CN118679181A

CN118679181A - Broad-spectrum neutralizing antibody for B-type coronavirus and its application

Info

Publication number: CN118679181A
Application number: CN202280091594.4A
Authority: CN
Inventors: 谢晓亮; 曹云龙
Original assignee: Beijing Changping Laboratory
Current assignee: Beijing Changping Laboratory
Priority date: 2022-02-14
Filing date: 2022-10-21
Publication date: 2024-09-20
Also published as: WO2023151312A1; TW202342513A; CN116621972A

Abstract

The invention relates to the field of immunology, in particular to a broad-spectrum neutralizing antibody for a coronavirus B and application thereof. In particular, the invention relates to a broad-spectrum neutralizing antibody or antigen binding fragment thereof for the B-type coronavirus and application of the antibody or fragment in diagnosis, prevention and treatment of various diseases caused by the B-type coronavirus infection.

Description

Broad-spectrum neutralizing antibody for B-type coronavirus and its application

Technical Field

Background

Coronaviruses (CoVs) are the largest RNA viruses identified so far that infect mammalian humans, mice, pigs, cats, dogs, bats, cattle and avian vertebrates, causing respiratory, intestinal, hepatic and neurological diseases in the host. The coronaviridae are divided into 4 genera, α, β, δ and γ coronaviruses, respectively, of which β is the most serious hazard to humans. The betacoronaviruses are divided into betacoronaviruses and into 5 subgenera, embecovirus, sarbecovirus, merbecovirus, nobecovirus and Hibecovirus. The subgenera of the B coronavirus (Sarbecovirus) comprises novel coronavirus (SARS-CoV-2) and variants thereof, severe acute respiratory syndrome coronavirus (SARS-CoV) and variants thereof, and SARS-related coronavirus (SARSr-CoV) which are classified as shown in figure 1 (DOI: 10.1038/s 41586-020-2294-9) in recent years.

Coronaviruses, which are single-stranded RNA viruses, are directly translated and replicated in cells and are more prone to mutation than DNA viruses. SARS-CoV-2 has produced a number of variants since the 2019 bottom outbreak, of which new crown variants (Variant of concern, VOCs) are listed as interesting by the world health organization, up to 5: 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). The variation strains have the defects that the structure of spike protein on the surface of the virus is changed due to the change of genetic sequences, thereby affecting the recognition capability of the virus by antibodies or the affinity of the virus to a receptor, causing the risks of reduced or invalid neutralization capability of the generated antibodies after vaccination, enhanced virus transmission capability, altered virus pathogenic mechanism and the like, and increasing the weight and the difficulty for epidemic prevention and epidemic resistance. The continuous variation is likely to bring greater epidemic risks in the future.

Therefore, there is a need to find more effective neutralizing antibodies against the broad spectrum of coronaviruses including the above variants, providing an effective means for diagnosing, preventing and/or treating variant infections, thereby helping to stabilize and eliminate epidemic situations.

The invention develops a set of unique technical routes by applying the techniques in a plurality of fields such as immunology, molecular biology, virology, protein structure analysis and the like, and obtains a series of broad-spectrum neutralizing antibodies of the coronavirus B. The antibodies are derived from the memory B cells of SARS-CoV infection healers inoculated with SARS-CoV-2 vaccine, have the characteristics of broad-spectrum high binding activity and broad-spectrum high neutrality of the coronavirus B, can efficiently neutralize pathogenic viruses including novel coronavirus, atypical coronavirus and the like, are particularly suitable for diagnosing, preventing and treating various diseases caused by the coronavirus B infection, and have important clinical values.

The invention successfully predicts the binding epitope and escape map of the neutralizing antibodies on the B coronavirus through an optimized high-flux yeast display technology, finds out antibody pairs based on the information, and verifies the antibody pairs on a frozen electron microscope, thereby reporting the sequences of the antibodies 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 complementarity determining regions of specific amino acid sequences.

In some embodiments, the heavy chain variable region and the light chain variable region comprise a particular amino acid sequence.

In some embodiments, the antibody or antigen binding fragment thereof comprises constant regions derived from a human immunoglobulin, e.g., a heavy chain constant region and a light chain constant region comprising a particular amino acid sequence.

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

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

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

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

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

(1) Culturing a host cell of the invention under conditions suitable for expression of a nucleic acid molecule or expression vector of the invention, and

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

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

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

In an eighth aspect, the present invention also provides the use of an antibody or antigen binding fragment thereof of the invention for the manufacture of a medicament for the prophylaxis and/or treatment of a disease caused by a coronavirus infection.

In some embodiments, the coronavirus B comprises a novel coronavirus (SARS-CoV-2) and variants thereof, severe acute respiratory syndrome coronavirus (SARS-CoV) and variants thereof, SARS-associated coronavirus (SARSr-CoV).

In some embodiments, the SARS-CoV-2 variant 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), armstrong (omacron, b.1.1.529), or any combination thereof.

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

In some embodiments, the detectable label is selected from the group consisting of an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound, luminol and derivatives thereof, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

In a tenth aspect, the invention also provides a kit comprising an antibody or antigen binding fragment thereof of the invention or a conjugate of the 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 comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and derivatives thereof, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

In an eleventh aspect, the present invention also provides a method for detecting the presence or level of a coronavirus b 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 binding of the antibody or antigen binding fragment or conjugate thereof to a target antigen in the sample.

In some embodiments, the presence of coronavirus b in the sample is indicated by detecting the binding.

In some embodiments, the level of coronavirus b in the sample is represented by detecting the magnitude of the binding.

In some embodiments, the sample is a blood sample (e.g., whole blood, plasma, or serum), fecal matter, oral or nasal secretions, or alveolar lavage from the subject.

In some embodiments, the subject is a mammal, e.g., a human.

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

In an eleventh aspect, the invention also provides the use of an antibody or antigen binding fragment thereof of the invention or a conjugate of the invention in the manufacture of a kit for detecting the presence or level of coronavirus b in a sample.

An antibody or antigen binding fragment thereof in the context of the present invention is understood to be a combination of more than one, for example 2,3,4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. Furthermore, the antibodies or antigen-binding fragments thereof of the invention may 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 BD 55-5514.

Drawings

FIG. 1 shows a subdivision of the subgenera of coronavirus type B (Sarbecovirus).

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

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

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

FIG. 5 shows the viral load of mouse tissues and organs in SARS-CoV-2 (Omicron BA.5) challenge experiments.

FIG. 6 shows the structure of a combined BD55-5514+BD55-5840 and BA.1 Spike cryomicroscope.

FIG. 7 shows the binding epitope for BD55-5514 on BA.1 RBD.

FIG. 8 shows the binding epitope for BD55-5840 on BA.1 RBD.

Detailed Description

1. Definition of the definition

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. 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 naturally or partially or fully synthetically (e.g., recombinantly) produced, including any fragment thereof comprising at least a portion of the variable region of an immunoglobulin molecule that retains the binding specificity of a full-length immunoglobulin. Thus, antibodies include any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding domain (antibody binding site). Antibodies include antibody fragments. As used herein, the term antibody includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, fab fragments, fab ' fragments, F (ab ') ₂ fragments, fv fragments, disulfide-linked Fv (dsFv), fd fragments, fd ' fragments, single chain Fv (scFv), single chain Fab (scFab), diabodies, anti-idiotype (anti-Id) antibodies, or antigen-binding fragments of any of the above. Antibodies provided herein include members of any immunoglobulin class (e.g., igG, igM, igD, igE, igA and IgY), any class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or subclass (e.g., igG2a and IgG2 b).

