit for detecting B-coronavirus or its S protein or S protein RBD, or the presence or level of cells infected with B-coronavirus in a sample, and/or for diagnosing whether a subject is infected with B-coronavirus.

In some embodiments, the kit detects B-coronavirus or its S protein or the RBD of S protein, or the presence or level of cells infected with B-coronavirus in the sample by the detection method as described above , and optionally diagnosing whether the subject is infected with B-coronavirus according to the test result.

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

In the above technical solutions of the present invention, such as pharmaceutical compositions, conjugates, kits, disease prevention and/or treatment methods or uses, detection methods, medicines or kit preparations, antibodies or antigen-binding fragments thereof should be understood as Can be more than one, such as 2 types, 3 types, 4 types, 5 types, 6 types, 7 types, 8 types, 9 types, 10 types, 11 types, 12 types, 13 types, 14 types, 15 types, 16 kinds, 17 kinds, 18 kinds, 19 kinds, 20 kinds, 21 kinds of combinations. Antibodies or antigen-binding fragments thereof of the present invention may also be combined with other antibodies or antigen-binding fragments thereof. The antibody or antigen-binding fragment thereof may be a combination of two specific antibodies or antigen-binding fragments thereof, such as a combination of BD55-5840 and BD55-5514. Moreover, the combination of antibodies or antigen-binding fragments thereof has more excellent binding activity and neutralizing effect on B-coronavirus.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention, rather than limiting the scope of the present invention. Various objects and advantages of this invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiment.

Unless otherwise specified, the molecular biology experiment methods and immunoassay methods used in the present invention are basically with reference to J.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M.Ausubel et al., Compiled Molecular Biology Experimental Guide, 3rd Edition, John Wiley & Sons, Inc., 1995 by the method described; restriction endonucleases were used in accordance with the conditions recommended by the product manufacturer. Those skilled in the art understand that the examples describe the present invention by way of example and are not intended to limit the scope of the claimed invention.

Example 1: Memory B cell isolation

Whole blood was collected from SARS-CoV-infected recovered patients vaccinated against SARS-CoV-2. Blood samples were first diluted with PBS (Invitrogen) containing 2% FBS (Gibco) and subjected to Ficoll (Cytiva) gradient centrifugation. After lysis and washing, cells were resuspended in PBS (Invitrogen) containing 2% FBS (Gibco) for downstream B cell isolation, or added to FBS containing 10% DMSO (Sigma-Aldrich) for long-term storage at -80°C. B cells were enriched by positive selection using CD19 ⁺ B cell isolation kit (STEMCELLS). Enriched B cells were stained with the following human antigens and antibodies in FACS buffer (1×PBS, 2% FBS, 1 mM EDTA): FITC anti-CD19 antibody (Biolegend), FITC anti-CD20 antibody (Biolegend), Brilliant Violet 421 anti-CD27 Antibody (Biolegend), PE/Cyanine7 anti-IgM and fluorophore-labeled RBD (SARS340 CoV-2 and SARS-CoV RBD, Sino Biological Inc.) and ovalbumin (Ova) were placed on ice for 30 minutes. Cells were stained with 7-AAD for 10 min before sorting, and single CD19 or CD20 ⁺ CD27 ⁺ IgM-Ova ^- RBD-PE ⁺ RBD-APC ⁺ B cells were sorted on an Astrios EQ (BeckMan Coulter) containing 30% FBS in the PBS. After flow cytometry sorting, the obtained cells were subjected to 5′-mRNA and single-cell V(D)J library preparation, which was further carried out based on the double-sided sequencing mode of 26bp (Barcode) + 91bp (insert sequence) on the HiSeq 2500 platform Illumina sequencing.

Example 2: Obtaining and Identification of Antibody Sequences

Raw FASTQ files were processed with reference to the human sequence GRCh38 database using Cell Ranger (version 6.1.1) software. Generate sequences using 'cellranger multi' or 'cellranger vdj' with default parameters. Protein sequences were then extracted and processed by IMGT/DomainGapAlign (version 4.10.2) to obtain annotations of V(D)J, CDR regions and mutation frequency. V gene amino acid mutation rate = mutation count/length of V gene peptide.

Example 3: Antibody Preparation and Purification

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 separately into expression vectors based on Gibson assembly, and the two plasmids were subsequently co-transfected into HEK293F cells. The secreted monoclonal antibody in the cell culture medium is then purified by Protein A affinity chromatography.