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 that comprises at least a portion of the variable region (e.g., one or more CDRs and/or one or more antibody binding sites) of the antibody that binds an antigen, and thus retains binding specificity as well as at least a portion of the specific binding capacity 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 an antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, fab ', F (ab ') ₂, single chain Fv (scFv), fv, dsFv, diabodies, fd, and Fd ' fragments, and others, including modified fragments (see, e.g., ,Methods in Molecular Biology,Vol 207：Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003);Chapter 1;p 3-25,Kipriyanov)., which may include multiple chains linked together, e.g., 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 antibody fragment that upon insertion into an antibody framework (e.g., by replacement of the corresponding region) attains immunospecific binding (i.e., exhibits a Ka antigen of at least or at least about 10 ⁷-10 ⁸M ^-1).

As used herein, "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to the other antibody molecules. This characteristic is in contrast to the characteristic of a polyclonal population of antibodies comprising antibodies having a plurality of different sequences. Monoclonal antibodies can be prepared by a number of well known methods (Smith et al (2004) J.Clin. Pathol.57, 912-917; and Nelson et al, J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalizing B cells, e.g., by fusion with myeloma cells to produce hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to produce antibodies from clonal populations of host cells in vitro 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 myeloma or lymphoma cells) produced by fusion of an antibody-producing lymphocyte and a non-antibody-producing cancer cell. As known to those of ordinary skill in the art, hybridomas can proliferate and continue to supply for the production of specific monoclonal antibodies. Methods for producing hybridomas are known in the art (see, e.g., harlow & Lane, 1988). When referring to the term "hybridoma" or "hybridoma cell," it also includes subclones and progeny cells of a hybridoma.

As used herein, "conventional antibody" refers to an antibody comprising two heavy chains (which may be labeled H and H ') and two light chains (which may be labeled L and L') and two antigen binding sites, wherein each heavy chain may be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen binding capacity (e.g., heavy chains including but not limited to VH chains, VH-CH1 chains, and VH-CH1-CH2-CH3 chains), and each light chain may be a full-length light chain or any functional region (e.g., light chains including but not limited to VL chains and VL-CL chains). Each heavy chain (H and H ') is paired with a light chain (L and L', respectively).

As used herein, a full length antibody is an antibody having two full length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH 4) and two full length light chains (VL-CL) and a hinge region, e.g., an antibody naturally produced by B cells by antibody secretion and an antibody synthetically produced with the same domains.

As used herein, dsFv refers to Fv with engineered intermolecular disulfide bonds that stabilize VH-VL pairs.

As used herein, fab fragments are antibody fragments obtained by digestion of full length immunoglobulins with papain, or fragments of the same structure synthetically produced, e.g., by recombinant methods. The Fab fragment comprises a light chain (comprising VL and CL) and another chain comprising the variable domain of the heavy chain (VH) and one constant region domain of the heavy chain (CH 1).

As used herein, a F (ab') ₂ fragment is an antibody fragment resulting from digestion of an immunoglobulin with pepsin at a pH of 4.0-4.5, or a fragment having the same structure, e.g., synthetically produced by recombinant methods. The F (ab') 2 fragment essentially comprises two Fab fragments, wherein each heavy chain portion comprises additional several amino acids, including cysteines forming disulfide bonds linking the two fragments.

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

As used herein, an scFv fragment refers to an antibody fragment comprising a variable light chain (V _L) and a variable heavy chain (V _H) covalently linked in any order by a polypeptide linker. The linker length is such that the two variable domains bridge substantially undisturbed. Exemplary linkers are (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 an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

By "humanized" antibody is meant a form of non-human (e.g., mouse) antibody that is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (e.g., fv, fab, fab ', F (ab') ₂ or other antigen-binding subsequence of the antibody) that contains minimal sequence derived from a non-human immunoglobulin. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the Complementarity Determining Regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.

Furthermore, in humanization, it is also possible to mutate amino acid residues within the CDR1, CDR2 and/or CDR3 regions of VH and/or VL, thereby improving one or more binding properties (e.g., affinity) of the antibody. Mutations, such as PCR-mediated mutations, can be introduced, and their effect on antibody binding or other functional properties can be assessed using 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, mutations within the CDRs typically do not exceed one or two. Thus, the humanized antibodies of the present invention also encompass antibodies comprising 1 or 2 amino acid mutations within the CDRs.

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

As used herein, a variable domain or variable region is a particular Ig domain of an antibody heavy or light chain that comprises amino acid sequences that vary between different antibodies. Each light chain and each heavy chain has one variable region domain VL and VH, respectively. The variable domains provide antigen specificity and are therefore responsible for antigen recognition. Each variable region comprises a CDR, which is part of an antigen binding site domain, and a Framework Region (FR).

As used herein, "antigen binding domain" and "antigen binding site" are synonymously used to refer to a domain within an antibody that recognizes and physically interacts with the cognate (cognate) antigen. The 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. Conventional antigen binding sites comprise loops linking antiparallel β chains within the variable region domain. The antigen binding site may comprise other portions of the variable region domain. Each conventional antigen binding site comprises 3 hypervariable regions from the heavy chain and 3 hypervariable regions from the light chain. The 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 one of a plurality of portions within each variable region that together form an antigen binding site of an antibody. Each variable region domain contains 3 CDRs, designated CDR1, CDR2, and CDR3. For example, the light chain variable region domain comprises 3 CDRs, designated VL CDR1, VL CDR2, and VL CDR3; the heavy chain variable region domain comprises 3 CDRs, designated VH CDR1, VH CDR2 and VH CDR3. The 3 CDRs in the variable region are discontinuous along the linear amino acid sequence but are contiguous in the folded polypeptide. The CDRs are located within loops connecting parallel chains of the β -sheet of the variable domain. As described herein, CDRs are known to those of skill in the art and can be identified 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, a Framework Region (FR) is a domain located within the variable region domain of an antibody within the β -sheet; the FR region is relatively more conserved than the hypervariable region 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 an amino acid sequence that is relatively more conserved than the amino acid sequence 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 comprises 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 hinge regions, while IgE and IgM comprise CH1, CH2, CH3 and CH4. The CH1 and CL domains extend the Fab arm of the antibody molecule, thus helping to interact with the antigen and turn the antibody arm. The antibody constant regions may serve effector functions such as, but not limited to, clearing antigens, pathogens, and toxins that the antibody specifically binds, such as by interacting with various cells, biomolecules, and tissues.

As used herein, a functional region of an antibody is an antibody portion comprising at least the VH, VL, CH (e.g., CH1, CH2, or CH 3), CL, or hinge region domain of the antibody, or at least the 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 part of the binding specificity of the complete VH domain (e.g., by retaining one or more CDRs of the complete VH domain) such that the functional region of the VH domain binds antigen alone or in combination with another antibody domain (e.g., a VL domain) or region thereof. The functional region of an exemplary VH domain is a region 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 an intact VL domain that retains at least a portion of the binding specificity of the intact VL domain (e.g., by retaining one or more CDRs of the intact VL domain) such that the functional region of the VL domain binds antigen alone or in combination with another antibody domain (e.g., VH domain) or region thereof. The functional region of an exemplary VL domain is a region comprising CDR1, CDR2, and/or CDR3 of the VL domain.

As used herein, "specific binding" or "immunospecifically binding" with respect to an antibody or antigen-binding fragment thereof is used interchangeably herein and refers to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with an alloantigen through non-covalent interactions between the antibody and the antibody binding site of the 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 binds the antigen with an affinity constant Ka of about or 1x 10 ⁷M ^-1 or 1x 10 ⁸M ^-1 or greater (or a dissociation constant (K _d) of 1x 10 ^-7 M or 1x 10 ^-8 M or less). Affinity constants can be determined by standard kinetic methods of antibody reaction, e.g., immunoassays, surface Plasmon Resonance (SPR) (Rich and Myszka (2000) curr. Opin. Biotechnol 11:54; englebiene (1998) analysis 123:1599), isothermal Titration Calorimetry (ITC), or other kinetic interaction assays known in the art (see, e.g., paul, ed., fundamental Immunology,2nd ed., RAVEN PRESS, new York, pages 332-336 (1989); see also U.S. Pat. No. 7,229,619) describing exemplary SPR and ITC methods for calculating binding affinity of antibodies. Instruments and methods for detecting and monitoring binding rates in real time 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 "competing" with respect to an antibody means that a first antibody or antigen-binding fragment thereof binds an epitope in a sufficiently similar manner to a second antibody or antigen-binding fragment thereof, whereby the binding result of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody as compared to the absence of the second antibody. Or in the case where the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody, this may, but need not, be the case. That is, the first antibody may inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits binding of another antibody to its cognate epitope or ligand, whether the same, higher or lower, the antibodies are said to "cross-compete" with each other for binding to their respective epitope. Competing and cross-competing antibodies are contemplated by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or fragment thereof), those skilled in the art will recognize, based on the teachings provided herein, that such competing and/or cross-competing antibodies are encompassed by the present invention and can be used in the methods disclosed herein.

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

"Isolated protein," "isolated polypeptide," or "isolated antibody" refers to the protein, polypeptide, or antibody: (1) not associated with components that accompany it in its natural state, (2) not containing other proteins from the same species, (3) expressed by cells from a different species, or (4) not occurring in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cell system that differs from the naturally derived cell of the polypeptide will be "isolated" from its naturally associated components. The protein may also be isolated such that it is substantially free of naturally associated components, i.e., 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. In general, one skilled in the art recognizes that single amino acid substitutions in the non-essential region 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 desired properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitution of an amino acid residue with an amino acid residue having a similar side chain, such as substitution with a residue that is physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids 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 twenty conventional amino acids referred to herein are written following conventional usage. See, e.g., ,Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991)),, incorporated by reference herein. In the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well 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 deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) that are typically linked together by phosphodiester bonds.

As used herein, an isolated nucleic acid molecule is a nucleic acid molecule that is isolated from other nucleic acid molecules that are present 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 the provided antibodies or antigen binding fragments.

Sequence "identity" has art-recognized meanings and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using the disclosed techniques. Sequence identity may be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. (see, e.g., ：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 identity between two polynucleotides or polypeptides, the term "identity" is well known to the skilled artisan (carrello, H. & Lipman, d., SIAM J APPLIED MATH 48:1073 (1988)).

As used herein, "operably linked" with respect to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, a promoter may 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. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. Vectors also include those comprising nucleic acids encoding polypeptides. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell, for amplifying the nucleic acid or for expressing/displaying the polypeptide encoded by the nucleic acid. Vectors typically remain episomal, but may be designed to integrate a gene or portion thereof into the chromosome of the genome. Vectors for artificial chromosomes are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.

As used herein, vectors also include "viral vectors" or "viral vectors". The vector of the virus is an engineered virus operably linked to a foreign gene to transfer (as a vehicle or shuttle) the foreign gene into a cell.

As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods of determining the amount of polypeptide produced from 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, an "expression vector" includes vectors capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, 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, an enhancer, a polyadenylation signal, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, that when introduced into an appropriate host cell results in expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic and/or prokaryotic cells as well as expression vectors that remain episomal or are integrated into the genome of a host cell.

By "codon optimization" is meant a method of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence with a more or most frequently used codon in the gene of the host cell (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; different species exhibit specific preferences for certain codons of a particular amino acid; codon preference (difference in codon usage between organisms) is often correlated with the translational efficiency of messenger RNA (mRNA) which is believed to depend on the nature of the translated codon and the availability of a particular transfer RNA (tRNA) molecule; the dominance of a selected tRNA within a cell generally reflects the codon most frequently used for peptide synthesis; thus, genes can be tailored to the optimal gene expression based on codon optimization in a given organism; codon usage tables can be readily obtained, e.g., at www.kazusa.orjp/codon a database ("available in 53kara; can be obtained by using these means of the database, and the like; see the methods of Namura et cetera) ,"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 that is used to receive, hold, replicate, and amplify a vector. Host cells may also be used to express the polypeptides encoded by the vectors. When the host cell is divided, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell may be a eukaryotic cell or a prokaryotic cell. 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 that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which is well known in the art (see, e.g., Remington′s Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania：Mack Publishing Company,1995), and includes, but is not limited to: for example, pH modifiers 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, osmotic pressure maintaining agents include, but are not limited to, sugar, naCl, and the like, absorption delaying agents include, but are not limited to, monostearates, and gelatin, diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), and the like, preservatives include, but are not limited to, various antibacterial and antifungal agents such as thiomersal, 2-phenoxyethanol, parabens, trichlorot-butanol, phenol, sorbic acid, and the like. Glycine), proteins (e.g., dried whey, albumin or casein) or degradation products thereof (e.g., lactalbumin hydrolysate), and the like. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (e.g., an 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 solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

As used herein, the term "preventing" refers to a method performed in order to prevent or delay the occurrence of a disease or disorder or symptom (e.g., SARS-CoV-2 infection) in a subject. As used herein, the term "treatment" refers to a method that is performed in order to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., no longer worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and diminishment of symptoms (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to an extension of survival compared to the expected survival (if not treated).

As used herein, "therapeutic effect" refers to the effect resulting from treatment of an individual that alters, generally improves or ameliorates symptoms of, or cures a disease or condition.

As used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to an 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 the onset or recurrence of a disease or symptom, reducing the likelihood of the onset or recurrence of a disease or symptom. The fully prophylactically effective dose need not occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

As used herein, a "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 after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, block or partially block 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., human) has, or is at risk of having, a SARS-CoV-2 infection or a disease associated with a SARS-CoV-2 infection (e.g., COVID-19).

As used herein, "Severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus, SARS-CoV-2)", which is known as "novel coronavirus" or "2019-nCov", belongs to the genus beta coronavirus, which is a enveloped single-stranded plus sense RNA virus. 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, like SARS-CoV, specifically combined with angiotensin transferase 2 (ACE 2) on host cell by receptor binding domain (Receptor binding domain, RBD) on S protein, and then grafted with membrane fusion and cell entry of virus, which plays a vital role in the process of virus infection of cells.