Example 4: Evaluation of broad-spectrum binding activity of antibodies

4.1 ELISA

The specific binding capacity of these antibodies or plasma was analyzed by ELISA binding determination. Briefly, after coating, blocking and washing the RBDs of 22 currently known sarbecovirus viruses (see Table 2) with 0.03 μg/mL and 1 μg/mL, 1 μg/mL antibody or serially diluted plasma samples were added to the ELISA plate.

Table 2: Gene name and NCBI accession number of B-coronavirus spike protein

After incubation and washing, the 96-well plate was incubated with diluted goat anti-human IgG 373(H+L)/HRP antibody (JACKSON). Then tetramethylbenzidine (TMB, Solarbio) was added for color development for 10 minutes, and 2M _H2SO4 was added to stop the development reaction, and the absorbance _was measured at 450 nm using a PerkinElmer Ensight HH3400 model microplate reader, and the EC50 was calculated by GraphPad Prism 8.0 software ( See Table 3).

4.2 Biolayer Interferometry Kinetic Experiment

use

The protein analysis system (Fortebio) was used for the biolayer interference analysis kinetic experiment, and the sensor used was protein A biosensor (Fortebio 18-5010). The sensors were first soaked in buffer for 10 minutes (buffer ForteBio 18-1105) to complete the sensor self-test step, followed by 30 seconds in the buffer to complete the baseline. In the antibody capture step, the sensor was immersed in antibody at a concentration of 2 μg/ml for 300 s, and the threshold was set at 0.4 nm. The sensor was then soaked in buffer for 120 seconds only to complete the baseline. Immediately following the antigen binding step, the sensors were soaked for 60 s in serial dilutions of the RBD protein. In the subsequent dissociation step, the sensor was switched to soak in buffer for 600 s. Finally, soak the sensor in the regeneration buffer (10 mM glycine hydrochloride, pH 1.5) for 30 seconds, then soak in the buffer for 30 seconds, and repeat 2 times to complete the regeneration step. Use software Data Acquisition 11.1 (Fortebio) to record data, software Data Analysis HT 11.1 (Fortebio) to carry out data analysis (see Table 4).

Example 5: Evaluation of broad-spectrum neutralizing activity of antibodies

Neutralization assays were performed using the pseudovirus method to screen broadly binding antibodies to assess the neutralizing capacity of antibodies and plasma. A series of two-fold dilutions of antibodies were incubated with seven VsV pseudoviruses of the sarbecovirus spike protein (labeled with luciferase) for 1 hr, and the mixture was incubated with Huh-7 cells. After culturing in a 37 °C incubator for 24 h, the cells were collected and lysed with a luciferase substrate (PerkinElmer), and then the luminescence intensity was measured by a microplate reader. IC50 was determined by a four-parameter nonlinear regression model (see Table 5).

Embodiment 6: Mouse challenge experiment--SARS-CoV-2 (Omicron BA.1) virus

1. Weight

Take the combination of antibody BD55-5514+BD55-5840 as an example to evaluate the antiviral effect of candidate drugs. Human ACE2 (hACE2) transgenic mice were divided into five groups with 5 mice in each group. SARS-CoV-2 (Omicron BA.1) virus was used to infect mice with a dose of 1*10 ⁵ TCID ₅₀ per mouse via intranasal route, and the prevention group was given intraperitoneal or intramuscular injection of drugs 24 hours before challenge, with a dose of 20 mg/ kg. The treatment group was given intraperitoneal injection of drugs 2 hours after challenge, the dose was 20 mg/kg. The control group was intraperitoneally injected with an equal volume of PBS 2 hours after challenge. The body weight of the mice was monitored at 0, 1, 2, and 3 days after the challenge, and the analysis of the body weight change curve (Fig. 2) showed that the body weight of the mice in each group decreased slightly on the 1 day after the challenge, and the weight of the mice in each group decreased slightly on the 2-3 days after the challenge. The body weight showed a rising trend, and all returned to the original body weight on the third day. There was no statistical difference in the weight change of mice among the experimental groups compared with the control group, which may be due to the low pathogenicity of BA.1 to hACE2 transgenic mice.