As used herein, the term "SARS-CoV-2" encompasses known isolates, including, for example, both the original strain (e.g., the first sequenced isolate GenBank: MN 908947.3) and subsequently discovered variants. In certain embodiments, the term "SARS-CoV-2" encompasses both isolates whose S protein does not comprise a mutation (e.g., as compared to reference strain MN 908947.3) and isolates that comprise a mutation in their S protein (e.g., an amino acid substitution as compared to reference strain MN908947.3, such as K417N, E484K, N501Y, L452R, T478K, or any combination thereof). In certain embodiments, the variant is preferably selected from isolates comprising mutations (e.g., amino acid substitutions, such as K417N, E484K, N501Y, L452R, T478K or any combination thereof) in their S proteins. In certain exemplary embodiments, the variant plant 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 omicon (omicon, b.1.1.529).

As used herein, the terms "novel coronavirus pneumonia" and "COVID-19" refer to pneumonia caused by SARS-CoV-2 infection, both of which have the same meaning and are used interchangeably.

As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment that binds to an antigenic protein on a virus, thereby preventing the maturation of virus-infected cells and/or virus progeny and/or the release of virus progeny, and an antibody or antibody fragment having neutralizing activity may prevent the amplification of a virus, thereby inhibiting or eliminating the infection by a virus.

2. Antibodies or antigen binding fragments thereof

The present invention provides antibodies or antigen binding fragments thereof comprising a heavy chain variable region and a light chain variable region comprising Complementarity Determining Regions (CDRs) of a particular amino acid sequence, or an amino acid sequence having 1 or 2 amino acid residue substitutions, deletions, or additions relative to a particular amino acid sequence.

In some embodiments, the heavy chain variable region and the light chain variable region comprise a particular amino acid sequence or variant thereof. Wherein the variant has a substitution, deletion, or addition of one or several amino acids compared to the sequence from which it is derived, e.g., a substitution, deletion, or addition of 1, 2,3, 4, or 5 amino acids, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; preferably, the substitution is a conservative substitution.

Information on the partial sequences to which the present invention relates is provided in Table 1 below, specifically 21 antibodies (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, VL CDR3 sequences numbered BD55-1239、BD55-3372、BD55-3500、BD55-3546、BD55-4637、BD55-5242、BD55-5263、BD55-5300、BD55-5386、BD55-5477、BD55-5483、BD55-5484、BD55-5514、BD55-5549、BD55-5558、BD55-5585、BD55-5591、BD55-5640、BD55-5697、BD55-5700、BD55-5840), respectively) 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 sequence

3. Preparation of antibodies

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

Antigen binding fragments of the invention may be obtained by hydrolysis of intact antibody molecules (see Morimoto et al, J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al, science 229:81 (1985)). Alternatively, these antigen binding fragments may be produced (reviewed in Hudson,Curr.Opin.Immunol.11：548-557(1999);Little et al.,Immunol.Today,21：364-370(2000)). directly from a recombinant host cell, e.g., fab' fragments may be obtained directly from the host cell; fab 'fragments can be chemically coupled to form the F (ab') ₂ fragment (Carter et al, bio/Technology,10:163-167 (1992)). Alternatively, fv, fab or F (ab') ₂ fragments may be isolated directly from recombinant host cell culture broth. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.

Thus, in another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding 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 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 heavy chain variable region of an antibody or antigen binding fragment thereof of the invention and a second nucleotide sequence encoding a light chain or light chain variable region of the antibody or antigen binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecules of the invention comprise a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

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

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

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

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

In another aspect, the invention provides a host cell transformed with a nucleic acid molecule of the invention or an expression vector of the invention. Such host cells include, but are not limited to, prokaryotic cells, such as bacterial cells (e.g., E.coli cells), and eukaryotic cells, such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., 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 of producing an antibody or antigen-binding fragment thereof of the invention, comprising culturing a host cell of the invention under conditions suitable for expression of a nucleic acid molecule or expression vector of the invention, and isolating and purifying the antibody or antigen-binding fragment thereof expressed by the nucleic acid molecule or expression vector.

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, and the like that are physiologically compatible. Preferably, the carrier and/or excipient is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody molecule, immunoconjugate, may be encapsulated in a material to protect the compound from acids and other natural conditions that may inactivate the compound.

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 ascorbyl palmitate, butylated Hydroxyanisole (BHA), butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelators such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

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

The prevention of the presence of microorganisms may be ensured by sterilization procedures or by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like. In many cases, it is preferred to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium oxide in the composition. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion in the composition of delayed absorption agents, for example, monostearates and gelatins.

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, except insofar as they are incompatible with the active compound, are possible in the pharmaceutical compositions of the present invention. Supplementary active compounds may also be incorporated into the compositions.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersant containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. For example, proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

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

The amount of active ingredient that can be combined with the carrier material to prepare a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier material to prepare a single dosage form is generally the amount of the composition that produces a therapeutic effect. Typically, this amount ranges 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% of the active ingredient, on a 100% basis, in combination with a pharmaceutically acceptable carrier.

The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased as needed for the emergency of the treatment situation. It is particularly 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 suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier. The specific description of dosage unit forms of the invention is limited to and directly depends on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) limitations inherent in the art for formulating such active compounds for use in the treatment of sensitivity in individuals.

For administration of antibody molecules, the dosage range is about 0.0001 to 100mg/kg, more typically 0.01 to 20mg/kg of the recipient body weight. For example, the dosage may be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, 10mg/kg body weight or 20mg/kg body weight, or in the range of 1-20 mg/kg. Exemplary treatment regimens require weekly, biweekly, tricyclically, weekly, monthly, 3 months, 3-6 months, or a slightly shorter initial dosing interval (e.g., weekly to tricyclically) followed by longer post dosing intervals (e.g., monthly to 3-6 months).

Alternatively, the antibody molecules of the invention may be administered as a sustained release formulation, in which case less frequent administration is required. Dosages and frequencies will vary depending on the half-life of the antibody molecule in the patient. Typically, human antibodies exhibit the longest half-life, followed by humanized, chimeric, and non-human antibodies. The dosage and frequency of administration will vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at less frequent intervals over a long period of time. Some patients continue to receive treatment for the remainder 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 a partial or complete improvement in the symptoms of the disease. Thereafter, the patient may be administered a prophylactic regimen.

The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response to the particular patient, composition and mode of administration without toxicity to the patient. The dosage level selected will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention being used, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of the treatment, other drugs, compounds and/or materials being used in combination with the particular compositions being used, the age, sex, weight, condition, general health and medical history of the patient undergoing treatment, and like factors well known in the medical arts.

The antibodies of the invention, or antigen-binding fragments thereof, or the pharmaceutical compositions of the invention, may be administered by one or more routes of administration using one or more methods well known in the art. Those skilled in the art will appreciate that the route and/or mode of administration will vary depending upon the desired result. Preferred routes of administration for the antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Alternatively, the antibodies of the invention or antigen-binding fragments thereof or the pharmaceutical compositions of the invention may be administered by a non-parenteral route, such as topical, epidermal or mucosal route, e.g., intranasal, oral, vaginal, rectal, sublingual or topical.

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

The therapeutic composition may 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 using a needleless subcutaneous injection device, such as those described in U.S. Pat. Nos. 5,399,163;5,383,851;5,312,335;5,064,413;4,941,880; 4,790,824; or 4,596,556. Examples of well known implants and modules that may be used in the present invention include: U.S. patent No.4,487,603, which discloses an implantable microinjection pump for dispensing a drug at a controlled rate; U.S. patent No.4,486,194, which discloses a therapeutic device for transdermal administration; U.S. Pat. No.4,447,233, which discloses a medical infusion pump for delivering a drug at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusion device for continuous delivery of a drug; U.S. Pat. No.4,439,196 discloses an osmotic drug delivery system having multiple compartments: and U.S. patent No.4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems and modules are known to those skilled in the art.

In some embodiments, the antibodies of the invention may be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) prevents 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, for example, in liposomes. As for the method of preparing liposomes, see, for example, U.S. Pat. nos. 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.ranade (1989) j.clin.pharmacol.29:685). Examples of targeting moieties include folic acid or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al); mannosides (Umezawa et al (1988) biochem. Biophys. Res. Commun. 153:1038); antibody (P.G.Bloeman et al (1995) FEBS Lett.357:140; M.Owais et al (1995) Antimicrob.Agents chemther.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, a step of; j. killion; j. Fidler (1994) Immunomethods 4:273.

In certain exemplary embodiments, the pharmaceutically acceptable carrier and/or excipient comprises a sterile injectable liquid (e.g., an 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 solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

5. Use for preventing and/or treating diseases

The antibody or the antigen binding fragment thereof can be used for neutralizing the B coronavirus in vitro or in a subject to block or inhibit the infection of cells by the B coronavirus, thereby achieving the purpose of preventing and/or treating the B coronavirus infection or diseases related to the B coronavirus infection of the subject.

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

In some embodiments, the SARS-CoV-2 comprises a variant. In some embodiments, the variant S protein contains mutations, such as amino acid substitutions, deletions, or additions. In some embodiments, the S protein of the variant comprises one or more amino acid substitutions selected from the group consisting of K417N, E484K, N501Y, L452R, T478K. In some embodiments, the SARS-CoV-2 variant 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), armstrong (omacron, 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 (e.g., another antiviral agent). The antibody of the invention or antigen-binding fragment thereof, or the pharmaceutical composition of the invention, and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.

In another aspect, the invention provides a method for neutralizing, blocking or inhibiting the binding of b-coronavirus to ACE2 receptor, or blocking or inhibiting the infection of cells by b-coronavirus, comprising using an antibody or antigen-binding fragment thereof or pharmaceutical composition of the invention. The methods are useful for neutralizing, blocking or inhibiting the binding of, or blocking or inhibiting the infection of cells by, b-coronavirus to ACE2 receptor in vitro or in a subject (e.g., human).

In some embodiments, the methods are used to neutralize virulence of, block or inhibit binding of, or block or inhibit infection of cells by, coronavirus b to ACE2 receptors in a sample. In some embodiments, the method comprises: a sample comprising coronavirus b is contacted with an antibody or antigen-binding fragment thereof or a pharmaceutical composition of the invention.

In another aspect, the invention relates to the use of an antibody or antigen binding fragment thereof of the invention for the manufacture of a medicament for the prevention and/or treatment of a disease caused by a coronavirus infection, said medicament being for one or more of the following:

(1) Neutralizing the coronavirus b in vitro or in a subject (e.g., human);

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

(3) Blocking or inhibiting infection of cells by coronavirus b; and/or

(4) For preventing and/or treating a coronavirus infection or a disease associated with a coronavirus infection in a subject (e.g., COVID-19).

The antibodies of the invention, or antigen-binding fragments thereof, or the pharmaceutical compositions of the invention, may be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and injectable concentrated solutions), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the invention should be sterile and stable under the conditions of manufacture and storage. One preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by the following methods: the antibody or antigen binding fragment thereof of the present invention is incorporated in the necessary amount in a suitable solvent, and optionally, simultaneously with other desired ingredients (including, but not limited to, pH modifiers, surfactants, adjuvants, ionic strength enhancers, isotonizing agents, preservatives, diluents, or any combination thereof), followed by filter sterilization. In addition, the sterile injectable solutions may be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for convenient storage and use. Such sterile lyophilized powders may be dispersed in a suitable carrier prior to use, such as water for injection (WFI), water for bacteriostatic injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), 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, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powder, ointment or drops), or nasal route. For many therapeutic uses, however, the preferred route/mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In some embodiments, the antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the invention are administered by intravenous injection or bolus injection.

In this context, the dosing regimen may be adjusted to obtain the optimal target response (e.g., therapeutic or prophylactic response). For example, the dosage may be administered in a single dose, may be administered multiple times over a period of time, or may be proportionally reduced or increased as the degree of urgency of the treatment situation.

6. Conjugate(s)

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

Thus, in some embodiments, the antibodies or antigen binding fragments thereof of the invention bear a detectable label.

In this context, a detectable label according to the invention may be any substance that is detectable by fluorescence, 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, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., ³H、 ¹²⁵I、 ³⁵S、 ¹⁴ C or ³² P), fluorescent dyes (e.g., fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., cy7, alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridine esters, luminol and derivatives thereof, ruthenium derivatives such as ruthenium terpyridyl), magnetic beads (e.g.,) A calorimetric label such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the label-modified avidin (e.g., streptavidin) described above.

In some embodiments, the detectable label can be suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In some embodiments, the detectable label may be selected from an enzyme (e.g., horseradish peroxidase, alkaline phosphatase, or β -galactosidase), a chemiluminescent reagent (e.g., an acridine ester compound, luminol and derivatives thereof, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein, such as FITC, TRITC, or PE), a radionuclide, or biotin.

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

7. Kit and detection application

The antibody or antigen binding fragment thereof of the present invention is capable of specifically binding to the RBD of the S protein of coronavirus b, and thus can be used to detect the RBD of coronavirus b or the S protein or S protein thereof, and optionally diagnose whether a subject is infected with coronavirus b based on the above detection result.

Thus, in another aspect, the invention provides a kit comprising an antibody or antigen-binding fragment thereof of the invention, 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 an antibody or antigen-binding fragment thereof of the invention; optionally, the secondary antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound, luminol and derivatives thereof, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

In some embodiments, the second antibody is specific for an antibody of the species (e.g., 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 (e.g., human immunoglobulin) antibody, such as 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 causing the detection of the corresponding detectable label. For example, when the detectable label is an enzyme, the kit may further comprise a chromogenic substrate for the corresponding enzyme, such as o-phenylenediamine (OPD), tetramethyl benzidine (TMB), ABTS, or luminol for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase. The kit may further comprise a pre-excitation and/or excitation liquid for chemiluminescence, for example when the detectable label is a chemiluminescent reagent, such as an acridine ester compound.

In another aspect, the invention provides a method of detecting the presence or level of RBD of coronavirus b or S protein thereof, or cells infected with coronavirus b in a sample comprising contacting the sample with an antibody or antigen binding fragment or conjugate thereof of the invention; detecting binding of the antibody or antigen binding fragment or conjugate thereof to a target antigen in the sample; wherein the presence of the coronavirus in the sample is indicated by detecting the binding, or the level of the coronavirus in the sample is indicated by detecting the level of the binding.

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

In some embodiments, the methods comprise using the conjugates of the invention.

In other embodiments, the methods comprise using 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 detecting the antibody or antigen-binding fragment thereof using a second antibody bearing a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound, luminol and derivatives thereof, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin).