2. Viral load

On the 3rd day after the challenge, the lung tissue and trachea of the mice were dissected and collected, and the viral ribonucleic acid (RNA) load in the samples was detected by qRT-PCR after homogenization. The results ( FIG. 3 ) showed that the viral loads in the lung tissue and trachea of the control group were higher, with an average of 4.91 (log10 copies/mL) and 3.82 (log10 copies/mL) respectively. Viral RNA copies were hardly detected in the lung tissue and trachea of the treatment group and the abdominal cavity prevention group, which were significantly different from the samples of the control group. The viral load of the lung tissue samples in the muscle prevention group was low, and the statistical results showed that it was significantly different from that of the control group (P<0.0001), and almost no viral RNA was detected in the trachea samples of the muscle prevention group. The viral load in the lung tissue and trachea samples of the control drug DXP-593 treatment group was reduced individually, but there was no statistical difference from the control group as a whole. The above experimental results showed that, compared with the control group, the treatment group, the abdominal cavity prevention group and the muscle prevention group could effectively reduce the viral load in the lung tissue and trachea of mice.

Embodiment 7: Mouse challenge experiment--SARS-CoV-2 (Omicron BA.5) virus

1. Weight

Still taking the combination of antibody BD55-5514+BD55-5840 as an example to evaluate the antiviral effect of candidate drugs. Human ACE2 (hACE2) transgenic mice were divided into four groups with 5 mice in each group. The SARS-CoV-2 (Omicron BA.5) virus was used to infect mice with a dose of 1*10 ⁵ TCID ₅₀ /mouse via intranasal route, and the prevention group was injected intraperitoneally or intramuscularly with a dose of 20mg/ kg. The treatment group was given intraperitoneal injection of drugs 2 hours after challenge, the dose was 20 mg/kg. The control group was intraperitoneally injected with an equal volume of PBS 2 hours after challenge. After the challenge, the body weight of the mice was continuously monitored every day, and the analysis of the body weight change curve (Figure 4) showed that the body weight of the mice in each group decreased slightly 1-2 days after the challenge, and the body weight showed a trend of recovery 3-4 days after the challenge. And on the 4th day, each group basically recovered to the original body weight. There was no statistical difference in the weight change of mice among the experimental groups compared with the control group, which may be due to the low pathogenicity of BA.5 to hACE2 transgenic mice.

2. Viral load

On the 4th day after the challenge, the lung tissue and trachea of the mice were dissected and collected, and the viral ribonucleic acid (RNA) load in the samples was detected by qRT-PCR after homogenization. The results ( FIG. 5 ) showed that the viral loads in the lung tissue and trachea of the control group were higher, with an average of 5.66 (log10 copies/mL) and 5.04 (log10 copies/mL) respectively. The viral load in the lung tissue and trachea of the peritoneal prophylaxis group was lower, with an average decrease of 3.10 and 5.04 lg compared with the control group, with a significant difference (P<0.0001). Compared with the control group, the viral load in the lung tissue and trachea of the abdominal cavity treatment group decreased by an average of 2.21 and 1.11 lg, and there was also 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 lg compared with the control group (P<0.01), while the viral load in the trachea of the muscle prevention group decreased individually, but there was no statistical difference between the group and the control group. The above experimental results show that, compared with the control group, both the intraperitoneal prevention group and the intraperitoneal treatment group can effectively reduce the viral load in the lung tissue and trachea of mice, while the muscle prevention group has a certain effect on reducing the viral load in the lung tissue .

Example 8: cryo-electron microscopy

We analyzed the cryo-electron microscope structure of antibody BD55-5514+BD55-5840 combined with BA.1 Spike, and proved that the epitopes of BD55-5514 and BD55-5840 do not conflict with each other and can bind to the S protein (Spike) at the same time. Among them, BD55-5840 can bind the receptor binding domain (RBD) in both "up" and "down" conformations, while BD55-5514 only binds the RBD in the "up" conformation.

The binding epitope of BD55-5514 is similar to that of ADG20 (Adintrevimab). The sites on BA.1 RBD that participate in the binding of BD55-5514 mainly include Asp405, Asn437, Asn439, Gly502, Val503, Gly504, Tyr508, etc., and the sites such as Pro373-Thr376, Val407-Arg408, Pro499, and Tyr501 are also close to the binding interface. However, the neutralization of BD55-5514 was not affected by Thr376Ala, Asp405Asn, Arg408Ser contained in BA.2, but was only affected by the mutations of Val503 and Gly504. These two sites are highly conserved and hardly occur in nature.

The binding epitope of BD55-5840 on RBD is similar to that of Ser309 (Sotrovimab), but the key binding site is different. The RBD residues appearing on the interface include Asp339, Glu340, Thr345, Arg346, Lys440, Leu441, Ser443, Lys444, etc. The mutations that mainly affect their binding are concentrated on Asp339-Glu340, Thr345-Arg346.

Neutralization experiments show that the pairing of BD55-5514 and BD55-5840 can provide the most difficult to escape broad-spectrum beta coronavirus lineage B (Sarbecovirus) neutralizing antibody drug against all current major new coronavirus variants, including All major branches of Omicron are extremely neutralizing.

Although the specific implementation of the present invention has been described in detail, those skilled in the art will understand that: according to all the teachings that have been published, various modifications and changes can be made to the details, and these changes are all within the protection scope of the present invention . The full scope of the invention is given by the claims appended hereto and any equivalents thereof.

Claims

An antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein,

(1) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 1,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 2, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 3;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 4,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 5, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 6; or

(2) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 9,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 10, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 11;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 12,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 13, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 14; or

(3) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 17,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 18, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 19 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 19;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 20,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 21, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 22; or

(4) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 25,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 26, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 27 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 27;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 28,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 29, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 30 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 30; or

(5) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 33,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 34 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 34, and

VH CDR3, it comprises the aminoacid sequence shown in SEQ ID NO:35 or comprises the aminoacid sequence that has 1 or 2 aminoacid residue substitutions, deletion or addition with respect to SEQ ID NO:35;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:36,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 37 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 37, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 38 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 38; or

(6) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 41,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 42 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 42, and

VH CDR3, it comprises the aminoacid sequence shown in SEQ ID NO:43 or comprises the aminoacid sequence that has 1 or 2 aminoacid residue substitutions, deletion or addition with respect to SEQ ID NO:43;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:44 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:44,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 45 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 45, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 46 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 46; or

(7) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 49 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 49,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:50 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:50, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:51 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:51;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:52 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:52,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:53 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:53, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:54 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:54; or

(8) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:57 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:57,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:58 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:58, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:59 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:59;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:60 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:60,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 61 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 61, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 62 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 62; or

(9) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:65 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:65,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:66 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:66, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:67 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:67;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:68 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:68,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 69 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 69, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 70 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 70; or

(10) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 73 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 73,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 74 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 74, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:75 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:75;

The light chain variable region comprises:

VL CDR1, it comprises the amino acid sequence shown in SEQ ID NO:76 or has 1 or 2 amino acid residue substitutions, deletions or the amino acid sequence of addition relative to SEQ ID NO:76,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 77 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 77, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 78 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 78; or

(11) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 81 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 81,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 82 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 82, and

VH CDR3, it comprises the aminoacid sequence shown in SEQ ID NO:83 or comprises the aminoacid sequence that has 1 or 2 aminoacid residue substitutions, deletion or addition with respect to SEQ ID NO:83;

The light chain variable region comprises:

VL CDR1, it comprises the amino acid sequence shown in SEQ ID NO:84 or has 1 or 2 amino acid residue substitutions, deletions or the amino acid sequence of addition relative to SEQ ID NO:84,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 85 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 85, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 86 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 86; or

(12) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 89 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 89,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 90 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 90, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:91 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:91;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:92 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:92,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 93 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 93, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 94 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 94; or

(13) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 97 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 97,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 98 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 98, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:99 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO:99;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 100 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 100,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 101 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 101, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 102 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 102; or

(14) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 105 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 105,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 106 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 106, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 107 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 107;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 108 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 108,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 109 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 109, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 110 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 110; or

(15) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 113 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 113,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 114 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 114, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 115 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 115;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 116 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 116,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 117 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 117, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 118 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 118; or

(16) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 121 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 121,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 122 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 122, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 123 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 123;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 124 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 124,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 125 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 125, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 126 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 126; or

(17) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 129 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 129,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 130 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 130, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 131 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 131;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 132 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 132,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 133 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 133, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 134 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 134; or

(18) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 137 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 137,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 138 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 138, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 139 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 139;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 140 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 140,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 141 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 141, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 142 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 142; or

(19) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 145 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 145,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 146 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 146, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 147 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 147;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 148 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 148,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 149 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 149, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 150 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 150; or

(20) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 153 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 153,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 154 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 154, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 155 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 155;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 156 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 156,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 157 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 157, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 158 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 158; or

(21) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 161 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 161,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 162 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 162, and

VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 163 or comprising an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 163;

The light chain variable region comprises:

VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 164 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 164,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 165 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 165, and

VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 166 or an amino acid sequence with 1 or 2 amino acid residue substitutions, deletions or additions relative to SEQ ID NO: 166.
The antibody or antigen-binding fragment thereof of claim 1, wherein

(1) the heavy chain variable region comprises a sequence shown in SEQ ID NO: 7 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 8 or a variant thereof; or

(2) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 15 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 16 or a variant thereof; or

(3) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 23 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 24 or a variant thereof; or

(4) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 31 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 32 or a variant thereof; or

(5) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 39 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 40 or a variant thereof; or

(6) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 47 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 48 or a variant thereof; or

(7) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 55 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 56 or a variant thereof; or

(8) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 63 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 64 or a variant thereof; or

(9) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 71 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 72 or a variant thereof; or

(10) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 79 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 80 or a variant thereof; or

(11) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 87 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 88 or a variant thereof; or

(12) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 95 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 96 or a variant thereof; or

(13) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 103 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 104 or a variant thereof; or

(14) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 111 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 112 or a variant thereof; or

(15) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 119 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 120 or a variant thereof; or

(16) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 127 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 128 or a variant thereof; or

(17) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 135 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 136 or a variant thereof; or

(18) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 143 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 144 or a variant thereof; or

(19) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 151 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 152 or a variant thereof; or

(20) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 159 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 160 or a variant thereof; or

(21) The heavy chain variable region comprises a sequence shown in SEQ ID NO: 167 or a variant thereof; the light chain variable region comprises a sequence shown in SEQ ID NO: 168 or a variant thereof;

wherein said variant has a substitution, deletion or addition of one or several amino acids, such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions, compared to the sequence from which it is derived, or have 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% Sequences with sequence identity; preferably, said substitutions are conservative substitutions.
The antibody or antigen-binding fragment thereof of claim 1 or 2, further comprising a constant region derived from human immunoglobulin;

Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g. IgG1, IgG2, IgG3 or IgG4) and the light chain of the antibody or antigen-binding fragment thereof comprises a human immunoglobulin derived from In the light chain constant region of a human immunoglobulin (such as kappa or lambda);

Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region as set forth in SEQ ID NO:169 and/or a light chain constant region as set forth in SEQ ID NO:170.
The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, bis Antibody (diabody), single domain antibody (sdAb), chimeric antibody, bispecific antibody or multispecific antibody.
An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-4, or the heavy chain variable region and/or light chain variable region thereof.
5. The isolated nucleic acid molecule of claim 5 operably linked to an expression control sequence.
An expression vector comprising the nucleic acid molecule of claim 5 or 6.
A host cell transformed with the nucleic acid molecule of claim 5 or 6 or the expression vector of claim 7.
A method of making an antibody or antigen-binding fragment thereof comprising,

(1) cultivating the host cell of claim 8 under conditions suitable for expression of said nucleic acid molecule or expression vector, and