In some embodiments, the methods may be used for diagnostic purposes, e.g., whether a subject is infected with a coronavirus b may be diagnosed based on the presence or level of the coronavirus b in a sample. In such embodiments, the sample may be a blood sample (e.g., whole blood, plasma, or serum), fecal matter, oral or nasal secretions, or alveolar lavage from the subject.

In some embodiments, the subject is a mammal, e.g., a human.

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

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

In some embodiments, the kit detects the presence or level of a coronavirus b or its S protein or RBD of an S protein, or cells infected with a coronavirus b in a sample by a detection method as described above, and optionally diagnoses whether the subject is infected with a coronavirus b based on the detection result.

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

In the above-described embodiments of the invention, e.g. pharmaceutical compositions, conjugates, kits, methods or uses for disease prevention and/or treatment, methods of detection, medicaments or kit preparation, antibodies or antigen binding fragments thereof are understood to be a combination of more than one, e.g. 2,3,4,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. The antibodies or antigen-binding fragments thereof of the 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 2 specific antibodies or antigen binding fragments thereof, such as a combination of BD55-5840 and BD 55-5514. And the combination of the antibodies or antigen binding fragments thereof has more excellent binding activity and neutralizing effect on the coronavirus B.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Unless otherwise indicated, molecular biology experimental methods and immunoassays used in the present invention are basically described in j.sambrook et al, molecular cloning: laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, fine-compiled guidelines for molecular biology experiments, 3 rd edition, john Wiley & Sons, inc., 1995; the use of restriction enzymes was in accordance with the conditions recommended by the manufacturer of the product. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

Example 1: memory B cell separation

Whole blood collection was performed on SARS-CoV infection recovery vaccinated with SARS-CoV-2 vaccine by first diluting the blood sample with PBS (Invitrogen) containing 2% FBS (Gibco) and performing Ficoll (Cytiva) gradient centrifugation. After lysis and washing, downstream B-cell isolation was performed by resuspension with PBS (Invitrogen) containing 2% FBS (Gibco) or long-term storage at-80℃in FBS with 10% DMSO (Sigma-Aldrich) added. B cells were enriched by positive selection using CD19 ⁺ B cell isolation kit (STEMCELLS). The enriched B cells were stained with the following human antigens and antibodies in FACS buffer (1×pbs, 2% fbs, 1mM EDTA): FITC anti-CD 19 antibody (bioleged), FITC anti-CD 20 antibody (bioleged), brilliant Violet 421 anti-CD27 Antibody (Biolegend), PE/Cyanine7 anti-IgM, and fluorophore-labeled RBDs (SARS 340 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 prior to sorting, and individual CD19 or CD20 ⁺CD27 ⁺IgM-Ova ^-RBD-PE ⁺RBD-APC ⁺ B cells were sorted into PBS containing 30% fbs at Astrios EQ (BeckMan Coulter). The obtained cells were subjected to 5' -mRNA and single cell V (D) J library preparation after flow cell sorting, and further illumina sequencing was performed based on the Hiseq 2500 platform using a double-sided sequencing mode of 26bp (Barcode) +91bp (insert).

Example 2: acquisition and identification of antibody sequences

The original FASTQ file was processed using CELL RANGER (version 6.1.1) software against the human sequence GRCh38 database. Sequences were generated using either "CELLRANGER MULTI" or "CELLRANGER VDJ" with default parameters. The protein sequence was then extracted and processed through IMGT/DomainGapAlign (version 4.10.2) to obtain annotations of V (D) J, CDR region and mutation frequency. V gene amino acid mutation rate = mutation count/V length of 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. Heavy and light chain sequences were cloned separately into expression vectors based on Gibson assembly, followed by co-transfection of the two plasmids into HEK293F cells. The secreted monoclonal antibodies in the cell culture medium were then purified by Protein a affinity chromatography.

Example 4: antibody broad-spectrum binding Activity assessment

4.1 ELISA

The specific binding capacity of these antibodies or plasma was assayed by ELISA binding assay. Briefly, after RBDs of 22 currently known sarbecovirus viruses (see Table 2) were coated, blocked and washed with 0.03 μg/mL and 1 μg/mL, 1 μg/mL antibodies or serial dilutions of plasma samples were added to ELISA plates.

Table 2: gene name of spike protein of coronavirus B and accession number on NCBI

After incubation and washing, 96-well plates were incubated with diluted coat anti-human IgG 373 (h+l)/HRP antibody (JACKSON). Then, after development for 10 minutes by adding tetramethylbenzidine (TMB, solarbio), the development reaction was stopped by adding 2M H ₂SO ₄, absorbance was measured at 450nm using a model PERKINELMER ENSIGHT HH3400 microplate reader, and EC50 was calculated by GRAPHPAD PRISM 8.0.0 software (see Table 3).

4.2 Biological layer interferometry kinetic experiments

UsingThe protein analysis system (Fortebio) performs a biological layer interference analysis kinetic experiment, and the adopted sensor is a protein A biosensor (Fortebio 18-5010). The sensor self-test step was first completed by immersing the sensor in the buffer for 10 minutes (buffer ForteBio 18-1105), and then the baseline was completed by immersing in the buffer for 30 seconds. In the antibody capturing step, the sensor was immersed in an antibody at a concentration of 2. Mu.g/ml for 300 seconds, and the threshold was set at 0.4nm. The sensor then completes the baseline by soaking in the buffer for only 120 seconds. Following the antigen binding step, the sensor was immersed in a serial dilution of RBD protein for 60 seconds. In the subsequent dissociation step, the sensor is turned to a buffer soak for 600 seconds. Finally, the sensor is soaked in regeneration buffer (10 mM glycine hydrochloride, pH 1.5) for 30 seconds, then soaked in the buffer for 30 seconds, and the regeneration step is completed by repeating 2 times. Data was recorded using software Data Acquisition 11.1 (Fortebio) and Data analysis was performed using software DATA ANALYSIS HT 11.1.1 (Fortebio) (see table 4).

Example 5: antibody broad-spectrum neutralization activity assessment

Broad-spectrum binding antibodies were screened using the pseudovirus method for neutralization assays to assess the neutralizing capacity of the antibodies and plasma. A series of two-fold diluted antibodies were incubated with seven VsV pseudoviruses (labeled with luciferase) with sarbecovirus spike proteins, respectively, for 1 hour, and the mixture was incubated with Huh-7 cells. After culturing in an incubator at 37℃for 24 hours, the cells were collected and lysed with a luciferase substrate (Perkinelmer), and then luminescence intensity measurement was performed by an enzyme-labeled instrument. IC50 was determined by a four-parameter nonlinear regression model (see table 5).

Example 6: mouse challenge experiment- -SARS-CoV-2 (Omicron BA.1) Virus

1. Weight of body

Taking the combination of antibodies BD55-5514+BD55-5840 as an example, the antiviral effect of candidate drugs was evaluated. Human ACE2 (hACE 2) transgenic mice were divided into five groups of 5 mice each. SARS-CoV-2 (Omicron BA.1) virus infects mice by nasal drip route at a dose of 1X 10 ⁵TCID ₅₀/dose, and the prophylaxis group injects drug into the abdominal cavity or muscle 24 hours before challenge at a dose of 20mg/kg. The treatment group was intraperitoneally injected with the drug 2 hours after the challenge, with a dose of 20mg/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 challenge, and analysis of the body weight change curve (fig. 2) showed that the body weight of each group of mice showed a slight decrease in the body weight at 1 day after challenge, and the body weight was in a trend of going back at 2-3 days after challenge, and had recovered to the original body weight at 3 days. The change in mouse body weight between the experimental groups was not statistically different from the control group, probably due to the low pathogenicity of ba.1 to hACE transgenic mice.