(2) Isolating and purifying the antibody or antigen-binding fragment thereof expressed from the nucleic acid molecule or expression vector.
A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-4, and a pharmaceutically acceptable carrier and/or excipient.
The pharmaceutical composition of claim 10, wherein said antibody or antigen-binding fragment thereof is more than one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 types, 12 types, 13 types, 14 types, 15 types, 16 types, 17 types, 18 types, 19 types, 20 types, 21 types.
The pharmaceutical composition of claim 11, wherein the antibody or antigen-binding fragment thereof is as described in numbers (13) and (21).
The pharmaceutical composition of claim 12, wherein said antibodies are BD55-5840 and BD55-5514.
A method for preventing and/or treating diseases caused by B-coronavirus infection, the method comprising administering to a subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1-4 or claim 10- 13 pharmaceutical compositions.
The method of claim 14, wherein said antibody or antigen-binding fragment thereof is more than one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 kinds, 13 kinds, 14 kinds, 15 kinds, 16 kinds, 17 kinds, 18 kinds, 19 kinds, 20 kinds, 21 kinds.
The method of claim 15, wherein said antibody or antigen-binding fragment thereof is as described in item (13) and (21).
The method of claim 16, wherein said antibodies are BD55-5840 and BD55-5514.
The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4 for the preparation of a medicament for the prevention and/or treatment of diseases caused by B-coronavirus infection.
The use of claim 18, wherein the antibody or antigen-binding fragment thereof is more than one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 kinds, 13 kinds, 14 kinds, 15 kinds, 16 kinds, 17 kinds, 18 kinds, 19 kinds, 20 kinds, 21 kinds.
The use of claim 19, wherein the antibody or antigen-binding fragment thereof is described in numbers (13) and (21).
The use of claim 20, wherein said antibodies are BD55-5840 and BD55-5514.
The method of any one of claims 14-17 or the use of any one of claims 18-21, wherein the beta-coronavirus includes novel coronavirus (SARS-CoV-2) and variants thereof, severe acute respiratory syndrome Coronavirus (SARS-CoV) and its variants, SARS-related coronavirus (SARSr-CoV).
The method or use of claim 22, wherein the SARS-CoV-2 mutant strain is selected from Alpha (Alpha, B.1.1.7), Beta (Beta, B.1.351), Gamma (Gamma, P.1), Delta (Delta, B.1.617.2), Omicron (Omicron, B.1.1.529) or any combination thereof.
The method of any one of claims 14-17 or claims 22-23, wherein the subject is a mammal, such as a human.
A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4, and a detectable label linked to the antibody or antigen-binding fragment thereof.
The conjugate of claim 25, wherein said antibody or antigen-binding fragment thereof is more than one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 types, 12 types, 13 types, 14 types, 15 types, 16 types, 17 types, 18 types, 19 types, 20 types, 21 types.
The conjugate of claim 26, wherein the antibody or antigen-binding fragment thereof is as described in numbering (13) and (21).
The conjugate of claim 27, wherein said antibodies are BD55-5840 and BD55-5514.
The conjugate according to any one of claims 25-28, wherein the detectable label is selected from enzymes (such as horseradish peroxidase or alkaline phosphatase), chemiluminescence reagents (such as acridinium esters, lucid minol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or biotin.
A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-4 or the conjugate according to any one of claims 25-29.
The kit of claim 30, wherein the kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1-4, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof.
The kit of claim 30 or 31, wherein said antibody or antigen-binding fragment thereof is more than one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11 kinds, 12 kinds, 13 kinds, 14 kinds, 15 kinds, 16 kinds, 17 kinds, 18 kinds, 19 kinds, 20 kinds, 21 kinds.
The kit of claim 32, wherein the antibody or antigen-binding fragment thereof is described in item numbers (13) and (21).
33. The kit of claim 33, wherein said antibodies are BD55-5840 and BD55-5514.
The kit of any one of claims 31-34, wherein the second antibody further comprises a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as an acridinium ester) luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or biotin.
A method for detecting the presence or level of betacoronavirus in a sample comprising:

(1) contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1-4 or the conjugate of any one of claims 25-29;

(2) detecting the binding of said antibody or antigen-binding fragment thereof or said conjugate to a target antigen in said sample;

Wherein, the detection of the combination represents the presence of B-coronavirus in the sample, or the detection of the strength of the combination represents the level of B-coronavirus in the sample.
The method of claim 36, wherein said antibody or antigen-binding fragment thereof is more than one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 kinds, 13 kinds, 14 kinds, 15 kinds, 16 kinds, 17 kinds, 18 kinds, 19 kinds, 20 kinds, 21 kinds.
The method of claim 37, wherein said antibody or antigen-binding fragment thereof is as described in item (13) and (21).
38. The method of claim 38, wherein said antibodies are BD55-5840 and BD55-5514.
The method of any one of claims 36-39, wherein the sample is a blood sample (eg, whole blood, plasma, or serum), feces, oral or nasal secretions, or alveolar lavage fluid from the subject.
40. The method of claim 40, wherein said subject is a mammal, such as a human.
The method of any one of claims 36-39, wherein said sample is not a sample from a subject, eg said sample is from a vaccine sample.
The antibody or antigen-binding fragment thereof according to any one of claims 1-4 or the conjugate according to any one of claims 25-29 is prepared for detecting the presence or level of B-coronavirus in a sample Use in the kit.
The use of claim 43, wherein the antibody or antigen-binding fragment thereof is more than one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 kinds, 13 kinds, 14 kinds, 15 kinds, 16 kinds, 17 kinds, 18 kinds, 19 kinds, 20 kinds, 21 kinds.
The use of claim 44, wherein the antibody or antigen-binding fragment thereof is described in numbers (13) and (21).
The use of claim 45, wherein said antibodies are BD55-5840 and BD55-5514.