2. Viral load

The lung tissue and trachea of the mice are dissected and collected on day 3 after the challenge, and the viral ribonucleic acid (RNA) load in the sample is detected by qRT-PCR method after homogenate. The results (FIG. 3) show that the viral loads in the lung tissue and trachea are higher for the control group, with average values of 4.91 (log 10 copy/mL) and 3.82 (log 10 copy/mL), respectively. Almost no viral RNA copy number was detected in lung tissue and trachea in the treated and peritoneal prophylaxis groups, with significant differences from the samples in the control group. The viral load of the lung tissue sample in the muscle prophylaxis group is low, and the statistical result shows that the viral load of the lung tissue sample is obviously different from that of the control group (P is less than 0.0001), and the viral RNA is hardly detected in the tracheal sample of the muscle prophylaxis group. The control drug DXP-593 treated group showed a respective reduction in viral load in lung tissue and tracheal samples, but no statistical difference from the control group as a whole. The above experimental results show that compared with the control group, the treatment group, the abdominal cavity prevention group and the muscle prevention group can effectively reduce the viral load in the lung tissue and the trachea of the mice.

Example 7: mouse challenge experiment- -SARS-CoV-2 (Omicron BA.5) Virus

1. Weight of body

The antiviral effect of candidate drugs was also evaluated using the combination of antibodies BD55-5514+BD55-5840 as an example. Human ACE2 (hACE 2) transgenic mice were divided into four groups of 5 mice each. SARS-CoV-2 (Omicron BA.5) virus infects mice by nasal drip at a dose of 1X 10 ⁵TCID ₅₀/mouse, and the prophylaxis group injects the drug into the abdominal cavity or muscle 24 hours before challenge at a dose of 20mg/kg. The treatment group was intraperitoneally injected with the drug 2 hours after the challenge, with a dose of 20mg/kg. The control group was intraperitoneally injected with an equal volume of PBS 2 hours after challenge. The body weight of the mice was continuously monitored daily after challenge, and analysis of the body weight change curve (fig. 4) showed that the body weight of each group of mice showed a slight decrease in 1-2 days after challenge, the body weight was in an upward trend 3-4 days after challenge, and each group was substantially restored to the original body weight on day 4. The change in mouse body weight between the experimental groups was not statistically different from the control group, probably due to the low pathogenicity of ba.5 for hACE transgenic mice.

2. Viral load

The lung tissue and trachea of the mice are dissected and collected on day 4 after the challenge, and the viral ribonucleic acid (RNA) load in the sample is detected by qRT-PCR method after homogenate. The results (FIG. 5) show that the viral loads in the lung tissue and trachea are higher for the control group, with average values of 5.66 (log 10 copy/mL) and 5.04 (log 10 copy/mL), respectively. The viral load in the lung tissue and trachea of the peritoneal prophylaxis group is lower, with an average 3.10 and 5.04lg reduction compared to the control group, with a significant difference (P < 0.0001). The average viral load in the lung tissue and trachea of the celiac treated group was reduced by 2.21 and 1.11lg compared to the control group, again with significant differences (P < 0.05). The average viral load of lung tissue in the muscle prophylaxis group was reduced by 2.89lg (P < 0.01) compared to the control group, whereas the viral load in the trachea in the muscle prophylaxis group was individually reduced, but the whole was not statistically different from the control group. Compared with a control group, the experimental result shows that the peritoneal cavity prevention group and the peritoneal cavity treatment group can effectively reduce the viral load in the lung tissue and the trachea of the mice, and the muscle prevention group has a certain effect on reducing the viral load in the lung tissue.

Example 8: freezing electron microscope

The cryo-electron microscope structure of the antibody BD55-5514+BD55-5840 combined with BA.1 Spike is analyzed, and the fact that the epitopes of BD55-5514 and BD55-5840 do not conflict with each other is proved, and the antibodies can be combined on the protein S (Spike) at the same time. Where BD55-5840 can bind both the Receptor Binding Domains (RBDs) in the "up" and "down" conformations, and BD55-5514 binds only the RBDs in the "up" conformation.

The binding epitope of BD55-5514 is similar to ADG20 (Adintrevimab). Sites on BA.1 RBD involved in binding BD55-5514 are mainly Asp405, asn437, asn439, gly502, val503, gly504, tyr508, etc., and sites Pro373-Thr376, val407-Arg408, pro499, tyr501, etc. are also close to the binding interface. However, neutralization of BD55-5514 was not affected by Thr376Ala, asp405Asn, arg408Ser contained in BA.2, but only by Val503 and Gly504 mutations. The conservation degree of the two sites is high, and the two sites hardly occur in nature.

The binding epitope on RBD for BD55-5840 is similar to Ser309 (Sotrovimab), but the key binding sites are different. RBD residues present at the interface are Asp339, glu340, thr345, arg346, lys440, leu441, ser443, lys444, etc., and mutations that primarily affect binding are concentrated at Asp339-Glu340, thr345-Arg 346.

Neutralization experiments show that BD55-5514 and BD55-5840, when paired, provide broad spectrum coronavirus strain B (Sarbecovirus) neutralizing antibody drugs that are most difficult to escape, and have extremely strong neutralizing capacity against all major strains of all major novel coronavirus variants at present, including Omicron.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

An antibody or an 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, which comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 1,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 2, and

VH CDR3, which comprises the amino acid sequence of SEQ ID NO: 3 or comprises an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 3;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:4,

VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:5, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:6; or

(2) The heavy chain variable region comprises:

VH CDR1, which comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:9,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 10, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 11;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 12,

VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 13, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 14; or

(3) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 17,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 18, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO: 19 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 19;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:20,

VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:21, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO:22, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:22; or

(4) The heavy chain variable region comprises:

VH CDR1, which comprises the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:25,

VH CDR2 comprising the amino acid sequence of SEQ ID NO:26 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:26, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO:27 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:27;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:28,

VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:29, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO:30, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added 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 having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:33,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:34, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO:35 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:35;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence of SEQ ID NO:36 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:36,

VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:37, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added 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 having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:41,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:42 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:42, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO:43 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:43;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO:44 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:44,

VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO:45 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:45, and

VL CDR3, which comprises the amino acid sequence of SEQ ID NO:46, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:46; or

(7) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence of SEQ ID NO:49 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:49,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:50 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:50, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO:51 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:51;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO:52 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:52,

VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO:53 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:53, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO:54, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added 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 having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:57,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:58 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:58, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO:59 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:59;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO:60 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:60,

VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO:61 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:61, and

VL CDR3, which comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:62; or

(9) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence of SEQ ID NO:65 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:65,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:66 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:66, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO:67 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:67;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO:68 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:68,

VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO:69 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:69, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO:70, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:70; or

(10) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence of SEQ ID NO:73 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:73,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:74 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:74, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO:75 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:75;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence of SEQ ID NO:76 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:76,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:77 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:77, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO:78, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added 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 having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:81,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:82 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:82, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO:83 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:83;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO:84 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:84,

VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO:85 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:85, and

VL CDR3, which comprises the amino acid sequence of SEQ ID NO:86, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added 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 having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:89,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:90 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:90, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO:91 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:91;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO:92 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:92,

VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO:93 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:93, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO:94, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:94; or

(13) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence of SEQ ID NO:97 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:97,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:98 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:98, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO:99 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO:99;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 100 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 100,

VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 101 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 101, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 102, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 102; or

(14) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 105,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 106 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 106, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO: 107 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 107;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence of SEQ ID NO: 108 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 108,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 109 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 109, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 110, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 110; or

(15) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 113,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 114 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 114, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO: 115 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 115;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence of SEQ ID NO: 116 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 116,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 117 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 117, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 118, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 118; or

(16) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence of SEQ ID NO: 121 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 121,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 122 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 122, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO: 123 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 123;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence of SEQ ID NO: 124 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 124,

VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 125 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 125, and

VL CDR3, which comprises the amino acid sequence of SEQ ID NO: 126, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 126; or

(17) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence of SEQ ID NO: 129 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 129,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 130 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 130, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO: 131 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 131;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 132,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 133 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 133, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 134, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 134; or

(18) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence of SEQ ID NO: 137 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 137,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 138 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 138, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO: 139 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 139;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 140 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 140,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 141 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 141, and

VL CDR3, which comprises the amino acid sequence of SEQ ID NO: 142, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 142; or

(19) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 145,

VH CDR2 comprising the amino acid sequence of SEQ ID NO: 146 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 146, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO: 147 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 147;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence of SEQ ID NO: 148 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 148,

VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 149 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 149, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 150, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 150; or

(20) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence of SEQ ID NO: 153 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 153,

VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 154 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 154, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO: 155 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 155;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 156,

VL CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 157 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 157, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 158, or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 158; or

(21) The heavy chain variable region comprises:

VH CDR1 comprising the amino acid sequence of SEQ ID NO: 161 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 161,

VH CDR2 comprising the amino acid sequence of SEQ ID NO: 162 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 162, and

VH CDR3 comprising the amino acid sequence of SEQ ID NO: 163 or comprising an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 163;

The light chain variable region comprises:

VL CDR1, which comprises the amino acid sequence of SEQ ID NO: 164 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 164,

VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 165 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 165, and

VL CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 166 or an amino acid sequence having 1 or 2 amino acid residues substituted, deleted or added relative to SEQ ID NO: 166.

The antibody or antigen-binding fragment thereof according to claim 1, wherein

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Wherein, the variant has one or several amino acid substitutions, deletions or additions, such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions, compared to the sequence from which it is derived, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; preferably, the substitutions are conservative substitutions.

The antibody or antigen-binding fragment thereof according to 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 light chain constant region derived from a human immunoglobulin (e.g., κ or λ);

Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region as shown in SEQ ID NO:169 and/or a light chain constant region as shown in SEQ ID NO:170.

The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antigen-binding fragment is selected from Fab, Fab', (Fab') ₂ , Fv, disulfide-linked Fv, scFv, 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, or the heavy chain variable region and/or light chain variable region thereof according to any one of claims 1 to 4.

The isolated nucleic acid molecule of claim 5, wherein the nucleic acid molecule is operably linked to an expression control sequence.

An expression vector comprising the nucleic acid molecule according to claim 5 or 6.

A host cell transformed by the nucleic acid molecule of claim 5 or 6 or the expression vector of claim 7.

A method for preparing an antibody or an antigen-binding fragment thereof, comprising,

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

(2) Isolating and purifying the antibody or antigen-binding fragment thereof expressed by 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 to 4, and a pharmaceutically acceptable carrier and/or excipient.

The pharmaceutical composition of claim 10, 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, 13, 14, 15, 16, 17, 18, 19, 20, 21.

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 the antibodies are BD55-5840 and BD55-5514.

A method for preventing and/or treating a disease caused by betacoronavirus 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 to 4 or the pharmaceutical composition according to claims 10 to 13.

The method of claim 14, wherein the antibodies or antigen-binding fragments thereof are more than one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21.

The method of claim 15, wherein the antibody or antigen-binding fragment thereof is as described in numbers (13) and (21).

The method of claim 16, wherein the antibodies are BD55-5840 and BD55-5514.

Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 for preparing a medicament for preventing and/or treating a disease caused by beta coronavirus infection.

The use of claim 18, wherein there is more than one antibody or antigen-binding fragment thereof, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21.

The use of claim 19, wherein the antibody or antigen-binding fragment thereof is as described in numbers (13) and (21).

The use of claim 20, wherein the antibodies are BD55-5840 and BD55-5514.

The method of any one of claims 14 to 17 or the use of any one of claims 18 to 21, wherein the betacoronavirus includes the novel coronavirus (SARS-CoV-2) and its variants, severe acute respiratory syndrome coronavirus (SARS-CoV) and its variants, and SARS-related coronavirus (SARSr-CoV).

The method or use of claim 22, wherein the SARS-CoV-2 variant is selected from Alpha (B.1.1.7), Beta (B.1.351), Gamma (Gamma, P.1), 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 according to any one of claims 1 to 4, and a detectable label linked to the antibody or antigen-binding fragment thereof.

The conjugate of claim 25, wherein the antibodies or antigen-binding fragments thereof are more than one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21.

The conjugate of claim 26, wherein the antibody or antigen-binding fragment thereof is as described in numbers (13) and (21).

The conjugate of claim 27, wherein the antibodies are BD55-5840 and BD55-5514.

The conjugate of any one of claims 25 to 28, wherein the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide or biotin.

A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the conjugate according to any one of claims 25 to 29.

The kit of claim 30, wherein the kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof.

The kit of claim 30 or 31, wherein the antibodies or antigen-binding fragments thereof are more than one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21.

The kit of claim 32, wherein the antibody or antigen-binding fragment thereof is as described in numbers (13) and (21).

The kit of claim 33, wherein the 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 (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide 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 according to any one of claims 1 to 4 or the conjugate according to any one of claims 25 to 29;

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

The detection of the binding represents the presence of betacoronavirus in the sample, or the detection of the strength of the binding represents the level of betacoronavirus in the sample.

The method of claim 36, wherein the antibodies or antigen-binding fragments thereof are more than one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21.

The method of claim 37, wherein the antibody or antigen-binding fragment thereof is as described in numbers (13) and (21).

The method of claim 38, wherein the antibodies are BD55-5840 and BD55-5514.

The method of any one of claims 36-39, wherein the sample is a blood sample (e.g., whole blood, plasma, or serum), feces, oral or nasal secretions, or bronchoalveolar lavage fluid from the subject.

The method of claim 40, wherein the subject is a mammal, such as a human.

The method of any one of claims 36 to 39, wherein the sample is not a sample from a subject, for example the sample is from a vaccine sample.

Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the conjugate according to any one of claims 25 to 29 in the preparation of a kit for detecting the presence or level of betacoronavirus in a sample.

The use of claim 43, wherein there is more than one antibody or antigen-binding fragment thereof, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21.

The use of claim 44, wherein the antibody or antigen-binding fragment thereof is as described in numbers (13) and (21).

The use of claim 45, wherein the antibodies are BD55-5840 and BD55-5514.